WO2019169179A1 - Augmenting efficacy of cancer therapies using probiotic based compositions - Google Patents

Augmenting efficacy of cancer therapies using probiotic based compositions Download PDF

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WO2019169179A1
WO2019169179A1 PCT/US2019/020121 US2019020121W WO2019169179A1 WO 2019169179 A1 WO2019169179 A1 WO 2019169179A1 US 2019020121 W US2019020121 W US 2019020121W WO 2019169179 A1 WO2019169179 A1 WO 2019169179A1
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cancer
lactobacillus
probiotic
formulation
probiotics
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PCT/US2019/020121
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French (fr)
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Kim SHAFER
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Shafer Kim
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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
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/255—Esters, e.g. nitroglycerine, selenocyanates of sulfoxy acids or sulfur analogues thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
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    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
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    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
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    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4873—Cysteine endopeptidases (3.4.22), e.g. stem bromelain, papain, ficin, cathepsin H
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/54—Mixtures of enzymes or proenzymes covered by more than a single one of groups A61K38/44 - A61K38/46 or A61K38/51 - A61K38/53
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4866—Organic macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics

Definitions

  • the invention pertains to the field of cancer therapy. Specifically, the invention pertains to methods and formulations of probiotics with digestive enzymes to increase efficacy of cancer therapies and/or for reducing the toxicity and adverse events associated with said cancer therapies.
  • chemotherapy-associated diarrhea occurs in 50-80% of cancer patients depending on the chemotherapy regimen that is administered [1, 2].
  • Some chemotherapy regimens for example, those utilizing irinotecan, fluorouracil, and leucovorin, can lead to potentially life-threatening gastrointestinal syndrome.
  • Adverse events include diarrhea (including Grades 3 and 4 severe diarrhea), nausea, vomiting, fatigue, malaise, leukopenia, and neutropenia can occur, to name a few. These adverse events can lead to dire medical consequences.
  • Diarrhea and nausea that are associated with cancer therapy can lead to dehydration, electrolyte imbalances, malnutrition, pain, and bleeding as a result of damge to the gastrointestinal tract.
  • patients have low immune function and inflammation.
  • Cancer treatments can therefore render patients more susceptible to various infections, which can themselves cause or exacerbate some of the abovementioned adverse events.
  • infections with Clostridium difficile are twice as common in hospitalized cancer patients as compared to other inpatients [3].
  • the antibiotics that are prescribed to treat these infections can themselves cause symptoms such as diarrhea; for example, for treating C. difficile, these antibodies may include vancomycin, fidaxomicin, or metronidazole.
  • the microbiome in the human gut is composed of 500-1000 distinct bacterial species and up to 10 14 total bacteria that are responsible for digestive health, including the assurance of nutrient and vitamin bioavailability and energy metabolism, and also have a significant impact on the immune system of a subject.
  • intestinal microorganisms may determine the outcome of cancer treatments and are also directly impacted by the treatments, often in a negative manner.
  • Gut dysbiosis whereby the composition of microorganisms is altered and often their diversity is reduced, predicted resistance to immunotherapeutic interventions such as checkpoint inhibitors in melanoma patients [5].
  • a low diversity of commensal microorganisms was also associated with immune suppression in cancer patients [6].
  • 5-fluoro uracil a first line agent for the treatment of metastatic colorectal agent, which is associated with severe colonic mucositis indicated by weight loss, diarrhea, bloody stool, shortened colon, and infiltration of inflammatory cells. 5-FU diminishes bacterial richness and diversity in the gut, leading to reduced overall abundance of important phyla involved in normal microbial metabolism.
  • Aspect 1 A method of increasing efficacy of a cancer therapy comprising administration of a probiotic composition.
  • Aspect 2 The method of aspect 1, wherein said probiotic composition comprises one of more probiotics.
  • Aspect 3 The method of aspect 2, wherein said probiotic composition is comprised of one or more probiotics selected from a group comprising of: a)
  • Bifidobacterium infantis b) Bifidobacterium bifidum; c) Lactobacillus acidophilus; d) Lactobacillus salivarius; e) Lactobacillus plantarum; f) Lactobacillus rhamnosus; g) Bifidobacterium longum; h) Lactobacillus casei; and i) Lactobacillus paracasei.
  • Aspect 4 The method of aspect 1, wherein said probiotic composition comprises the addition of digestive enzymes to said probiotics
  • Aspect 5 The method of aspect 4, wherein said digestive enzymes are selected from a group comprising of: a) amylase; b) glucoamylase; c) lipase; d) bromelain; e) maltase; f) lactase; g) hemicellulose; h) xylanase; i) papain, and j) invertase.
  • Aspect 6 The method of aspect 1, wherein said cancer therapy is one or more therapies selected from a group comprising of: a) immunotherapy; b)
  • Aspect 7 The method of aspect 1, wherein augmentation of efficacy of said cancer therapy is reduction of toxicity associated with said cancer therapy.
  • Aspect 8 The method of aspect 1, wherein said probiotic composition is administered in a manner to augment immunogenic death of tumor cells.
  • Aspect 9. The method of aspect 8, wherein said immunogenic death of tumor cells is associated with release of HMGB-l from tumors.
  • Aspect 10 The method of aspect 8, wherein said immunogenic death of tumor cells is elicited by radiation therapy and augmented by treatment with said probiotic composition.
  • Aspect 11 The method of aspect 10, wherein said radiation therapy is capable of triggering an abscopal effect.
  • Aspect 12 The method of aspect 11, wherein said abscopal effect is amplified by administration of said probiotic composition.
  • Aspect 13 The method aspect 12, wherein said abscopal effect comprises one or more of the following biological events: a) recognition of non-viable cancer cells by antigen presenting cells; b) uptake of immunogenic agents from said non-viable cancer cells by said antigen presenting cells; c) presentation of peptides derived from said immunogenic agents by said antigen presenting cells to T cells; and d) activation of T cells capable of recognizing and killing identical or substantially similar cells to said non- viable cancer cells.
  • Aspect 14 The method of aspect 13, wherein said non-viable cancer cells are the result of exposure to radiation capable of inducing DNA damage.
  • Aspect 15 The method of aspect 13, wherein said non-viable cancer cells are the result of exposure to radiation capable of inducing ceramide signalling.
  • Aspect 16 The method of aspect 13, wherein said non-viable cancer cells are the result of exposure to radiation capable of inducing caspase activation.
  • Aspect 17 The method of aspect 13, wherein said non-viable cancer cells are the result of exposure to chemotherapy capable of inducing DNA damage.
  • Aspect 18 The method of aspect 13, wherein said non-viable cancer cells are the result of exposure to chemotherapy capable of inducing DNA damage.
  • Aspect 19 The method of aspect 13, wherein said antigen presenting cells are monocytes.
  • Aspect 20 The method of aspect 13, wherein said antigen presenting cells are macrophages.
  • Aspect 21 The method of aspect 13, wherein said antigen presenting cells are B cells.
  • Aspect 22 The method of aspect 13, wherein said antigen presenting cells are endothelial cells.
  • Aspect 23 The method of aspect 13, wherein said antigen presenting cells are dendritic cells.
  • Aspect 24 The method of aspect 13, wherein said dendritic cells are myeloid dendritic cells.
  • Aspect 25 The method of aspect 13, wherein said dendritic cells are lymphoid dendritic cells.
  • Aspect 26 The method of aspect 13, wherein said immunogenic agents are agents capable of acting as tumor antigens.
  • Aspect 27 The method of aspect 13, wherein said immunogenic agents are capable of stimulating a“danger signal” to the immune system.
  • Aspect 28 The method of aspect 27, wherein said danger signal comprises activation of a toll like receptor.
  • Aspect 29 The method of aspect 28, wherein said toll like receptor is selected from a group comprising of TLR 1-9.
  • Aspect 30 The method of aspect 13, wherein said presentation of peptides is accomplished by loading of peptides into MHC I or MHC II.
  • Aspect 31 The method of aspect 30, wherein said peptides presented on MHC I are involved in stimulation of CD8 T cells.
  • Aspect 32 The method of aspect 30, wherein said peptides presented on MHC I are involved in stimulation of CD4 T cells.
  • Aspect A method of preventing hematological toxicity of a cancer therapy comprising administration of a probiotic composition.
  • Aspect 34 The method of aspect 33, wherein said probiotic composition comprises one of more probiotics.
  • Aspect 35 The method of aspect 34, wherein said probiotic composition is comprised of one or more probiotics selected from a group comprising of: a)
  • Bifidobacterium infantis b) Bifidobacterium bifidum; c) Lactobacillus acidophilus; d) Lactobacillus salivarius; e) Lactobacillus plantarum; f) Lactobacillus rhamnosus; g) Bifidobacterium longum; h) Lactobacillus casei; and i) Lactobacillus paracasei.
  • Aspect 36 The method of aspect 33, wherein said probiotic composition comprises the addition of digestive enzymes to said probiotics
  • Aspect 37 The method of aspect 36, wherein said digestive enzymes are selected from a group comprising of: a) amylase; b) glucoamylase; c) lipase; d) bromelain; e) maltase; f) lactase; g) hemicellulose; h) xylanase; i) papain, and j) invertase.
  • said cancer therapy is selected from a group comprising of: a) radiotherapy; b) chemotherapy; c) immunotherapy; d) hyperthermia; and e) surgery.
  • Aspect 39 The method of aspect 33, wherein said hematopoietic toxicity is neutropenia.
  • Aspect 40 The method of aspect 33, wherein said hematopoietic toxicity is anemia.
  • Aspect 41 The method of aspect 33, wherein said hematopoietic toxicity is thrombocytopenia.
  • Aspect 42 The method of aspect 33, wherein said hematopoietic toxicity is pancytopenia.
  • Aspect 43 The method of aspect 33, wherein said hematopoietic toxicity is lymphopenia.
  • Aspect 44 The method of aspect 1 and 33, wherein said probiotic composition is a pharmaceutical composition comprising: a formulation of probiotics comprising: Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei, and Lactobacillus paracasei; and, a formulation of digestive enzymes comprising: amylase, glucoamylase, lipase, bromelain, maltase, lactase, hemicellulase, xylanase, papain, and invertase.
  • a formulation of probiotics comprising: Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus
  • Aspect 45 The method of aspect 44, wherein the form of composition is selected from the group consisting of a pharmaceutically acceptable: pill, a tablet, a caplet, a capsule, powder, a suspension, a gel, and a liquid.
  • Aspect 46 The method of aspect 45, wherein said probiotics are present in a total weight of 116.20 mg.
  • Aspect 47 The method of aspect 45, wherein said digestive enzymes are present in a total weight of 272.65 mg.
  • Aspect 48 The method of aspect 44, wherein said formulations of probiotics and digestive enzymes are contained in a plurality of capsules.
  • Aspect 49 The method of aspect 48, wherein said formulations of probiotics and digestive enzymes are present in the same, single, capsule.
  • Aspect 50 The method of aspect 44, wherein said probiotics are present in the following amounts: Bifidobacterium Infantis - between 5-7 billion cfu,
  • Aspect 51 The method of aspect 50, wherein said probiotics are present in the following amounts: Bifidobacterium Infantis - 6 billion cfu, Bifidobacterium Longum
  • Aspect 52 The method of Aspect 44, wherein said enzymes are present in the following amounts: hemicellulase - between 5-11 mg, xylanase - between 2-6 mg, amylase - between .5 - 2.5 mg, glucoamylase - between 30 - 70 mg, maltase - between 8
  • Aspect 53 The method of Aspect 52, wherein said enzymes are present in the following amounts: hemicellulase - 8 mg, xylanase - 3.9 mg, amylase - 1.33 mg, glucoamylase - 50 mg, maltase - 10 mg, papain - 1.7 mg, bromelain - 18 mg, lipase - 25 mg, invertase - 1.5 mg, and lactase - 9.5 mg.
  • Aspect 54 The method of aspects 1 and 33, wherein said probiotic composition is a pharmaceutical composition comprising: (a) probiotic formulation comprising Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus rhamnosus GG,
  • Bifidobacterium longum Lactobacillus casei, Lactobacillus paracasei, and (b) a formulation of digestive enzymes.
  • Aspect 55 The method of aspect 54, wherein said digestive enzymes are selected from the group consisting of: a) proteases; b) Carbohydrate-digesting enzymes; c) Fiber-digesting enzymes; and, d) lipases.
  • Aspect 56 The method of aspect 55, wherein said carbohydrate-digesting enzymes are selected from the group consisting of: a) Amylase; b) Glucoamylase; c) Lactase; d) Invertase; and, e) Maltase.
  • Aspect 57 The method of aspect 55, wherein said fiber-digesting enzymes are selected from the group consisting of: a) Xylanase; and b) Hemicellulase.
  • Aspect 58 The method of aspect 54, wherein the form of pharmaceutical composition is selected from the group consisting of: a pill, a tablet, a caplet, a capsule, powder, a suspension, a gel, and, a liquid.
  • Aspect 59 The method of aspect 54, wherein said formulations of probiotics and digestive enzymes are formulated in the same single formulation.
  • Aspect 60 The method of aspect 59, wherein said single formulation is a capsule.
  • Aspect 61 The method of aspect 55, wherein said formulation of digestive enzymes comprises at least two proteases.
  • a probiotic formulation having: Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius,
  • Lactobacillus plantarum Lactobacillus rhamnosus, Bifidobacterium longum,
  • Lactobacillus casei Lactobacillus paracasei; in conjunction with a formulation of digestive enzymes having: amylase, glucoamylase, lipase, bromelain, maltase, lactase, hemicellulase, xylanase, papain, and invertase that is useful in the preparation of a medicament for lowering cancer therapy associated toxicities in a subject in need.
  • Aspect 63 The probiotic formulation of aspect 62, wherein said formulation of probiotics and digestive enzymes are combined in capsules.
  • Aspect 64 The probiotic formulation of aspect 62, wherein said probiotics and digestive enzymes are formulated to be administered orally.
  • Aspect 65 The probiotic formulation of Aspect 62, wherein said probiotics are present in the following amounts: Bifidobacterium Infantis - between 5-7 billion cfu, Bifidobacterium Longum -between 750 million and 2 billion cfu, Bifidobacterium Bifidum -between 3-5 billion cfu, Lactobacillus Rhamnosus - between 5-7 billion cfu, Lactobacillus Acidophilus -between 1-3 billion cfu, Lactobacillus salivarius -between 1- 3 billion cfu, Lactobacillus plantarum - between 1-3 billion cfu, Lactobacillus Casei - between 750,000 million-2 billion cfu, Lactobacillus paracasei - between 1-3 billion cfu.
  • Aspect 66 The probiotic formulation of Aspect 65, wherein said probiotics are present in the following amounts: Bifidobacterium Infantis - 6 billion cfu, Bifidobacterium Longum - 1 billion cfu, Bifidobacterium Bifidum - 4 billion cfu, Lactobacillus Rhamnosus - 6 billion cfu, Lactobacillus Acidophilus - 2 billion cfu, Lactobacillus salivarius - 2 billion cfu, Lactobacillus plantarum - 2 billion cfu,
  • Aspect 67 The probiotic formulation of Aspect 66, wherein said enzymes are present in the following amounts: hemicellulase - between 5-11 mg, xylanase - between 2-6 mg, amylase - between .5 - 2.5 mg, glucoamylase - between 30 - 70 mg, maltase - between 8 - 12 mg, papain - between .5 - 3mg, portease, such as bromelain - between 13-23 mg, lipase - between 18- 32 mg, invertase - between .5-3 mg, lactase - between 8.0-11 mg.
  • Aspect 68 The probiotic formulation of Aspect 67, wherein said enzymes are present in the following amounts: hemicellulase - 8 mg, xylanase - 3.9 mg, amylase - 1.33 mg, glucoamylase - 50 mg, maltase - 10 mg, papain - 1.7 mg, bromelain - 18 mg, lipase - 25 mg, invertase - 1.5 mg, and lactase - 9.5 mg.
  • Aspect 69 The probiotic formulation of aspect 62, wherein said formulation is administered together with a chemotherapeutic agent.
  • Aspect 70 The probiotic formulation of aspect 69, wherein said
  • chemotherapeutic agent is capable of augmenting expression of immunogenic molecules on a cancer cell or plurality of cancer cells.
  • Aspect 71 The probiotic formulation of aspect 70, wherein said
  • chemotherapeutic agents are selected from a group comprising of: a) alkylating agents; b) plant alkaloids; c) antitumor antibiotics; d) antimetabolites; e) topoisomerase inhibitors; and f) ribonucleotide reductase inhibitors.
  • Aspect 72 The probiotic formulation of aspect 71, wherein said
  • chemotherapeutic agent is vinblastine at a concentration and duration sufficient to augment HLA I expression.
  • Aspect 73 The probiotic formulation of aspect 71, wherein said
  • chemotherapeutic agent is cyclophosphamide at a concentration and duration sufficient to augment HLA I expression.
  • Aspect 74 The probiotic formulation of aspect 71, wherein said
  • chemotherapeutic agent is topotecan at a concentration and duration sufficient to augment HLA I expression.
  • Aspect 75 The probiotic formulation of aspect 71, wherein said
  • chemotherapeutic agent is paclitaxel at a concentration and duration sufficient to augment HLA I expression.
  • Aspect 76 The probiotic formulation of aspect 62 wherein said formulation is administered to protect from toxicity effects of chemotherapy.
  • Aspect 77. The probiotic formulation of aspect 76, wherein said toxicity effect of chemotherapy is gastrointestinal toxicity.
  • Aspect 78 The probiotic formulation of aspect 76, wherein said toxicity effect of chemotherapy is hematological toxicity.
  • Aspect 79 The probiotic formulation of aspect 76, wherein said toxicity effect of chemotherapy is cardiac toxicity.
  • Aspect 80 The probiotic formulation of aspect 76, wherein said toxicity effect of chemotherapy is gastrointestinal toxicity.
  • Aspect 81 The probiotic formulation of aspect 76, wherein said toxicity effect of chemotherapy is vascular toxicity.
  • Aspect 82 The probiotic formulation of aspect 76, wherein said toxicity effect of chemotherapy is nephrotoxicity toxicity.
  • Aspect 83 The probiotic formulation of aspect 76, wherein said toxicity effect of chemotherapy is neurological toxicity.
  • Aspect 84 The probiotic formulation of aspect 76, wherein said toxicity effect of chemotherapy is alopecia.
  • Aspect 85 The probiotic formulation of aspect 76, wherein said toxicity is severity of grade 2 or higher diarrhea resulting from an anti-cancer chemotherapy in a subject in need thereof.
  • Aspect 86 The probiotic formulation of aspect 76, wherein said
  • chemotherapy comprises antibody therapy with or without small-molecule chemotherapy.
  • Aspect 87 The probiotic formulation of aspect 76, wherein said
  • chemotherapy comprises bevacizumab, cetuximab or panitumumab antibody therapy with or without small-molecule chemotherapy.
  • Aspect 88 The probiotic formulation of aspect 76, wherein said probiotic formulation is administered in a regimen comprising daily administration of said formulation for 2 to 10 days, and said chemotherapy cycle is 8 to 24 days.
  • chemotherapy comprises administration of one or more compounds selected from the group consisting of antimetabolites, alkylating agents, anticancer antibiotics, microtubule targeting agents, topoisomerase inhibitors, alkaloids, antibodies, pyrimidine analogs, purine analogs, folate antagonists, epidipodophyllotoxins, DNA damaging agents, antiplatelet agents, platinum coordination complexes, hormones, hormone analogs, aromatase inhibitors, anti-angiogenic compounds, growth factor inhibitors, angiotensin receptor blockers, nitric oxide donors, antisense oligonucleotides, cell cycle inhibitors, differentiation inducers, mTOR inhibitors, mitochondrial dysfunction inducers, chromatin disruptors.
  • compounds selected from the group consisting of antimetabolites, alkylating agents, anticancer antibiotics, microtubule targeting agents, topoisomerase inhibitors, alkaloids, antibodies, pyrimidine analogs, purine analogs, folate antagonists, epidipodophyllotoxins, DNA
  • Aspect 90 The probiotic formulation of aspect 76, wherein said
  • chemotherapy comprises administration of one or more compounds selected from the group consisting of 5-fluorouracil (5-FU), floxuridine, capecitabine, gemcitabine, cytarabine, irinotecan, doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, mitoxantrone, topotecan, lapatinib, oxaliplatin, cisplatin, carboplatin, folinic acid, methothrexate, erlotinib, sorafenib, and lapatinib.
  • 5-FU 5-fluorouracil
  • floxuridine floxuridine
  • capecitabine gemcitabine
  • gemcitabine gemcitabine
  • cytarabine irinotecan
  • chemotherapy comprises administration of oxaliplatin or irinotecan.
  • chemotherapy comprises administration of oxaliplatin or irinotecan in combination with cetuximab, bevacizumab, and/or panitumumab.
  • chemotherapeutic agent(s) is administered at least during the first two consecutive days from the beginning of each chemotherapy cycle.
  • Aspect 94 The probiotic formulation of aspect 76, wherein said
  • chemotherapy is administered as FOLFOX or FOLFIRI chemotherapy regimen.
  • Aspect 95 The probiotic formulation of aspect 65, wherein said formulation is administered in 6 to 8 doses per day.
  • Aspect 96 The probiotic formulation of aspect 66, wherein said formulation is administered in 6 to 8 doses per day.
  • Aspect 97 The probiotic formulation of aspect 67, wherein said formulation is administered in 6 to 8 doses per day.
  • Aspect 98 Use of a probiotic formulation in the preparation of a medicament for improving the efficacy of a cancer therapy comprising in a subject with cancer scheduled to undergo or is undergoing treatment for cancer by administering to said subject said probiotic formulation in an amount sufficient to improve the efficacy of the cancer therapy.
  • Aspect 99 Use of a probiotic formulation in the preparation of a
  • Fig. 1A is a bar graph showing that as the reactors acidify during changes in microbial activity, base is added.
  • Fig. 1B is a bar graph showing that as the reactors acidify during changes in microbial activity, base is added.
  • Figure 2 depicts bar graphs showing analysis of acetate, propionate, and butyrate concentrations in the SHIME.
  • Figure 3 depicts bar graphs showing the effects of a curative (CUR) and preventive (PREV) administration of the probiotic with digestive enzymes supplement as compared to a control SHIME (CTRL) on luminal Lactobacillus and Bifidobacterium levels in the proximal (PC) and distal colon (DC).
  • CUR curative
  • PREV preventive
  • Figure 4 depicts bar graphs showing the effect of a curative (CUR) and preventive (PREV) administration of the probiotic with digestive enzymes supplement as compared to a control SHIME (CTRL) on luminal Bacteroidetes, and Finnicutes levels in the proximal (PC) and distal colon (DC).
  • CUR curative
  • PREV preventive
  • Figure 5 depicts a bar graph showing the differences in microbial compositions at the phylum level in the proximal and distal colons caused by the probiotic with digestive enzymes supplement.
  • Figure 6A depicts a bar graph showing modulation of IL-6 in an in vitro model of intestinal inflammation by metabolites from SHIME reactors treated with the probiotic with digestive enzymes supplement.
  • Figure 6B depicts a bar graph showing modulation of IL-10 in an in vitro model of intestinal inflammation by metabolites from SHIME reactors treated with the probiotic with digestive enzymes supplement.
  • This invention teaches methods and compositions for augmenting the efficacy of cancer therapeutic drugs using formulations comprising probiotic compositions combined with digestive enzymes. This invention also teaches methods and compositions for preventing, improving or alleviating adverse events that are associated with cancer therapeutic drugs in a subject in need of treatment.
  • probiotic (based) formulation and“probiotic (based) composition” may refer to a formulation comprising probiotics or to a formulation containing probiotics combined together with digestive enzymes.
  • formulation and “composition” encompass said probiotics or said probiotics with digestive enzyme ingredients that are mixed together into a delivery vehicle for administration to or consumption by a subject in need thereof.
  • administration or consumption of a probiotic based formulation by said subject may occur orally via swallowing of a capsule, tablet, or soft gel, orally via swallowing of a powder that may be dissolved or distributed in food or water, orally via gavage, sublingually via a troche, or rectally as a suppository.
  • each probiotic microorganism in said probiotic based formulation is measured by activity per gram(g), which is expressed as colony forming units (CFU).
  • each digestive enzymes in said probiotic based formulation is measured by enzyme activity per gram (g).
  • the activities of each digestive enzyme are measured using methods known in the art, as follows: Hemicellulase units (HU) for hemicellulase, Xylanase unit (XU) for xylanase, Alpha - amylase Dextrinizing units (DU) for amylase, amyloglucosidase (AGU) for glucoamylase, degrees Diastatic power (DP) for maltase, enzyme activity or units (TP) for papain, Gelatin Digestion units (GDU) for bromelain, Fungi Lipase-Intemational FIP standard (FIP) for lipase, Summer units (SU) for invertase, lactase units (ALU) for lactase.
  • HU Hemicellulase units
  • XU Xylanase unit
  • AGU amyloglucos
  • cancer therapeutic drug(s) “cancer therapeutic agent(s)”,“cancer drug(s)”,“cancer therapy”,“oncology drug(s)”,“treatment agent(s)”, and the like, are used synonymously to refer to the cancer treatment received by a subject in need thereof, as is recognized in the art.
  • these cancer treatments may include one or more of the following treatment categories: a) immunotherapy; b) chemotherapy; c) surgery; d) radiation therapy; e) hyperthermia; f) radiation; g) targeted therapy; h) hormone therapy; i) stem cell transplantation.
  • Use of these terms also signifies that one or more of the abovementioned treatment categories may be administered to said subject.
  • “subject” refers to a human (e.g. a subject with cancer and/or a subject taking one or more cancer therapeutic drugs and/or one or more antibiotics) and, more broadly speaking, may include any mammal. Accordingly, aspects of the present invention may be practiced in human clinical medicine as well as in veterinary medicine.
  • the phrase“augmentation of efficacy” or“improved efficacy” of a cancer therapeutic agent includes enhancement of one or more of the therapeutic effect(s) of said cancer drug.
  • the therapeutic effect of a cancer drug can refer to inhibition of tumor growth, induction of tumor regression, suppression of metastasis, inhibition of metastasis, suppression of proliferation, induction of programmed and/or non-programmed cell death.
  • cancer inhibiting drugs can be observed by one or more of the following clinical endpoints: a) Overall survival (OS); b) Progression-free survival (PFS); c) Time to progression (TTP); d) Time to treatment failure (TTF); c) Event- free survival; d) Objective response rate (ORR); e) Duration of response (DR); f) Improvement in immunoscore; and g) Decrease in cancer- associated biomarkers including but not limited to tumor-associated antigens (TAAs).
  • OS Overall survival
  • PFS Progression-free survival
  • TTP Time to progression
  • TTF Time to treatment failure
  • Event- free survival d
  • Objective response rate ORR
  • DR Duration of response
  • f Improvement in immunoscore
  • TAAs tumor-associated antigens
  • said subject may be afflicted with one or more types of cancer including but not limited to the following: brain, melanoma, bladder, breast, cervix, colon, rectal, head and neck, kidney, lung, non- small cell lung, mesothelioma, ovary, prostate, sarcoma, stomach, uterus, as well as carcinomas, including spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, carcinoma villosum, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepid
  • Sarcomas include, chondrosarcoma, fibrosarcoma, lymphosarcoma, melano sarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilns' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblast
  • One embodiment of the present invention teaches a method of improving the efficacy of a cancer therapy comprising the following: (a) Identifying a subject with cancer who plans to undergo or is undergoing treatment for cancer; (b) administering to said subject a formulation comprising a probiotic based formulation, which is preferably a formulation comprised of probiotics with digestive enzymes.
  • said formulation of probiotics with digestive enzymes comprises of one or more probiotics selected from a group comprising of: a) Bifidobacterium inf antis, b) Bifidobacterium bifidum, c) Lactobacillus acidophilus, d) Lactobacillus salivarius; e) Lactobacillus plantarum, f) Lactobacillus rhamnosus, g) Bifidobacterium longum, h) Lactobacillus casei; and i) Lactobacillus paracasei.
  • probiotics selected from a group comprising of: a) Bifidobacterium inf antis, b) Bifidobacterium bifidum, c) Lactobacillus acidophilus, d) Lactobacillus salivarius; e) Lactobacillus plantarum, f) Lactobacillus rhamnosus, g) Bifidobacterium longum,
  • Said formulation of probiotics with digestive enzymes may also be comprised of one or more digestive enzymes selected from a group comprising: a) amylase; b) glucoamylase; c) lipase; d) bromelain; e) maltase; f) lactase; g) hemicellulose; h) xylanase; i) papain, and j) invertase.
  • one or more digestive enzymes selected from a group comprising: a) amylase; b) glucoamylase; c) lipase; d) bromelain; e) maltase; f) lactase; g) hemicellulose; h) xylanase; i) papain, and j) invertase.
  • a preferred composition for improving the efficacy of cancer therapy in a subject comprises the following active ingredients: a) Bifidobacterium infantis; b) Bifidobacterium bifidum, c) Lactobacillus acidophilus, d) Lactobacillus salivarius; e) Lactobacillus plantarum; f) Lactobacillus rhamnosus; g) Bifidobacterium longum; h) Lactobacillus casei and i) Lactobacillus paracasei j) amylase; k) glucoamylase; 1) lipase; m) bromelain; n) maltase; o) lactase; p) hemicellulose; q) xylanase; r) papain, and s) invertase.
  • this compositon comprises between 100-500 mg by weight of digestive enzymes and between 50-250 mg by weight digestive enzymes per dose.
  • said formulation comprises 272.65 mg by weight of digestive enzymes and 116.20 mg of probiotics per dose.
  • this formulation is contained in capsules for oral administration.
  • treatment cycle is used herein to refer to a period of time between the initial administration of an anti-cancer agent and its repeat administration.
  • the cycle of the FOLFOX4 chemotherapy includes 14 days, wherein anti-cancer agents are administered only for the first 2 days of the cycle as follows: Day 1: oxaliplatin 85 mg/m.sup.2 IV infusion and leucovorin 200 mg/m.sup.2 IV infusion both given over 120 minutes at the same time in separate bags, followed by 5-FU 400 mg/m.sup.2 IV bolus given over 2-4 minutes, followed by 5-FU 600 mg/m.sup.2 IV infusion as a 22 -hour continuous infusion; Day 2: leucovorin 200 mg/m.sup.2 IV infusion, followed by 5-FU 400 mg/m.sup.2 IV bolus given over 2-4 minutes, followed by 5-FU 600 mg/m.sup.2 IV infusion as a 22-hour continuous infusion.
  • Day 1 oxaliplatin 85 mg/m.sup.2 IV infusion and leucovorin 200 mg/m.sup.2 IV infusion both given over 120 minutes at the
  • the cycle of the FOLFIRI chemotherapy includes 14 days, wherein anti-cancer agents are administered only for the first 2 days of the cycle as follows: irinotecan (180 mg/m.sup.2 IV over 90 minutes) concurrently with folinic acid (400 mg/m.sup.2 [or 2. times.250 mg/m.sup.2] IV over 120 minutes), followed by fluorouracil (400-500 mg/m.sup.2 IV bolus) then fluorouracil (2400-3000 mg/m.sup.2 intravenous infusion over 46 hours).
  • Bevacizumab is usually given intravenously every 14 days, although the frequency can be dose dependent (for example 5 mg/kg by intravenous infusion every two weeks or 7.5 mg/kg by intravenous infusion every three weeks).
  • it is given in combination with the chemotherapy drug 5-FU (5-fluorouracil), leucovorin, and oxaliplatin or irinotecan.
  • 5-FU 5-fluorouracil
  • leucovorin oxaliplatin or irinotecan.
  • One recommended dose and schedule for cetuximab is 400 mg/m.sup.2 administered intravenously as a l20-minute infusion as an initial dose, followed by 250 mg/m.sup.2 infused over 30 minutes weekly, preferably in combination with FOLFIRI.
  • said probiotic based formulation for example, consisting of probiotics with digestive enzymes
  • said probiotic based formulation may be given for a duration that surpasses the therapeutic regimen that said subject undergoes.
  • administration of said probiotic based formulation may also be initiated during any of the treatment cycles with a given cancer therapeutic agent.
  • said probiotic based formulation will be administered on a daily basis thereafter.
  • the probiotic based formuations may be adminsiterd to a subject in need thereof a doses of 2-10 capsules per day and on a daily basis.
  • this formulation is administered in 6-8 doses per day and on a daily basis.
  • the probiotics based formuation is taken at a dose of 6 capsules per day; specifically, 2 capsules taken in the morning, 2 capsules at midday, and 2 capsules at night on days when cancer treatment agents are not being administered to said subject.
  • Eight capsules may be taken daily on each day that a cancer treatment agent is being administered to said subject; specifically, 2 capsules taken in the morning, 2 capsules at midday, 2 capsules at night, 1 additional capsule taken within 2 hours prior to the cancer treatment agent is administered, and 1 additional capsule taken within two hours after the cancer treatment agent has been administered.
  • This dosing regimen can be continued for the duration of cancer treatment cycles and irrespective of the specific cancer drug(s) being administered to said subject.
  • said probiotic based formulation contains said probiotics having the following activity/gram: between 25-150 billion colony forming units (cfu) for Bifidobacterium inf antis, between 50-150 billion cfu for Bifidobacterium longum, between 350-550 billion cfu for Bifidobacterium bifidum, between 100-300 billion cfu for Lactobacillus rhamnosus, between 100-300 billion cfu for Lactobacillus acidophilus, between 200-400 billion for Lactobacillus salivarius, between 300-500 billion cfu for Lactobacillus plantarum, between 200-400 billion cfu for Lactobacillus casei, and between 300-500 billion cfu Lactobacillus paracasei.
  • cfu colony forming units
  • said probiotic based formulation comprises digestive enzymes having the following activity/gram: between 300,000-500,000 HCU for hemicellulase, between 100,000-300,000 XU for xylanase, between 100,000-300,000 DU for amylase, between 900-1,100 AGU for glucoamylase, between 10,000-30,000 DP for maltase, between 900,000, 1,100,000 TU for papain, between 2,000-4,000 gdu for bromelain, between 150-300 FIP for lipase, between 100,000-300,000 SU for invertase, between 75,000-150,000 ALU for lactase.
  • digestive enzymes having the following activity/gram: between 300,000-500,000 HCU for hemicellulase, between 100,000-300,000 XU for xylanase, between 100,000-300,000 DU for amylase, between 900-1,100 AGU for glucoamylase, between 10,000-30,000 DP for maltas
  • the invention provides the ability to augment dosage of chemotherapy or other oncology drugs in order to achieve killing of tumor cells without achieving dose limiting toxicity.
  • the invention provides means of providing multiple therapies that would otherwise not be possible due to toxicities, in other embodiments the invention provides means of suppressing cytokine release syndrome associated with cancer therapies, particularly immunotherapies, in other embodiments the invention provides means of reducing adverse events associated with administration of checkpoint inhibitors through selectively enhancing tolerance towards self antigens while not suppressing or minimally suppressing antitumor immunity.
  • the invention teaches administration of probiotics and/or combinations of probiotics and digestive enzymes, collectively referred to herein as probiotic based compositions.
  • the present invention teaches methods for prevention or management of cancer treatment-related adverse events comprising the following: (a) Identifying a subject with cancer who has plans to undergo, is undergoing or has undergone treatment for cancer; (b) Identifying that said subject also exhibits symptoms of cancer treatment-related adverse events or is at-risk for experiencing cancer treatment-related adverse events (b) administering to said subject a formulation comprising probiotics with digestive enzymes.
  • “prevention” or“management” of said adverse event(s) may refer to initiation of administration of a probiotic based formulation prior to initiation or re-initiation of treatment with a cancer drug or at some time during the treatment with a cancer drug (i.e. whe treatment with the cancer drug has already commenced).
  • said cancer treatment-related adverse events may be identified as one or more of the following: a) Nausea; b) Vomiting; c) Diarrhea; d) Abdominal pain; e) Bloating; f) Ulcers in the gastrointestinal tract; g) Mucositis in the gastrointestinal tract; h) Esphagitis; i) Infusion-related reactions; j) Fatigue; k) Malaise; 1) Cytokine release syndrome; m) Serum sickness; n) Alopecia; o) Anemia; p) Leukocytosis; q) Viral infections; r) Bacterial infections; s) Yeast infections t) Acid reflux; u) Neutropenia; v) Lymphopenia; w) Thrombocytopenia; x) Leaky gut; y) Cardiomyopathy; and z) Neuropathy.
  • a preferred composition for prevention or management of cancer treatment-related adverse events in a subject comprises the following active ingredients: a) Bifidobacterium infantis b) Bifidobacterium bifidum c) Lactobacillus acidophilus ; d) Lactobacillus salivarius e) Lactobacillus plantarum f) Lactobacillus rhamnosus, g) Bifidobacterium longum h) Lactobacillus cased, and i) Lactobacillus paracasev, j) amylase; k) glucoamylase; 1) lipase; m) bromelain; n) maltase; o) lactase; p) hemicellulose; q) xylanase; r) papain, and s) in
  • this compositon comprises between 100-500 mg by weight of digestive enzymes and between 50-250 mg by weight digestive enzymes per dose.
  • said formulation comprises 272.65 mg of digestive enzymes and 116.20 mg of probiotics per dose.
  • this formulation is contained in capsules for oral administration.
  • dosing regimens for prevention or management of adverse event(s) in a subject may be similar or identifical to those used to improve the efficacy of cancer therapy in a subject as per the descriptions provided herein.
  • said probiotic based formulation(s) described herein can be used for reducing existing inflammation in a subject with cancer, wherein said inflammation may be caused by the cancer disease process and/or treatment agents used to treat cancer or infections.
  • said probiotic based formulation(s) described herein can be used for modulating the immune system in a subject with cancer, wherein said immune system may require modulation due to the cancer disease process and/or treatment agents used to treat cancer or infections.
  • said probiotic based formulation(s) described herein can be used for modulating the microbiome in the gut.
  • the microbiome refers to the microorganisms that are present in the gut, which can be assessed based on sequencing tehniques and analytical techniques that are known in the art.
  • the two main approaches for analyzing the microbiome 16S ribosomal RNA (rRNA) gene amplicons and shotgun metagenomics are widely practiced.
  • rRNA 16S ribosomal RNA
  • shotgun metagenomics are widely practiced.
  • these techniques may be performed using stool samples acquired before and after a specific treatment, which, in this case, may be before and after administration of a probiotics-based formulation to a subject in need thereof.
  • the probiotic based formulation(s) described herein are intended to treat, reverse, reduce or prevent dysbiosis of the gut microbiome. This may also be referred to generally as“modulating” the microbiome.
  • the term “dysbiosis” can refer to one or more of (i) an increase in the proportion of a first species (bacterial or archaeal species) or strain in the microbiota (eg, gut microbiota) (eg, Bacteriodes fragalis or thetaiotamicron ); (ii) an increase in the relative proportion of first and second species (eg, Bacteriodes fragalis versus Clostridium pere, or Streptococcus thermophilus verus Escherichia coli or Lactococcus lactis ), first and second strains of the same species, or first and second phyla which are different from each other (eg, Bacteriodetes versus Firmicutes); (iii) an addition of a first species (bacterial or archae
  • dysbiosis of the gut microbiome is treated, reversed, reduced and/or prevented by administering to a subject in need thereof a probiotic based composition
  • a probiotic based composition comprising the following: a) Bifidobacterium infantis, b) Bifidobacterium bifiidunv, c) Lactobacillus acidophilus, d) Lactobacillus salivarius; e) Lactobacillus plantarum, f) Lactobacillus rhamnosus, g) Bifidobacterium longum, h) Lactobacillus cased, and i) Lactobacillus paracasev, j) amylase; k) glucoamylase; 1) lipase; m) bromelain; n) maltase; o) lactase; p) hemicellulose; q) xylanase; r) papain, and s) invert
  • said formulation comprises 272.65 mg of digestive enzymes and 116.20 mg of probiotics per dose. In one embodiment, between 2-10 doses are administered daily. In a preferred embodiment, 6-8 doses are administered daily.
  • said formulation of probiotics with digestive enzymes is contained in capsules for oral administration to a subject in need thereof, wherein one dose is present in each capsule. In the context of the invention, said formulation is administered for a duration of a minimum of three weeks; preferably for at least three months.
  • said probiotic based composition comprising probiotics with digestive enzymes is administered to a subject in need thereof for modulating the ratios of Bacteroidetes to Firmicutes phyla in the gut of said subject.
  • This ratio is known in the art to have significance with regard to the health of the microbiome and the overall wellness of a subject.
  • modification of the presence of phyla can serve as confirmation of the bioactivity of the probiotics present in said formulation. Said modification can be measured by evaluating the composition of the microbiome from stool samples using methods known in the art, for example, 16S rRNA based sequencing methods, that may be performed before and after administration of said probiotic based composition.
  • changes in the genera and species within the Firmicutes and Bacteroidetes phyla can also be monitored.
  • Examples within the Firmicutes phylum include but are not limited to the following: Clostridium clusters IV and XlVa, the Veillonellaceae family, and Lactobacillus species.
  • Examples within Bacteroidetes phlyum including but are not limited to Bacteroides species.
  • other phyla, species and genera can also be evaluated in order to determine the effects of the probiotic based composition for modulating the gut microbiome; for example, Akkermansia muciniphila (phylum Verrucomicrobia).
  • modulation of the ratios and quantities of microorganisms belonging to the Firmicutes, Bacteroidetes or other phyla may also modulate the metabolic and fermentation activities in the gut, which may in turn modify the immune status, susceptibility to infections, manifestion of adverse events in response to cancer therapeutics, and/or the efficacy of cancer therapeutics.
  • said probiotic based composition comprising probiotics with digestive enzymes is administered to a subject in need thereof for increasing the presence of Lactobacillus and/or Bifidobacterium genera in the gut of said subject.
  • said probiotic based formulation is administered to a subject in need thereof prophylactically for modulating the gut microbiome.
  • prophylactically refers to initiation administration of said probiotic based formulation at a time prior to administration of one or more cancer therapeutic agents and/or an antibiotics. Administration of said probiotic based formulation may then continue throughout and following the administration of one or more cancer therapeutic agents and/or an antibiotics.
  • said probiotic based formulation will be administered for a total duration of at least three or six months.
  • said probiotic based formulation is administered to a subject in need thereof subsequent to administration of one or more cancer therapeutic agents or antibiotics for the purpose of modulating the gut microbiome. Administration of said probiotic based formulation may then continue throughout and following the administration of one or more cancer therapeutic agents and/or an antibiotics.
  • said probiotic based formulation will be administered for at least three or six months.
  • the probiotic based formulation may require testing for bioactivity of its active ingredients prior to administration to said subject.
  • Methods known in the art can be utilized for determining the activity of probiotic microorganisms.
  • he SHIME® model allows to culture the complex gut microbiota over a longer period under representative conditions of the different intestinal regions. Therefore, the SHIME® does not only allow to obtain detailed information about the fermentation profile of the test products, but importantly also about the localization of the intestinal fermentation activity.
  • the SHIME® allows for performance of mechanistic research as the gut microbiome is fully stable prior to treatment. This is achieved by applying a two- week stabilization period with strict control of the environmental conditions (e.g.
  • the human fecal inoculum evolves to a stable in vitro microbiota that is representative for the different colon regions of interest. This is crucial because a microbiota will inevitably alter after being transferred from an in vivo environment to a laboratory model.
  • the probiotic based composition may be added to the SHIME®.
  • the first two reactors are of the fill-and-draw principle to simulate different steps in food uptake and digestion, with peristaltic pumps adding a defined amount of SHIME feed (140 mL 3x/day) and pancreatic and bile liquid (60 mL 3x/day), respectively to the stomach and small intestine compartment and emptying the respective reactors after specified intervals.
  • the last three compartments simulate the large intestine.
  • These reactors are continuously stirred, they have a constant volume and pH control. Retention time and pH of the different vessels are chosen in order to resemble in vivo conditions in the different parts of the colon. Upon inoculation with fecal microbiota, these reactors simulate the ascending, transverse and descending colon. Inoculum preparation, retention time, pH, temperature settings and reactor feed composition were previously described by [11].
  • the probiotic based composition may be added to the SHIME® together with a chemotherapeutic agent and/or an antibiotic.
  • a chemotherapeutic agent and/or antibiotic can be added without the probiotic based composition.
  • the probiotic based composition can be added to the SHIME® reactors prior to addition of a chemotherapeutic agent and/or an antibiotic. Subsequently, activity in the SHIME® reactors can be measured based on acid/base consumption, concentrations of short-chain fatty acids in the reactors, concentration of lactate in the reactors, and the microbial community composition using quantitative PCR methods known in the art.
  • the SHIME® method can be used to assess the bioactivity of a probiotic based composition in the presence of a chemotherapeutic agent or an antibiotic. Additionaly, the SHIME® study can be utilized to gauge the optimum time for administration of the probiotic based composition for its microbiome modulating effects to occur (i.e. before or during chemotherapy and/or antibiotic administration).
  • probiotic based compositions or “probiotic based formulations” may refer to probiotics comprising two or more live microorganisms from the genera Lactobacillus or Bifidobacterium.
  • nine probiotic microorganisms from the genera Lactobacillus or Bifidobacterium are present in the composition
  • the following probiotic species are present in the composition: Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei, Lactobacillus paracasei
  • the terms“probiotic(s) based compositions” or“probiotic(s) based formulations” may also refer to two or more probiotics with two or more digestive enzymes.
  • said mixtures or blends of probiotics with digestive enzymes are prepared or manufactured in a manner that they are co-adminisetered to a subject in need thereof.
  • said digestive enzymes present in said formulation include enzymes that are have activity for digesting carbohydrates, proteins, and fats.
  • 10 digestive enzymes are present in the formulation.
  • the following digestive enzymes are present in the composition: a) amylase; b) glucoamylase; c) lipase; d) bromelain; e) maltase; f) lactase; g) hemicellulose; h) xylanase; i) papain, and j) invertase.
  • said probiotic based composition is contained in a capsule.
  • said capsule contains probiotics and digestive enzymes as active ingredients and inactive ingredients that include one or more of the following substances: a) Inulin; b) Rice extract; c) Hydroxypropyl methylcellulose.
  • said probiotic based composition is administered to a subject in need thereof by the oral route of administration.
  • said capsule may be broken open and the contents of said capsules may be mixed into applesauce for subjects who have difficulty swallowing intact capsules.
  • said probitioc based composition is adminsered to a subject with cancer who is taking antibotics or who may need antibiotics before, during or aftr the course of treatment with cancer drugs.
  • said subject may have an infection or be at-risk for an infection. It is known in the art, that infections in cancer patients often occr due to their immune suppressed status, which is due to their disease and/or secondary to certain cancer drug treatments.
  • Said antibiotics include but are not limited to the following: Aminocoumarins (such as Novobiocin, Albamycin, Coumermycin and Clorobiocin), Aminoglycosides (such as Amikacin, Apramycin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin and Spectinomycin), Ansamycins (such as Geldanamycin, Herbimycin, Rifaximin and Streptomycin), Carbapenems (such as Ertapenem, Daripenem, Cilastatin ('Imipenem') and Meropenem), Cephalosporins (such as Cefadroxil, Cefazolin, Cefalothin ('Cefalotin'), Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefoperazone, Cefotaxime, Ce
  • This invention teaches formulations of probiotics with digestive enzymes that can serve as anti-diarrhea agents in subjects with cancer for preventing or improving cancer drug-associated diarrhea.
  • Another embodiment of this invention pertains to formulations of probiotics with digestive enzymes that can serve to prevent or allevaite antibiotic-associated diarrhea in a subject with cancer in need thereof.
  • Another preferred embodiment of this invention pertains to formulations of probiotics with digestive enzymes that can serve as antiemetic agents in a subject with cancer for the purpose of preventing or improving cancer drug-associated nausea or vomiting.
  • Another embodiment of the invention pertains to formulations of probiotics with digestive enzymes that serve to prevent or treat antibiotic-associated nausea or vomiting in a subject with cancer.
  • Another embodiment of the invention pertains to formulations of probiotics with digestive enzymes that serve to prevent or treat alopecia in a subject with cancer.
  • Another embodiment of the invention pertains to a formulation of probiotics with digestive enzymes that serves to prevent or treat cardiotoxicity in a subject with cancer.
  • cardiotoxicity in said subject may include clinical syndromes including but not limited to reduced ejection fraction, dilated cardiomyopathy, and congestive heart failure.
  • Another embodiment of the invention pertains to a formulation of probiotics with digestive enzumes that serves to prevent or treat neurotoxicity in a subject with cancer.
  • neurotoxicity in a said subject may include clinical syndromes including but not limited to depression, encephalopathy, cerebellar ataxia, acute neuromuscular syndrome, thrombotic microangiopathy, blurred vision, dizziness, headaches, delayed progressive encephalopathy, optic neuropathy, Parkinsonism, leukocencephalopathy, and acute chemical meningitis.
  • immune suppression refers to reduction in numbers of immune cells.
  • immune suppression refers to activity of immune cells.
  • immune cells include neutrophils, eosinophils, basophils, natural killer cells, natural killer T cells, gamma delta T cells, monocytes, macrophages, dendritic cells, B cells, CD4 T cells, CD8 T cells, and innate lymphoid cells.
  • immune cell activities include cytokine production, phagocytosis, cytotoxic activity, and antigen presenting activity.
  • a formulation of probiotics with digestive enzymes is used for treating cytokine release syndrome, serum sickness, or sepsis in a subject with cancer. Said formulation may be used in conjunction with supportive care or other medications used to address said cytokine release syndrome, serum sickness or sepsis.
  • a subject receiving treatment with chimeric antigen receptor (CAR) T cells is administered a probiotic based formulation for several days prior to infusion of CAR T cells for the prevention of cytokine release syndrome.
  • CAR chimeric antigen receptor
  • compositions of probiotics with digestive enzymes for improving efficacy of cancer therapy, for reducing toxicities of cancer therapy, and/or for allowing for higher concentrations of cancer therapeutic agents to be used in a subject with cancer.
  • the invention also teaches compositions of probiotics with digestive enzymes administered to a subject with cancer in order to improve the subject’s tolerability to a cancer treatment for the purpose of allowing continued or extended administration of cancer therapeutic agents to said subject. For example, increased numbers of cycles of chemotherapy may be tolerated or additional cancer therapeutic agents may be administed to said subjects.
  • the specific probiotic based formulations when used in combination and administered to a subject can be utilized to improve the health of a subject undergoing cancer therapy; specifically, for subjects afflicted with toxicities associated with cancer therapy including hypotension, cardiovascular disease, disorders related to the vascular system, metabolic syndrome, disorders of the digestive tract, hematological toxicities, hepatotoxicities, and neural toxicities.
  • Said probiotics based formulation can also be administered to a subject as a to improve quality of life of said subject and of specific organ systems in said subject in the absence of any specific cancer therapy as a prophylactic.
  • the terms“overall wellness” or “quality of life’ may be used interchangeably in the context of the present invention.
  • Quality of life may be defined subjectively by said subject; for example, based on self- reporting of energy levels, presence or absence of malaise or other measures.
  • quality of life may be defined by a health care professional or clinical study personnel using a validated questionnaire.
  • One non-limiting example of a questionnaire is the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire.
  • EORTC European Organization for Research and Treatment of Cancer
  • efficacy of said probiotic based formulation for improving quality of life in a subject in need thereof is evaluated by a health care professional or clinical study personnel using Kamofsky Performance Status, an assessment tool for functional impairment that is known in the art.
  • An object of the invention is to provide a composition that relieves symptoms of chemotherapy induced toxicity.
  • An object of the invention is to provide a composition that relieves symptoms of radiotherapy induced toxicity.
  • An object of the invention is to provide a composition that relieves symptoms of immunotherapy induced toxicity.
  • An object of the invention is to provide a composition that relieves symptoms of hyperthermia induced toxicity.
  • An object of the invention is to provide a composition that relieves symptoms of surgery induced toxicity.
  • An object of the invention is to provide a probiotic based formulation that replaces and replenishes the bacteria that are beneficial to the human body in a subject in need thereof, after such bacteria are reduced or destroyed by intervention with a cancer therapy.
  • An object of the invention is to provide a probiotic based formulation to improve digestion and nutrient absorption in a subject with cancer.
  • An object of the invention is to provide the inventive composition in a form that has a long shelf life.
  • An object of the invention is to provide the inventive composition in pill form and to provide a pill that reaches the digestive tract prior to being absorbed.
  • An object of the invention is to provide a probiotic based formulation that allows a subject in need thereof to ingest foods that otherwise result in adverse reactions by the body (for example, any reaction related to food intolerance or allergic responses), subsequent to which the patient was treated with a cancer therapy or a plurality of cancer therapies.
  • Chemotherapeutic agents are well known in the art. Specific non-limiting examples of chemotherapeutic agents are provided throughout the specification and include, for example, FOLFOX (a chemotherapy regimen for treatment of colorectal cancer, which comprises administration of folinic acid (leucovorin), fluorouracil (5-FU), and oxaliplatin) and FOLFIRI (a chemotherapy regimen for treatment of colorectal cancer, which comprises administration of folinic acid (leucovorin), fluorouracil (5-FU), and irinotecan), as well as administration of targeted monoclonal antibody therapy (e.g., bevacizumab, cetuximab, or panitumumab) alone or in combination with chemotherapeutic agents.
  • FOLFOX a chemotherapy regimen for treatment of colorectal cancer, which comprises administration of folinic acid (leucovorin), fluorouracil (5-FU), and irinotecan
  • FOLFIRI a chemotherapy regimen for treatment of colorectal cancer
  • Bevacizumab (AVASTIN ® ) is a monoclonal antibody that targets a protein called VEGF that affects tumor blood vessel growth. It can cause debilitating side effects such as high blood pressure, bleeding, poor wound healing, blood clots, and kidney damage.
  • the probiotic based composition is used to enhance therapeutic efficacy of passively administered antibodies (such as Herceptin®, Rituximab®, and Cetuximab®), and/or augment cancer killing/inhibitor activity of endogenous antibodies in a subject.
  • passively administered antibodies such as Herceptin®, Rituximab®, and Cetuximab®
  • Antibodies exist in three formats; monoclonal (mAbs), oligo/polyclonal and antibody-drug conjugates.
  • mAbs represent an effective therapeutic modality and are important to the treatment paradigm of various diseases. Recent insights into the detailed mechanism of mAbs link their strong disease fighting potential to the immune system.
  • mAbs designed to inhibit specific checkpoints in the immune system have demonstrated strong immune responses and therapeutic benefit in patients.
  • Immune cells express proteins that are immune checkpoints that control and down-regulate the immune response. These are best defined in T lymphocytes and include PD-l, CTLA-4, TIM-3 and LAG3.
  • Tumor cells express the ligands to these receptors. When T cells bind the ligand to these proteins on the tumor cells, the T cell is turned off and does not attempt to attack the tumor cell.
  • These agents, typically mAbs, that block these proteins are known as checkpoint inhibitor drugs.
  • the degree of efficacy of these therapies is heavily reliant on the immune system of patients, many of whom are severely immuno-compromised.
  • checkpoint inhibitor drugs include the following: a) Inhibitors of Programmed Death 1 (PD-l, CD279), such as nivolumab (OPDIVO.RTM., BMS-936558, MDX1106, or MK-34775), and pembrolizumab (KEYTRODA.RTM., MK-3475, SCH-900475, lambrolizumab, CAS Reg. No. 1374853-91-4), as well as the PD-l blocking agents described in U.S. Pat. Nos.
  • PD-l Inhibitors of Programmed Death 1
  • CD279 Inhibitors of Programmed Death 1
  • nivolumab OPDIVO.RTM., BMS-936558, MDX1106, or MK-34775
  • pembrolizumab KEYTRODA.RTM., MK-3475, SCH-900475, lambrolizumab, CAS Reg. No. 1374853-91-4
  • WO 03042402 WO 2008156712, WO 2010089411, WO 2010036959, WO 2011066342, WO 2011159877, WO 2011082400, and WO 2011161699;
  • Inhibitors of Programmed Death— Ligand 1 include antibodies such as BMS-936559, MPDL3280A), MEDI4736, MSB0010718C, and MDX1105-01); also including: atezolizumab, durvalumab and avelumab;
  • Inhibitors of CTLA-4 such as ipilimumab (YERVOY.RTM., MDX-010, BMS-734016, and MDX-101), tremelimumab, antibody clone BNI3 (Abeam), RNA inhibitors, including those described in WO
  • the checkpoint inhibitor is an inhibitor of a checkpoint protein selected from the group of PD-l, PD-L1, and CTLA-4.
  • the checkpoint inhibitor is selected from the group of an anti-PD-l antibody, and anti-PD-Ll antibody, and an anti- CTLA-4 antibody.
  • the anti-PD-l antibody is selected from the group of nivolumab, pembrolizumab, and lambrolizumab.
  • the anti-PD-Ll antibody is selected from the group of as BMS-936559, MPDL3280A, MEDI4736, MSB0010718C, and MDX1105-01.
  • the anti-PD-Ll antibody is selected from the group of durvalumab, atezolizumab, and avelumab.
  • the anti-CTLA-4 antibody is selected from the group of ipilimumab and tremelimumab.
  • the checkpoint inhibitor is selected from the group consisting of nivolumab, pembrolizumab, lambrolizumab, BMS-936559, MPDL3280A, MEDI4736, MSB0010718C, MDX1105-01, durvalumab, atezolizumab, avelumab, ipilimumab, and tremelimumab.
  • the checkpoint inhibitor is selected from the group consisting of nivolumab, pembrolizumab, lambrolizumab, durvalumab, atezolizumab, avelumab, ipilimumab, and tremelimumab. In some embodiment, In one embodiment, the checkpoint inhibitor is selected from the group consisting of nivolumab, pembrolizumab, durvalumab, atezolizumab, and avelumab.
  • non-limiting examples of mAbs include the following: abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elot
  • the mAbs may be joined to another entity; for example, mAbs joined to a chemotherapy drug or to a radiolabeled particle, or they may be bispecific monoclonal antibodies that are made up of two different mAbs.
  • the probiotic based compositions disclosed herein can be administered to a subject in need thereof as a means of enhancing immune stimulatory efficacy of dendritic cell therapy.
  • the dendritic cell approach is designed to indirectly stimulate a patient’s T- cells by leveraging the role of dendritic cells in presenting antigens to T-cells.
  • Cancer vaccines are the most common application of dendritic cells.
  • the only FDA-approved dendritic cell therapy is PROVENGE, which entails collecting monocytes from the patient, maturing them into dendritic cells,“loading” ex vivo with the patient’s cancer antigens, and then re-infusing in the patient.
  • a probiotic based composition is is utilized to increase dendritic cell anticancer activity.
  • said probiotic mix is utilized to increase the number of dendritic cells infiltrating tumors.
  • the 5-year survival rate of patients with T-cell infiltration also was significantly better than that of patients without such infiltration (83% versus 61%; P ⁇ 0.05). Similar trends were observed in patients with adenocarcinoma; 5-year survival rates for patients with LC infiltration and those without LC infiltration were 49% and 25%, respectively (P ⁇ 0.025). The survival rates for patients with T-cell infiltration and those without were 50% and 33%, respectively (P ⁇ 0.1).
  • An analysis of patterns of failure of radiation therapy demonstrated that the favorable prognosis in LC infiltration was attributable mainly to improvement of local control rates, but that in T-cell infiltration was not. T-cells infiltrated into tumor specifically in the patients with LC infiltration in both cell types.
  • probiotic based compositions are administered to a subject with cancer for enhancing infiltration of immune cells, including antigen presenting cells, into tumors subsequent to radiation and/or chemotherapy.
  • patients treated with cancer therapy are administered a blend of probiotics; specifically, Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum,, Lactobacillus casei, Lactobacillus paracasei, in combination with a blend of digestive enzymes; specifically, amylase, glucoamylase, lipase, bromelain, maltase, lactase, hemicellulase, xylanase, papain, and invertase.
  • probiotics specifically, Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium
  • Identifying a patient in need can be done by any conventional detection method used in oncology practice, non-exclusively including blood tests, or identifying and assessing risk factors for cardiovascular disease, such as smoking, drinking, lack of exercise, weight of patient, age, family history, etc.
  • T cells recognize diseased cells by receptors engaging with antigens that are present on or inside the diseased cells.
  • CAR-T therapy entails genetically engineering T- cells to express synthetic CARs that direct T-cells to antigens on the surface of cancer cells.
  • TCR therapy modifies T-cells to express high-affinity tumor specific TCRs that recognize intra-cellular antigens that must be presented on the surface of target cells.
  • CAR-T and TCR therapies have demonstrated impressive anti-tumor activity in a narrow spectrum of hematologic cancers and garnered significant attention by research institutions and biopharmaceutical companies.
  • a probiotic based composition is administered to patients prior to CAR-T cell therapy for the purpose of off-setting probable toxicity caused by the treatment.
  • a probiotic with digestive enzymes formuaiton is administered to patients after CAR T cell therapy, either upon presentation of adverse events or before such adverse events are clinically manifested.
  • the probiotic based composition is used to reduce toxicity and augment efficacy of various cancer therapies, for example chemotherapeutic agents, radiotherapeutic agents, or drugs such as atovaquone, azacitidine, bexarotene, boceprevir, bosentan, bosutinib, brentuximab vedotin, carbidopa-levodopa, carglumic acid, decitabine, eribulin mesylate, foscarnet, metformin, ofatumumab, pomalidomide, prelatrexate solution, ropivacaine, rosiglitazone, sirolimus, temsirolimus, and valganciclovir.
  • the probiotic mixture and mixtures described in the invention mitigate or prevent the decrease in the production of red blood cells caused by the surgery or second drug.
  • the“probiotic composition” (which may also exist as a drug or pharmaceutical) may comprise at least two ingredients for administration to a subject with cancer.
  • the two ingredients may include at least one probiotic ingredient.
  • the composition may include at least two different probiotic ingredients or at least one probiotic ingredient and at least one digestive enzyme ingredient, or other ingredients in various combinations.
  • the composition may be substantially, if not completely, devoid of artificial flavors, colorings or preservatives.
  • the supplement may be developed for human or other animal consumption by swallowing or other ingestion technique. In a situation where the composition is developed for consumption by swallowing, the composition may be enclosed within a capsule or other form known to facilitate swallowing.
  • probiotics may be defined as live microorganisms thought to be healthy for the host organism; digestive enzymes may be defined as enzymes that break down polymeric macromolecules into their smaller building blocks in order to facilitate their absorption by the body; dietary supplements may be defined as a preparation intended to supplement the diet and provide nutrients that may be missing or may not be consumed in sufficient quantities in a human's diet.
  • the probiotic ingredients of the composition may be present in an effective dose.
  • the probiotic ingredients may total at least 6 x 1 Q9 colony forming units (cfu) and may include at least 13 x 1Q9 cfu of probiotics or more.
  • the probiotic ingredients total at least 13 x 1Q9 cfu of probiotics. In a more preferred aspect the probiotic ingredients total at least 14 x 1Q9 cfu of probiotics.
  • a colony forming unit (cfu) is generally accepted as a measure of viable bacterial or fungal numbers. Such quantity of probiotic ingredient may facilitate providing a consumer with an effective dose of probiotics at the time of ingestion, as the inventor has realized that probiotics may be destroyed during storage due to undesirable environments (e.g., temperature extremes) and other reasons.
  • the probiotic ingredients may comprise a probiotic blend including one or more of the following: Lactobacillus rhamnosus GG, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus salivarius, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium bifidum.
  • the composition includes at least one probiotic from each of the strains listed above.
  • Each probiotic ingredient present in the probiotic based composition may be present in any desired quantity. In one aspect each probiotic ingredient of the composition may be present in an amount up to 1.5 x 10 9 cfu.
  • each probiotic ingredient of the probiotic based composition may be present in an amount equal to or greater than 1 x 10 9 cfu, and in a preferred aspect when combined the nine probiotic ingredients may total as much as, or more than, 13 x 10 9 cfu of probiotic ingredients. Preferably the amounts are equal to or are more than 14 x 10 9 cfu. In the example, these quantities may be measured at the time of manufacture.
  • GI gastrointestinal
  • Lactobacillis acidophilis is a prominent strain of probiotic for the small intestine
  • Bifidobacterium bifidum is a prominent strain of probiotic for the large intestine
  • those supplemental ingredients and other of the respective probiotic ingredients and the digestive enzymes
  • a blister pack for storing the capsule assists in preserving the potency of the ingredients such that the combination of the composition with the capsule in a protected blister package assists with appropriate and effective delivery (location and potency).
  • location and potency associated with appropriate and effective delivery
  • Digestive enzymes of the composition may be present in an effective dose to supplement existing quantities of enzymes and improve digestion of ingested food and absorption of the nutrients within the ingested food.
  • the digestive enzyme ingredients may comprise any enzyme that is useful in the digestion of ingested food.
  • inventors has developed a particularly effective probiotic based formulation also containing digestive enzymes comprising some or all of the following: amylase, glucoamylase, lipase, bromelain, maltase, lactase, hemicellulose, xylanase, papain, and invertase.
  • digestive enzymes comprising some or all of the following: amylase, glucoamylase, lipase, bromelain, maltase, lactase, hemicellulose, xylanase, papain, and invertase.
  • a capsule may enclose the probiotic based composition to facilitate increasing the shelf-life of the composition, swallowing of the composition, timing a release of the composition after ingestion and other considerations.
  • the capsule may be a gelatin capsule, vegetable- based (e.g., vegetable cellulose) capsule or other type of capsule. If the capsule is a vegetable-based capsule, the capsule may facilitate releasing the probiotics at a desirable location within the digestive tract. Preferably the capsule is a vegetable-based capsule.
  • An exemplary embodiment of the composition includes a probiotic blend and a digestive enzyme blend enclosed within a vegetable cellulose capsule.
  • capsules comprising the composition may be and are preferably stored in blister packs. That is, the blister packs may seal the capsule from a surrounding environment and thus, extend the life of the effective ingredients of the composition.
  • the method of using the composition may be used as desired by the subject in need thereof or as determined by the oncologist or other health practitioner who advises said subject.
  • the probiotics based composition is self-administered by said subject on a daily. Continuous daily use of the probiotic based composition may result in greater and sustained benefits for the subject.
  • the composition may include at least one of the probiotics of the Lactobacillus species and at least one probiotic of the Bifidobacterium species together with a digestive enzyme and contained in a vegetable-based capsule for at least 2 x 10 9 colony forming units.
  • the capsule is stored in a blister pack.
  • the blend includes at least some additional probiotic ingredients as noted above and at least 6 x 10 9 colony forming units assuming 1 billion cfu per probiotic at time of manufacture.
  • the composition may include, for instance, Lactobacillus acidophilus, Lactobacillus rhamnosus CG, Bifidobacterium infantis, and Bifidobacterium bifidum, together with a digestive enzyme and contained in a capsule.
  • the foregoing composition may include others from the above list of probiotics for at least 9 x 10 9 colony forming units, and stored in a blister pack or other sealed package.
  • the foregoing composition may include others from the above list of probiotics for at least 13 x 10 9 colony forming units, and stored in a blister pack or other sealed package.
  • the composition may include a greater amount of Lactobacillus probiotic as compared to Bifidobacterium probiotic.
  • said formulations of probiotics with digestive enzymes can contain said probiotics having the following activity/gram: between 25-150 billion colony forming units (cfu) for Bifidobacterium infantis, between 50-150 billion cfu for Bifidobacterium longum, between 350-550 billion cfu for Bifidobacterium bifidum, between 100-300 billion cfu for Lactobacillus rhamnosus, between 100-300 billion cfu for Lactobacillus acidophilus, between 200-400 billion for Lactobacillus salivarius, between 300-500 billion cfu for Lactobacillus plantarum, between 200-400 billion cfu for Lactobacillus casei, and between 300-500 billion cfu for Lactobacillus paracasei.
  • cfu colony forming units
  • said formulation of probiotics with digestive enzymes contains digestive enzymes having the following activity/gram: between 300,000-500,000 HCU for hemicellulase, between 100,000-300,000 XU for xylanase, between 100,000-300,000 DU for amylase, between 900-1,100 AGU for glucoamylase, between 10,000-30,000 DP for maltase, between 900,000, 1,100,000 TU for papain, between 2,000-4,000 GDU for bromelain, between 150-300 FIP for lipase, between 100,000-300,000 SU for invertase, between 75,000-150,000 ALU for lactase.
  • digestive enzymes having the following activity/gram: between 300,000-500,000 HCU for hemicellulase, between 100,000-300,000 XU for xylanase, between 100,000-300,000 DU for amylase, between 900-1,100 AGU for glucoamylase, between 10,000-30,000 DP for mal
  • a formulation comprising probiotics or probiotics with digestive enzymes may be utilized in subjects with cancer who are scheduled to undergo, are currently undergoing, and/or have undergone treatment with one or more types of chemotherapy. Selection of chemotherapy will depend on the subject to be treated, the type of cancer, and the intent of therapy. Selection of a subject for treatment with said probiotic based formulation may be performed prophylactically. Alternatively, the current invention may be practiced by selection of a subject for treatment with said probiotic based formulation during or after the course of chemoetherapy drug treatment.
  • chemotherapeutic agents that are known in the art and include but are not limited to: methotrexate, taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, etoposides, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel, doxorubicin, epirubicin, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole
  • imatinib mesylate herbimycin A, genistein, erbstatin, and lavendustin A.
  • the anti-cancer agent can be, but is not limited to, a drug listed: Alkylating agents Nitrogen mustards: Cyclophosphamide Ifosfamide Trofosfamide Chlorambucil Nitrosoureas: Carmustine (BCNU) Lomustine (CCNU) Alkylsulphonates: Busulfan Treosulfan Triazenes: dacarbazine Platinum containing Cisplatin compounds: Carboplatin Aroplatin Oxaliplatin Plant Alkaloids Vinca alkaloids: Vincristine Vinblastine Vindesine Vinorelbine Taxoids: Paclitaxel Docetaxel DNA Topoisomerase Inhibitors Epipodophyllins: Etoposide Teniposide Topotecan 9- aminocamptothecin Camptothecin Crisnatol mitomycins: Mitomycin C Anti-metabolites Anti-folates: DHFR inhibitors: Methotrexate Trimetrexate IMP dehydrogena
  • Angiostatin (plasminogen fragment) antiangiogenic antithrombin III Angiozyme ABT-627 Bay 12-9566 Benefin Bevacizumab BMS-275291 cartilage-derived inhibitor (CDI) CAI CD59 complement fragment CEP-7055 Col 3 Combretastatin A-4 Endostatin (collagen XVIII fragment) Fibronectin fragment Gro-beta Halofuginone Heparinases Heparin hexasaccharide fragment HMV833 Human chorionic gonadotropin (hCG) IM-862 Interferon alpha/beta/gamma Interferon inducible protein (IP- 10) Interleukin- 12 Kringle 5 (plasminogen fragment) Marimastat Metalloproteinase inhibitors (TIMPs) 2-Methoxyestradiol MMI 270 (CGS 27023A) MoAb IMC-1C11
  • Additional anti-cancer agents that may be used in the methods of the present invention include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; ce
  • the present invention is based on the discovery that the occurrence of chemotherapy-induced diarrhea is prevented or its severity is reduced upon administration of the probiotic with digestive enzyme compositions described herein.
  • the invention is based on the unexpected finding that the administration of said probiotic compositions provides a protective effect against chemotherapy-induced diarrhea that extends long after the composition is administered. The effect is especially pronounced for diarrhea of Grade II as determined by National Cancer Institute Common Toxicity Criteria for Diarrhea (CTCAE v.4.03).
  • the invention provides a method for preventing or reducing the occurrence of grade 2 or higher diarrhea resulting from an anti-cancer chemotherapy in a subject in need thereof, which method comprises administering to the subject an effective amount of a probiotic based composition. More generally speaking, the invention provides a method for treating gastrointestinal mucositis, or otherwise preventing or reducing gastrointestinal damage and/or dysfunction associated with an anti-cancer chemotherapy in a subject in need thereof, which method comprises administering to the subject a therapeutically effective amount of probiotic based composition for a plurality of consecutive days, preferably commencing at the start of the chemotherapy cycle or prior to the chemotherapy cycle.
  • anti-cancer drugs that can be used include, but are not limited to: 20- epi-l,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- dorsalizing morphogenetic protein- 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-
  • the composition(s) of the invention including probiotics, and probiotics together with enzymes are administered in the form of a nutraceutical.
  • Nutraceuticals whether in the form of a liquid extract or dry composition, are edible and may be eaten directly by humans or mammals.
  • Said nutraceuticals are preferably provided to humans in the form of additives or nutritional supplements for example they may be administered in the form of tablets of the kind sold in health food stores, or as ingredients in edible solids, more preferably processed food products such as cereals, breads, tofu, cookies, ice cream, cakes, potato chips, pretzels, cheese, and in drinkable liquids such as beverages such as milk, soda, sports drinks, and fruit juices.
  • a method for enhancing the nutritional value of a food or beverage by intermixing the food or beverage with a nutraceutical in an amount that is effective to enhance the nutritional and probiotic or immune modulatory and/or cancer therapy augmentative value of the food or beverage.
  • a flavoring agent is added.
  • Preferred flavoring agents include sweeteners such as sugar, com syrup, fructose, dextrose, maltodextrose, cyclamates, saccharin, phenyl-alanine, xylitol, sorbitol, maltitol, and herbal sweeteners such as Stevia.
  • probiotics useful for the practice of the invention include soft drinks, a fruit juice or a beverage comprising whey protein, health teas, cocoa drinks, milk drinks and lactic acid bacteria drinks.
  • Probiotic bacteria may be administered together with agents known to enhance efficacy and retention of probiotics, including
  • various extracts and plant powders are incorporated into our compositions, depending on the desired properties according to the end use of said compositions.
  • compositions according to the present invention can be characterized in that in addition to the discussed prebiotics and phytosterols and lecithins the said further plant extracts or powders are one or more of those of Panax ginseng (red, Korean ginseng), Panax ginseng (white, Chinese ginseng), Rhodiola rosea (golden root), Panax quinquefolium (American ginseng), Eleutherococcus senticosus (Siberian ginseng), Cynara scolymus (artichoke), Uncaria tomentosa (Cat's claw), Lepidium meyenii (maca, Peruvian ginseng), Paullinia cupana (guarana), Croton lechleri (Sangre de Grado), Whitania somnifera (ashwagandha, Indian ginseng), Panax japonicus (Japanese ginseng), Panax vietnamensis (Vietnamese ginseng), Panax trifolius, Panax pseudo
  • plant extracts and plant powders are capable to potentiate the bioactivity of these compositions based on prebiotics, phytosterols, lecithins, vitamins and minerals. In given cases it also adds other prebiotics to the aforementioned prebiotic mixtures. These can result in more pronounced bioactivities as prebiotics and also in the chosen other bioactivity directions.
  • chemotherapy may be divided into the following classes: 1) Alkylating agents. These drugs kill cells that are not in cell cycle [13, 14], and include mustard gas derivatives such as Mechlorethamine, Cyclophosphamide, Chlorambucil, Melphalan [15], and Ifosfamide. Ethylenimines such as Thiotepa and Hexamethylmelamine, Alkylsulfonates such as Busulfan, Hydrazines. Triazines such as Altretamine, Procarbazine, dacarbazine and Temozolomide. Nitrosureas such as Carmustine, Lomustine and Streptozocin.
  • mustard gas derivatives such as Mechlorethamine, Cyclophosphamide, Chlorambucil, Melphalan [15], and Ifosfamide. Ethylenimines such as Thiotepa and Hexamethylmelamine, Alkylsulfonates such as Busulfan, Hydrazines. Triazines such
  • Alkylating agents are one of the original classes of chemotherapies that where historically developed [17, 18].
  • Plant Alkaloids These are chemotherapeutic drugs that are extracted from certain types of plants. The plant alkaloids are cell-cycle specific. This means they attack the cells during various phases of division.
  • the vinca alkaloids are made from the periwinkle plant (catharanthus rosea).
  • the taxanes are made from the bark of the Pacific Yew tree (taxus).
  • the vinca alkaloids and taxanes are also known as antimicrotubule agents.
  • the podophyllotoxins are derived from the May apple plant.
  • Camptothecan analogs are derived from the Asian "Happy Tree” (Camptotheca acuminata). Podophyllotoxins and camptothecan analogs are also known as topoisomerase inhibitors, which are used in certain types of chemotherapy. Vinca alkaloids include Vincristine, Vinblastine and Vinorelbine. Taxanes include Paclitaxel and Docetaxel. Podophyllotoxins include Etoposide and Tenisopide. Camptothecan analogs include Irinotecan and Topotecan. 3) Antitumor Antibiotics. This type of chemotherapy is generated from species of the soil fungus Streptomyces. These drugs act during multiple phases of the cell cycle and are considered cell-cycle specific.
  • Antitumor antibiotics include Anthracyclines such as Doxorubicin, Daunorubicin, Epirubicin, Mitoxantrone, and Idarubicin.
  • Anthracyclines are generally a class of compounds that have the structural core of anthracene. They often are highly effective chemotherapeutics and therefore are used for the treatment of many cancers, including leukemias, lymphomas, breast, uterine, ovarian, bladder cancer, and lung cancers and are often used in childhood cancer treatment regimens.
  • Some anthracycline drugs include doxorubicin, daunorubicin, idarubicin, and epirubicin.
  • anthracyclines have been reported to work by inhibiting DNA and RNA synthesis; promoting free radical formation through redox cycling, with iron promoting the conversion of superoxide into hydroxyl radicals; inhibiting topoisomerases (e.g., topoisomerases Il.alpha. and/or Il.beta.); and evicting histones from open chromosomal areas.
  • Chromomycins such as Dactinomycin and Plicamycin. Additional antitumor antibiotics include Mitomycin and Bleomycin.
  • Antimetabolites This type of chemotherapy resembles normal substances within the cell. When the cells incorporate these substances into the cellular metabolism, they are unable to divide. Antimetabolites are cell-cycle specific.
  • Antimetabolites are classified according to the substances with which they interfere. Antimetabolites include the folic acid antagonist Methotrexate, the pyrimidine antagonists 5-Fluorouracil, Foxuridine, Cytarabine, Capecitabine, and Gemcitabine, the purine antagonists 6-Mercaptopurine and 6-Thioguanine, the adenosine deaminase inhibitors Cladribine, Fludarabine, Nelarabine and Pentostatin. 5) Topoisomerase Inhibitors. These chemotherapeutic agents that interfere with the action of topoisomerase enzymes (topoisomerase I and II).
  • topoisomerase enzymes control the manipulation of the structure of DNA necessary for replication. Ironotecan and topotecan are considered topoisomerase I inhibitors, whereas amsacrine, etoposide, etoposide phosphate, and teniposide are considered topoisomerase II inhibitors. 6) Alternative types of chemotherapy. Hydroxyurea is considered a ribonucleotide reductase inhibitor. Mitotane is considered an adrenocortical steroid inhibitor. Asparaginase and Pegaspargase are enzymatic types of chemotherapy. Estramustine is considered an antimicrotubule drug and members of the retinoid family of chemotherapies include Bexarotene, Isotretinoin, Tretinoin (ATRA).
  • the various probiotic, and probiotic/enzyme mixtures described herein are intended for human consumption and thus the processes for obtaining them are preferably conducted in accordance with Good Manufacturing Practices (GMP) and any applicable government regulations governing such processes. Especially preferred processes utilize only naturally derived solvents.
  • GMP Good Manufacturing Practices
  • the so-called “medical foods” are not meant to be used by the general public and are not available in stores or supermarkets. Medical foods are not those foods included within a healthy diet to decrease the risk of disease, such as reduced-fat foods or low-sodium foods, nor are they weight loss products.
  • a physician prescribes a medical food when a patient has special nutrient needs in order to manage a disease or health condition, and the patient is under the physician's ongoing care.
  • the label must clearly state that the product is intended to be used to manage a specific medical disorder or condition.
  • An example of a medical food is nutritionally diverse medical food designed to provide targeted nutritional support for patients with chronic inflammatory conditions. Active compounds of this product are for instance one or more of the compounds described herein.
  • the present invention thus relates to the use of an immuno-modulating properties of probiotics as related to prevention and/treatment of pregnancy complications.
  • said probiotics can be used in the preparation of a medicament, a vaginal suppository, medical food or nutraceutical to induce immune tolerance or immune modulation.
  • compositions according to the present invention comprise prebiotic components selected from fructose polymers GF.sub.n and F.sub.m, either containing a glucose (G) end-group, or without this glucose end-group and one or more component of a group of prebiotics consisting of modified or unmodified starch and partial hydrolysates thereof, partially hydrolysed inulin, natural oligofructoses, fructo- oligosaccharides (FOS), lactulose, galactomannan and suitable partial hydrolysates thereof, indigestible polydextrose, acemannan, various gums, indigestible dextrin and partial hydrolysates thereof, trans-galacto-oligosaccharides (GOS), xylo-oligosaccharides (XOS), beta-glucan and partial hydrolysates thereof, together if desired with phytosterol/phytostanol components and their suitable esters, and if desired other plant extracts, mineral components, vitamins
  • the fructose polymers of GF.sub.n or F.sub.m structures are linear fructose polymers having either a glucose (G) and -group, or being without this glucose and -group.
  • Oligofructoses are consisted of 3 to 10 carbohydrate units.
  • chicory inulin contains 10 to 60 carbohydrate units, typically with 27 carbohydrates (fructoses with our without one glucose end-group and a fructose chain).
  • Other plants may produce different fructans. These fructans are capable to increase the number of colonized and planktonic bacteria in the large intestine.
  • the local chemical carcinogenesis can also be the result of the formation of secondary bile acids.
  • These secondary bile acids are often formed upon the action of enzymes produced by resident Clostridia.
  • the chance of secondary bile acid formation can also be reduced. This can be demonstrated by measuring the faecal primary/secondary bile acid ratio.
  • prebiotics can be selected from a group of prebiotics consisting of various gums (guar gum, xanthan gum, locust been gum), carob seed flour, oat bran, rice bran, barley, modified or unmodified starch and suitable partial hydrolysates thereof, partially hydrolysed inulin, natural or synthetic/biosynthetic oligofructoses, fructo- oligosaccharides (FOS), lactulose, galactomannan and suitable hydrolysates thereof, indigestible polydextrose, indigestible dextrin and partial hydrolysates thereof, trans- galacto-oligosaccharides (GOS), xylo-oligosaccharides (XOS), acemannan, lentinan or beta-glucan and partial hydrolysates thereof, polysaccharides P and K (PSP, PSK), tagatose, various fungal oligosaccharides and polysaccharides, together with other components.
  • gums
  • probiotic compositions are utilized to enhance efficacy of immunotherapy.
  • Said immunotherapy may be utilized alone, or in combination with other types of cancer therapies such as chemotherapy, surgery, radiation therapy, or hyperthermia.
  • the basis of immunotherapy is activation of a competent T cell response. It is known that T cell responses are controlled in large part by activation of said T cells by antigen presenting cells.
  • probiotic compositions are utilized to augment efficacy of antigen presentation.
  • said probiotic compositions are utilized as in vivo activators of antigen presentation by stimulation of dendritic cell activity.
  • said probiotic compositions are administered after a patient is treated with dendritic cell therapy.
  • DCs dendritic cells
  • Numerous animal models have demonstrated that in the context of neoplasia dendritic cells (DCs) can bind to and engulf tumour antigens that are released from tumor cells, either alive or dying, and cross-present these antigens to T cells in tumour-draining lymph nodes. This results in the generation of tumour- specific immune responses that have been demonstrated to inhibit tumor growth or in some cases induced transferrable immunological memory.
  • DCs recognize tumors using the same molecular means that they would use to recognize apoptotic cells, or cells that are stressed.
  • One set of signals are molecules released from apoptotic cells, which are highly released by tumors, these include the nucleotides UTP and ATP, fractalkine, lipid lysophosphatidylcholine, and sphingosine 1- phosphate [19].
  • Signals from stressed cells, such as tumor cells include externalization of phosphatidylserine onto the outside of the cell membrane, calreticulin, avB5 integrin, CD36 and lactadherin.
  • dendritic cells actively promote tumor immunity in that patients with dendritic cell infiltration of tumors generally have a better prognosis [20-23].
  • Provenge is a cellular product derived from autologous peripheral blood mononuclear cell (PBMC) derived dendritic cells that have been grown using a chimeric protein comprised of GM-CSF and the prostate specific antigen, prostatic acid phosphatase [24, 25]. In the pivotal trial, this DC based therapeutic resulted in extension of survival by 4.1 months [25]. Prior to approval of Provenge, numerous clinical trials using DC were performed in prostate cancer, which will be discussed below.
  • PBMC peripheral blood mononuclear cell
  • probiotic compositions are performed with the intent of activating efficacy of dendritic cell therapy.
  • dendritic cell therapy in various types of cancers has previously been reported and incorporated by reference from the following publications melanoma [29-80], soft tissue sarcoma [81], thyroid [82-84], glioma [85-106], multiple myeloma ,[107-115], lymphoma [116-118], leukemia [119-126], as well as liver [127-132], lung [133-146], ovarian [147-150], and pancreatic cancer [151-153].
  • the probiotic with digestive enzymes formulation is used to enhance T cell immunity in a subject with cancer.
  • T cells are immune effectors against tumors, possessing ability to directly kill via CD8 cytotoxic cells [154- 156], or indirectly killing tumors by activation of macrophages through interferon gamma production [157-159]. Additionally, T cells have been shown to convert protumor M2 macrophages to Ml [160].
  • the probiotic with digestive enzymes formulation is used to enhance the functions of macrophages in a subject with cancer
  • Macrophages are key components of the innate immune system which play a principal role in the regulation of inflammation as well as physiological processes such as tissue remodeling [161, 162].
  • the diverse role of macrophages can be seen in conditions ranging from wound healing [163-166], to myocardial infarction [167-173], to renal failure [174- 177] and liver failure [178].
  • Ml macrophages are described as the pro-inflammatory sub-type of macrophages induced by IFN-. gamma and LPS. They produce effector molecules (e.g., reactive oxygen species) and pro- inflammatory cytokines (e.g., IL-12, TNF-. alpha and IL-6) and they trigger Thl polarized responses [181].
  • effector molecules e.g., reactive oxygen species
  • pro-inflammatory cytokines e.g., IL-12, TNF-. alpha and IL-6
  • manipulation of macrophages is performed using a probiotic with digestive enzyme formulation in combination with agents and approaches known to modulate macrophage activity.
  • Said macrophage activity may include pro-angiogenic activities or anti-angiogneic and cytotoxic activities.
  • Macrophages can play a tumor inhibitory, as well as a tumor stimulatory role. Initial studies supported the role of macrophages in mediating antibody dependent cellular cytotoxicity in tumors [182-189], and thus being associated with potentiation of antitumor immune responses.
  • Macrophages also possess the ability to directly recognize tumors by virtue of tumor expressed“eat-me” signals, which include the stress associated protein calreticulin [190, 191], which binds to the low-density lipoprotein receptor-related protein (LRP) on macrophages to induce phagocytosis [192].
  • Tumors protect themselves by expression of CD47, which binds to macrophage SIRP-l and transduces an inhibitory signal [193].
  • Blockade of CD47 using antibodies results in remission of cancers mediated by macrophage activation [194-198].
  • macrophages play an important role in induction of antitumor immunity.
  • the duality of macrophages in growth of tumors may be seen in studies of “inverse hormesis” in which low concentrations of antibodies targeting the tumor specific marker sialic acid N-glycolyl-neuraminic acid (Neu5Gc) actually leads to enhanced tumor growth in a macrophage dependent manner [212].
  • inverse hormesis in which low concentrations of antibodies targeting the tumor specific marker sialic acid N-glycolyl-neuraminic acid (Neu5Gc) actually leads to enhanced tumor growth in a macrophage dependent manner [212].
  • a probtioic with digestive enzyme formulation is used to generate Ml macrophages in a subject with cancer.
  • M2 macropahges are typically augmenters of tumor growth whereas Ml usually inhibit tumor growth [213, 214].
  • other agents may be used to modulate M2 to Ml transition of tumor associated macrophages including RRx-OOl [215], the bee venom derived peptide melittin [216], CpG DNA [217, 218], metformin [219], Chinese medicine derivative puerarin [220], rhubarb derivative emodin [221], dietary supplement chlorogenic acid [222], propranolol [223], poly ICLC [224], BCG [225], Agaricus blazei Murill mushroom extract [226], endotoxin [227], olive skin derivative maslinic acid [228], intravenous immunoglobulin [229], phosphotidylserine targeting antibodies [230], dimethyl sulfoxide [231], surfactant protein A [232], Zoledronic acid [233], bacteriophages [234]
  • a probtioic with digestive enzyme formulation is used to generate activated and/or tumor-cytotoxic T cells in a subject with cancer.
  • a probiotic with digestive enzyme formulation is used to reduce the prevalence of regulatory T (Treg) cells in a subject with cancer, as can be measured based on the presence of said cells in the peripheral blood of said subject with cancer using techniques that are known in the art.
  • Treg regulatory T
  • the importance of T cells in cancer is illustrated by positive correlation between tumor infiltrating lymphocytes and patient survival [235-239].
  • positive correlations between responses to various immunotherapies has been made with tumor infiltrating lymphocyte density [240, 241].
  • Increased T cell activity is associated with reduction in T regulatory (Treg) cells.
  • Treg cells have improved tumor control.
  • Agents that inhibit Treg cells include arsenic trioxide [242], cyclophosphamide [243-245], triptolide [244], gemcitabine [246], and artemether [247].
  • a probiotic with digestive enzyme formulation is utilized to augment the anticancer effects of metformin, and/or metformin together with chemotherapy.
  • metformin for the treatment of cancer and its activies is known in the art and includes macrophage Ml polarization and activation of CD8 T cells.
  • TAM tumor-associated macrophage
  • TAMs-conditioned medium and a coculture system were performed, which demonstrated an inhibitory effect of metformin on endothelial sprouting and tumour cell proliferation promoted by M2-polarized RAW264.7 macrophages [248] . From the CD8 perspective, Metformin enabled normal but not T-cell-deficient SCID mice to reject solid tumors.
  • CD8(+) tumor-infiltrating lymphocytes TILs
  • CD8(+) TILs capable of producing multiple cytokines were mainly PD-l(-)Tim-3(+), an effector memory subset responsible for tumor rejection.
  • metformin and cancer vaccine improved CD8(+) TIL multifunctionality.
  • Example 1 Clinical evaluation of a probiotic based formulation in patients with colorectal cancer.
  • the following example is a clinical study designed to evaluate the efficacy of a probiotic based formulation comprising a blend of probiotic and digestive enzymes contained in capsules.
  • This example teaches methods of use for a probiotic with digestive enzyme formulation disclosed in the present invention.
  • this example also teaches methods of using a probiotic with digestive enzyme formulation for improving the efficacy of a chemotherapy drug and/or for preventing and treating adverse events associated with chemotherapy drug treatment of a cancer patient.
  • the Investigation Product is a formulation comprising capsules that contain 9 probiotic microorganisms of the genera Lactobacillus and Bifidobacterium , as well as 10 digestive enzymes, and is being tested for its efficacy as part of a treatment regimen for patients with colon cancer.
  • DBR consists of capsules containing a proprietary blend of probiotics (116.20 mg total weight); specifically, Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei, Lactobacillus paracasei, and digestive enzymes (272.65 mg total weight); specifically, amylase, glucoamylase, lipase, bromelain, maltase, lactase, hemicellulose, xylanase, papain, and invertase.
  • the inactive ingredients are: inulin, rice extract, and hydroxypropyl methycellulose.
  • the Invesigational Product is manufactured in the United States under GMP conditions and is NSF certified. Placebo capsules will have the same packaging and labelling as the Investigational Product.
  • the capsules will be taken orally at 6 capsules daily (2 capsules in the morning, noon, and night) on days when chemotherapy is not administered. Eight capsules will be taken daily on each day of chemotherapy treatment (one additional capsule to be taken within two hours before chemotherapy and another additional capsule to be taken within two hours after the treatment session has been completed).
  • Cohort 2 Placebo supplementation for patients with stage IV colorectal cancer that are undergoing chemotherapy with FQLFQX .
  • Placebo capsules will be taken orally at 6 capsules daily on days when chemotherapy is not administered and 8 capsules daily on each day of scheduled chemotherapy treatment (one additional capsule to be taken within two hours before therapy and another additional capsule within two hours after the treatment session has been completed).
  • the capsules will be taken orally at 6 capsules daily on days when chemotherapy is not administered and 8 capsules daily on each day of scheduled chemotherapy treatment (one additional capsule to be taken within two hours before therapy and another additional capsule within two hours after the treatment session has been completed).
  • Placebo capsules will be taken orally at 6 capsules daily on days when chemotherapy is not administered and 8 capsules daily on each day of scheduled chemotherapy treatment (one additional capsule to be taken within two hours before therapy and another additional capsule within two hours after the treatment session has been completed).
  • CRP C-reactive protein
  • the duration of therapy with DBR for enrolled patients is 5 months.
  • Microbiome testing will demonstrate changes in the ratios of Firmicutes to Bacteroidetes that are associated with continued intake of the Investigational Product when analysis is performed at baseline (i.e. prior to Investigational Product administration, 3 months, and 5 months of the study).
  • Example 2 Evaluation of the bioactivity of a probiotic based formulation and its impact on a chemotherapeutic agent and an antibiotic
  • the following example is an in vitro study that was conducted to evaluate the activity of a probiotic based formulation comprising a blend of probiotic and digestive enzymes. This example validates the specific activity of a formulation disclosed in the present invention.
  • the supplement restored the Bacteroidetes to Firmicutes ratios in the colon reactors, increased the diversity of microbiota, and induced the production of microbial metabolites that elicited anti inflammatory cytokines in an in vitro model of intestinal inflammation.
  • preventative administration of the supplement resulted in full recovery of the gut microbial community after cessation of 5-FU and vancomycin treatment.
  • the probiotic with digestive enzymes supplement used herein comprises capsules is manufactured using proprietary methods and contains a blends of probiotics (116.20 mg total weight); specifically, Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei, Lactobacillus paracasei, and digestive enzymes (272.65 mg total weight); specifically, amylase, glucoamylase, lipase, bromelain, maltase, lactase, hemicellulose, xylanase, papain, and invertase.
  • the capsules used herein are the same formulation that would be administered in vivo.
  • the product was tested at an in vitro dose of 3 capsules/day before chemotherapy/antibiotic treatments, and 4 capsules/day during and after chemotherapy/antibiotic treatment (to be described below). This corresponds to an in vivo dosage of 6 capsules/day and 8 capsules/day before and after chemotherapy/antibiotic treatment, respectively.
  • the SHIME Set-un [00242] Briefly, the SHIME system consists of a series of double-jacketed vessels, simulating the digestive compartments that are initially inoculated with a fecal sample from a healthy adult donor using methods described previously [11].
  • the typical reactor setup consists of a succession of five reactors simulating the different parts of the human gastrointestinal tract.
  • the first two reactors simulate different steps in food uptake and digestion, with peristaltic pumps adding a defined amount of SHIME feed (140 mL 3x/day) and pancreatic and bile liquid (60 mL 3x/day), respectively to the stomach (VI) and small intestine (V2) compartment and emptying the respective reactors after specified intervals [11].
  • the last three compartments simulate the large intestine.
  • These reactors are continuously stirred, and they have a constant volume and pH control. Retention time and pH of the different vessels are chosen in order to resemble in vivo conditions in the different parts of the colon.
  • these reactors Upon inoculation with fecal microbiota, these reactors simulate the ascending (V3), transverse (V4) and descending (V5) colon.
  • Control Arm Chemotherapy and antibiotics; no supplement given.
  • Curative Arm Chemotherapy and antibiotics; probiotic with digestive enzymes supplement added at the same time as the other agents.
  • Preventative Arm Chemotherapy and antibiotics; probiotic with digestive enzymes supplement added prior to the other agents.
  • a two-week stabilization period allowed the microbial community to differentiate in the different reactors depending on the local environmental conditions.
  • the basic nutritional matrix was provided to the SHIME to support diversity of the gut microbiota originally present in the fecal inoculum.
  • the third arm of the SHIME setup (preventive arm; PREV), already received 3 capsules/day during the stabilization period (corresponding to an in vivo dose of 6 capsules/day).
  • lactate the precursor of SCFA
  • concentrations of lactate, the precursor of SCFA were also monitored using a d- lactate/l-lactate kit (R-Biopharm, Mannheim, Germany), according to the manufacturer's protocols.
  • SHIME experiments the following groups were quantified via quantitative PCR (qPCR; once/week): Bacteroidetes phylum, Firmicutes phylum, Lactobacillus spp. and Bifidobacterium spp. as previously reported [251].
  • 16S ribosomal RNA (rRNA) sequencing was used to analyze samples from the SHIME reactors to identify and compare the microorganisms using similar methods as published elsewhere [252].
  • the Illumina sequencing method was used to amplify microbial sequences until a saturation level was reached. Information on a broad spectrum of OTUs was obtained (>100 different of the most dominant OTUs), however, the results were presented as proportional values versus the total amount of sequences within each sample, thus providing semi-quantitative results.
  • the methodology used primers that span 2 hypervariable regions (V3-V4) of the 16S rDNA. Using a paired sequencing approach, sequencing of 2x250 bp resulted in 424 bp amplicons.
  • the Simpson reciprocal index was calculated as a measure of diversity and evenness of the microbiota as described previously [253].
  • An increase in the Simpson reciprocal index reflects a diversity increase, with 1 being the lowest possible number, and the number of bacterial species/OTUs present in the sample being the maximal number. The index will approach the maximal value when the OTU distribution is more even. The higher the index, the larger the diversity and the larger the evenness.
  • Caco-2 cells The co-culture experiment was performed as previously described [254] H3riefly, Caco-2 cells (HTB-37; American Type Culture Collection) were seeded in 24-well semi-permeable inserts (0.4 pm pore size) at a density of 1 x 10 5 cells/insert. Caco-2 monolayers were cultured for 14 days, with three medium changes/week, until a functional cell monolayer was obtained. Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 25 mM glucose and 4 mM glutamine and supplemented with 10 mM HEPES and 20% (v/v) heat-inactivated (HI) fetal bovine serum (FBS).
  • DMEM Dulbecco's Modified Eagle Medium
  • HI heat-inactivated
  • FBS heat-inactivated fetal bovine serum
  • THP-1 cells THP1 cells (InvivoGen) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium containing 11 mM glucose and 2 mM glutamine, supplemented with 10 mM HEPES, 1 mM sodium pyruvate and 10% (v/v) HI-FBS. THP1 cells were seeded in 24- well plates at a density of 5 x 10 5 cells/well and treated with 100 ng/mL of PMA for 48 hours (h). PMA induces the differentiation of the cells into macrophage-like cells.
  • RPMI Roswell Park Memorial Institute
  • Caco-2/THP-l co-cultures To mimic the interface between host immune cells and the fermentation products of the gut microbiome, in vitro experiments were conducted based on previous studies by Satsu and colleagues [255]. In this setup, the colonic suspensions collected from the SHIME are brought in contact with the apical side of the co-cultures (i.e. Caco-2 cells). The effects observed on the basolateral chamber where the THP-l cells reside are mediated indirectly by signals produced by the Caco-2 cells and/or by the transport of micro- and macro-molecules.
  • the apical compartment containing Caco-2 cells was filled with sterile- filtered (0.22 pm) colonic SHIME suspensions (diluted 1:5 (v/v) in Caco-2 complete medium). Cells were also treated apically with 12 mM Sodium butyrate (NaB) (Sigma- Aldrich) as a positive control in experiments establishing the system.
  • the basolateral compartment containing THP1 cells was filled with Caco-2 complete medium. Cells were also exposed to Caco-2 complete medium in both chambers in control experiments.
  • THP- 1 cells were stimulated with Caco-2 complete medium containing 500 ng/mL of ultrapure LPS (. Escherichia coli K12, InvivoGen). Cells were also stimulated at the basolateral side with LPS in combination with 1 pM hydrocortisone (HC) (Sigma-Aldrich) and medium without LPS (LPS-) in control experiments. Cultures were incubated at 37 degrees Celcius in a humidified atmosphere of air/C0 2 (95:5, v/v).
  • HC hydrocortisone
  • LPS- medium without LPS
  • the basolateral supernatants were collected for cytokine measurement (human IL-6 and IL-10) by Luminex. multiplex (Affymetrix-eBioscience) according to the manufacturers’ instructions. All treatments were done in triplicate.
  • the probiotic with digestive enzymes formulation increases fermentation activity in the simulated gut microenvironment:
  • the consumption of acid and base reflects the overall microbial activity in the SHIME reactors representing the proximal and distal colon. Generally speaking, there are distinct bacterial populations that are native to the proximal and distal colon regions, reflecting the different requirements for digestion in each segment. Ligures 1A and 1B depict the average weekly acid/base consumption during the control and treatment periods (i.e. before and after 5-LU/vancomycin treatment).
  • the treatment period consisted of one week of 5-LU/vancomycin followed by three weeks without these agents during which time recovery from 5-FU/vancomycin could be monitored and compared for the experimental treatment arms, as follows: 1) Control arm: receiving no supplement; 2) Preventative arm that had already been receiving the probiotic with digestive enzymes supplement all through the previous stabilization and control periods; and. 3; The curative arm that commenced supplementation with the probiotic with digestive enzymes supplement only at the start of 5-FU/vancomycin administration.
  • SCFA The abundant SCFA, acetate, propionate and butyrate, are generated by fermentation of dietary fibers by gut microbiota. SCFA have a plethora of health-promoting effects through their interactions with metabolite- sensing G protein-coupled receptors on the gut epithelium and on immune cells. In these experiments, we monitored the production of these three SCFA in the proximal and distal colon reactors, comparing pre- and post-5- FU/vancomycin treatments in the control (non-supplemented), curative, and preventative arms that were treated with the probiotic with digestive enzymes supplement.
  • Chemotherapy treatment resulted in a decrease in acetate levels in both the proximal and distal colon reactors. Although the recovery of acetate did not occur in the proximal colon, preventative treatment with the probiotic with digestive enzymes supplement increased acetate in both control and 5-FU/vancomycin treatment periods in the distal colon, suggesting that the pretreatment with the supplement offsets the adverse impact of the drugs on microbial metabolism.
  • Figure 2 also shows the results for butyrate production in the SHIME reactors, a primary product of Clostridium clusters IV and XlVa (phylum Firmicutes).
  • butyrate is largely metabolized by intestinal epithelial cells where it serves as an energy source as well as a homeostatic factor for normal colonic cell turnover and repair processes.
  • the expected result was obtained whereby 5-FU/vancomycin treatment strongly decreased butyrate levels in both the proximal and distal colon reactors.
  • Supplementation of the reactors with curative and preventative administration of the probiotic with digestive enzymes supplement resulted in improved butyrate production in the proximal and distal colon.
  • the differences between the curative and preventative arms were not statistically different in either of the reactors.
  • Figure 3 show the effects of a curative (CUR) and preventive (PREV) administration of the probiotic with digestive enzymes supplement as compared to a control SHIME (CTRF) on luminal Factobacillus (left panels) and Bifidobacterium (right panels) levels (16S rDNA copies/mF) in the proximal (PC; top panels) and distal colon (DC; bottom panels).
  • CUR curative
  • PREV preventive
  • CTRF control SHIME
  • the data are represented for the control weeks (Cl, C2)andtreatment weeks (TR1, TR2, TR3, and TR4. It should be noted that 5-FU/vancomycin was administered to the system in TR1 and discontinued in TR2- TR4.
  • Figure 4 shows the effect of a curative (CUR) and preventive (PREV) administration of the probiotic with digestive enzymes supplement as compared to a control SHIME (CTRL) on luminal Bacteroidetes, and Finnicutes levels (16S rDNA copies/mL) in the proximal (PC; top panels) and distal colon (DC; bottom panels).
  • CUR curative
  • PREV preventive
  • the data are represented for the control weeks (Cl, C2) and treatment weeks (TRI, TR2, TR3, and TR4. It should be noted that 5-FU/vancomycin was administered to the system in TRI and discontinued in TR2-TR4.
  • Preventative administration of the probiotic with digestive enzymes supplement was being administered throughout the control periods (Cl and C2), while curative supplementation of the probiotic with digestive enzymes formulation was initiated and maintained at TR1-TR4. () indicates statistically significant differences relative to the preceding period, while different letters indicate a statistical difference between different treatments; p ⁇ 0.05.
  • l6S-targeted Illumina sequencing was used whereby amplified 16S rRNA marker gene sequences are clustered into taxonomic units of bacteria.
  • the Simpson diversity index was calculated. The lowest possible value of the index is 1, representing a community consisting of only one Operational Taxonomic Unit (OTU). The highest possible value is the total number of OTUs, and the higher the index, the larger the diversity and the larger the evenness.
  • OTU Operational Taxonomic Unit
  • Table 1 shows the Simpson Diversity Index results to evaluate the impact of the probiotic with digestive enzymes supplement on diversity changes in the microbiota in the SHIME that are caused by 5-FU and vancomycin.
  • the supplement given preventatively had the most significant impact on increasing the microbial diversity.
  • the highest diversity was observed in the negative control (i.e. no supplement) following recovery from 5-FU and vancomycin (TR4).
  • Curative treatment with the supplement also increased the microbial diversity in both the proximal and distal colons.
  • FIG. 5 shows the abundance(%) of the dominant phyla in the lumen of the proximal (PC) and distal colon (DC) of the SHIME upon a curative (CUR) and preventive (PREV) administration of the probiotic with digestive enzymes supplement as compared to control (CTRL) the end of the control week 2 (C) and treatment week 4 (TR) period.
  • CUR curative
  • PREV preventive
  • preventative treatment prior to 5-FU/vancomycin treatment did not cause any major changes at the phylum level in the proximal colon.
  • preventative supplementation resulted in increases in abundance of Actinobacteria and reductions in Bacteroidetes and Proteobacteria.
  • the probiotic with digestive enzymes formulation modulates cytokine production in a model of intestinal inflammation:
  • THP-l cells derived from acute leukemia, differentiate into macrophages upon culture with phorbol l2-myristate l3-acetate (PMA), and can then be activated toward a highly pro-inflammatory phenotype upon treatment with lipopolysaccharide (LPS).
  • PMA phorbol l2-myristate l3-acetate
  • LPS lipopolysaccharide
  • Caco-2 cells were placed on top of PMA- treated THP-l cells, on the apical and basolateral sides of culture chambers, respectively.
  • Table 2 outlines the corresponding statistical analyses performed using two- way ANOVA with Dunnett's multiple comparisons test. Significance is depicted where (*), (**), (***) and (****) represent p ⁇ 0.05, p ⁇ 0.01, p ⁇ 0.001 and p ⁇ 0.0001, respectively.
  • Ns non- specific; PC: proximal colon; DC: distal colon.
  • a murine model will be implemented to evaluate the effect of a probiotic with digestive enzymes formulation on ovarian failure caused by chemotherapy.
  • C57BL6 mice Weight 20-25 g
  • Age 4-6 weeks will be allotted randomly to three different groups, and each had 6 mice; group 1; the control group (no chemotherapy; placebo supplement), group 2; the chemotherapy group (chemotherapy; placebo supplement), group 3 (treatment arm; the mice will be given chemotherapy and a probiotic with digestive enzymes supplement).
  • the supplement will be a formulation containing a proprietary blends of probiotic; specifically, Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei, Lactobacillus paracasei, and digestive enzymes; specifically, amylase, glucoamylase, lipase, bromelain, maltase, lactase, hemicellulose, xylanase, papain, and invertase.
  • probiotic specifically, Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus case
  • the probiotics with digestive enzymes formulation will be diluted at a concentration of (65 pg/mL) in the drinking water of the mice.
  • a placebo capsule will be diluted into the drinking water.
  • a combination chemotherapy (CTX) of busulfan (12 mg/kg) and cyclophosphamide (70 mg/kg) (Sigma Aldrich., St. Louis, MO, USA) will beadministered intra-peritoneal to mice in groups 2 and 3.
  • mice receiving chemotherapy will become infertile and posses enhanced levels of follicle stimulating hormone (FSH).
  • FSH follicle stimulating hormone
  • Mice that received the probiotic with digestive enzymes supplement will be compared in terms of the percentages of animals who developed ovarian failure vs. controls, as well as with respect to the blood concentrations of FSH. The results will show that fewer of the chemotherapy-treated mice that receive the probiotic with digestive enzyme formulation demonstrate an elevation of FSH levels and the FSH levels will be statistically lower in this group vs. the placebo group that will receive chemotherapy.
  • Lacy, M.Q., et al., Idiotype-pulsed antigen-presenting cells following autologous transplantation for multiple myeloma may be associated with prolonged survival.
  • Hus, L, et al., Vaccination ofB-CLL patients with autologous dendritic cells can change the frequency of leukemia antigen-specific CD8+ T cells as well as CD4+CD25+FoxP3+ regulatory T cells toward an antileukemia response.

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Abstract

Methods, compositions, and protocols are disclosed for increasing the efficacy of cancer therapies using formulations comprised of probiotics and digestive enzymes. In preferred embodiments, said formulations of probiotics with digestive enzymes are comprised of one or more of the following: Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei, Lactobacillus paracasei, and also a blend of digestive enzymes; including but not limited to amylase, glucoamylase, lipase, bromelain, maltase, lactase, hemicellulase, xylanase, papain, and invertase.

Description

AUGMENTING EFFICACY OF CANCER THERAPIES USING PROBIOTIC
BASED COMPOSITIONS
Cross-Reference to Related Applications
[001] This application claims the benefit of priority to United States Provisional
Appliation No. 62/636,728, filed February 28, 2018, the contents of which are
incorporated herein by reference.
Field of the Invention
[002] The invention pertains to the field of cancer therapy. Specifically, the invention pertains to methods and formulations of probiotics with digestive enzymes to increase efficacy of cancer therapies and/or for reducing the toxicity and adverse events associated with said cancer therapies.
Background of the Invention
[003] Despite improvements in current cancer treatments with respect to clinical endpoints such as progression-free survival, overall survival, and objective response rates, the majority of the treatment regimens that patients endure are associated with high levels of toxicity. For example, chemotherapy-associated diarrhea occurs in 50-80% of cancer patients depending on the chemotherapy regimen that is administered [1, 2]. Some chemotherapy regimens, for example, those utilizing irinotecan, fluorouracil, and leucovorin, can lead to potentially life-threatening gastrointestinal syndrome. Adverse events include diarrhea (including Grades 3 and 4 severe diarrhea), nausea, vomiting, fatigue, malaise, leukopenia, and neutropenia can occur, to name a few. These adverse events can lead to dire medical consequences. Diarrhea and nausea that are associated with cancer therapy can lead to dehydration, electrolyte imbalances, malnutrition, pain, and bleeding as a result of damge to the gastrointestinal tract. As a consequence, patients have low immune function and inflammation. Cancer treatments can therefore render patients more susceptible to various infections, which can themselves cause or exacerbate some of the abovementioned adverse events. For example, infections with Clostridium difficile are twice as common in hospitalized cancer patients as compared to other inpatients [3]. In addition, the antibiotics that are prescribed to treat these infections can themselves cause symptoms such as diarrhea; for example, for treating C. difficile, these antibodies may include vancomycin, fidaxomicin, or metronidazole. Research efforts have focused on evaluating the prophylactic application of specific antibiotics for cancer patients, which are aimed at weighing the risks vs. benefits of such an approach [4]. One one hand, the severity of treatment-associated diarrhea means that it must be treated aggressively, making a preventative approach desirable. On the other hand, overuse of antibiotics also carries risks. Novel agents for preventing or dealing with cancer drug-associated adverse events including new anti-diarrhea and antiemetic agents are therefore sought. These agents should ideally be capable of improving or mitigating the adverse events caused by the cancer drugs themselves as well as for reducing the need for antibiotic use or improving the adverse events associated with said antibiotics.
[004] The microbiome in the human gut is composed of 500-1000 distinct bacterial species and up to 1014 total bacteria that are responsible for digestive health, including the assurance of nutrient and vitamin bioavailability and energy metabolism, and also have a significant impact on the immune system of a subject. Of relevance to the present invention, intestinal microorganisms may determine the outcome of cancer treatments and are also directly impacted by the treatments, often in a negative manner. Gut dysbiosis, whereby the composition of microorganisms is altered and often their diversity is reduced, predicted resistance to immunotherapeutic interventions such as checkpoint inhibitors in melanoma patients [5]. A low diversity of commensal microorganisms was also associated with immune suppression in cancer patients [6]. Addtionally, the prevalence and relative proportions of specific types of microorganisms in the gut are also altered in response to certain cancer drugs as well as by antibiotics. Certain therapeutic drugs that are used to treat cancer cause changes in the microbiome that compromise energy metabolism, cause inflammation, and underlie the adverse events and poor quality of life of patients undergoing drug treatments. One example is 5-fluoro uracil (5-FU), a first line agent for the treatment of metastatic colorectal agent, which is associated with severe colonic mucositis indicated by weight loss, diarrhea, bloody stool, shortened colon, and infiltration of inflammatory cells. 5-FU diminishes bacterial richness and diversity in the gut, leading to reduced overall abundance of important phyla involved in normal microbial metabolism. In fact, causal relationships are established between 5-FU-induced perturbations of the gut microbiota, inflammation in the intestine, and adverse events experienced by patients [7]. [005] Despite the emerging knowledge of the roles of probiotics in the immune system, in additional to their widespread use as nutritional supplements, most probiotics are not studied for their specific bioactivity in the gut and the vast majority of probiotic formulations are not evaluated for use for specific disease indications. The impact of probiotics on the gut microbiome may vary widely. Both the safety and efficacy of probiotics have been raised as issues, particualry for use by subjects who are immunocompromised [8, 9]. The present invention also teaches specific formulations of probiotics combined with digestive enzymes for use in oncology and methods of use for subjects in need thereof.
Summary of the Invention
[006] Various aspects of the invention are described below.
[007] Aspect 1. A method of increasing efficacy of a cancer therapy comprising administration of a probiotic composition.
[008] Aspect 2. The method of aspect 1, wherein said probiotic composition comprises one of more probiotics.
[009] Aspect 3. The method of aspect 2, wherein said probiotic composition is comprised of one or more probiotics selected from a group comprising of: a)
Bifidobacterium infantis; b) Bifidobacterium bifidum; c) Lactobacillus acidophilus; d) Lactobacillus salivarius; e) Lactobacillus plantarum; f) Lactobacillus rhamnosus; g) Bifidobacterium longum; h) Lactobacillus casei; and i) Lactobacillus paracasei.
[0010] Aspect 4. The method of aspect 1, wherein said probiotic composition comprises the addition of digestive enzymes to said probiotics
[0011] Aspect 5. The method of aspect 4, wherein said digestive enzymes are selected from a group comprising of: a) amylase; b) glucoamylase; c) lipase; d) bromelain; e) maltase; f) lactase; g) hemicellulose; h) xylanase; i) papain, and j) invertase.
[0012] Aspect 6. The method of aspect 1, wherein said cancer therapy is one or more therapies selected from a group comprising of: a) immunotherapy; b)
chemotherapy; c) surgery; d) radiation therapy; and e) hyperthermia.
[0013] Aspect 7. The method of aspect 1, wherein augmentation of efficacy of said cancer therapy is reduction of toxicity associated with said cancer therapy.
[0014] Aspect 8. The method of aspect 1, wherein said probiotic composition is administered in a manner to augment immunogenic death of tumor cells. [0015] Aspect 9. The method of aspect 8, wherein said immunogenic death of tumor cells is associated with release of HMGB-l from tumors.
[0016] Aspect 10. The method of aspect 8, wherein said immunogenic death of tumor cells is elicited by radiation therapy and augmented by treatment with said probiotic composition.
[0017] Aspect 11. The method of aspect 10, wherein said radiation therapy is capable of triggering an abscopal effect.
[0018] Aspect 12. The method of aspect 11, wherein said abscopal effect is amplified by administration of said probiotic composition.
[0019] Aspect 13. The method aspect 12, wherein said abscopal effect comprises one or more of the following biological events: a) recognition of non-viable cancer cells by antigen presenting cells; b) uptake of immunogenic agents from said non-viable cancer cells by said antigen presenting cells; c) presentation of peptides derived from said immunogenic agents by said antigen presenting cells to T cells; and d) activation of T cells capable of recognizing and killing identical or substantially similar cells to said non- viable cancer cells.
[0020] Aspect 14. The method of aspect 13, wherein said non-viable cancer cells are the result of exposure to radiation capable of inducing DNA damage.
[0021] Aspect 15. The method of aspect 13, wherein said non-viable cancer cells are the result of exposure to radiation capable of inducing ceramide signalling.
[0022] Aspect 16. The method of aspect 13, wherein said non-viable cancer cells are the result of exposure to radiation capable of inducing caspase activation.
[0023] Aspect 17. The method of aspect 13, wherein said non-viable cancer cells are the result of exposure to chemotherapy capable of inducing DNA damage.
[0024] Aspect 18. The method of aspect 13, wherein said non-viable cancer cells are the result of exposure to chemotherapy capable of inducing DNA damage.
[0025] Aspect 19. The method of aspect 13, wherein said antigen presenting cells are monocytes.
[0026] Aspect 20. The method of aspect 13, wherein said antigen presenting cells are macrophages.
[0027] Aspect 21. The method of aspect 13, wherein said antigen presenting cells are B cells.
[0028] Aspect 22. The method of aspect 13, wherein said antigen presenting cells are endothelial cells. [0029] Aspect 23. The method of aspect 13, wherein said antigen presenting cells are dendritic cells.
[0030] Aspect 24. The method of aspect 13, wherein said dendritic cells are myeloid dendritic cells.
[0031] Aspect 25. The method of aspect 13, wherein said dendritic cells are lymphoid dendritic cells.
[0032] Aspect 26. The method of aspect 13, wherein said immunogenic agents are agents capable of acting as tumor antigens.
[0033] Aspect 27. The method of aspect 13, wherein said immunogenic agents are capable of stimulating a“danger signal” to the immune system.
[0034] Aspect 28. The method of aspect 27, wherein said danger signal comprises activation of a toll like receptor.
[0035] Aspect 29. The method of aspect 28, wherein said toll like receptor is selected from a group comprising of TLR 1-9.
[0036] Aspect 30. The method of aspect 13, wherein said presentation of peptides is accomplished by loading of peptides into MHC I or MHC II.
[0037] Aspect 31. The method of aspect 30, wherein said peptides presented on MHC I are involved in stimulation of CD8 T cells.
[0038] Aspect 32. The method of aspect 30, wherein said peptides presented on MHC I are involved in stimulation of CD4 T cells.
[0039] Aspect A method of preventing hematological toxicity of a cancer therapy comprising administration of a probiotic composition.
[0040] Aspect 34. The method of aspect 33, wherein said probiotic composition comprises one of more probiotics.
[0041] Aspect 35. The method of aspect 34, wherein said probiotic composition is comprised of one or more probiotics selected from a group comprising of: a)
Bifidobacterium infantis; b) Bifidobacterium bifidum; c) Lactobacillus acidophilus; d) Lactobacillus salivarius; e) Lactobacillus plantarum; f) Lactobacillus rhamnosus; g) Bifidobacterium longum; h) Lactobacillus casei; and i) Lactobacillus paracasei.
[0042] Aspect 36. The method of aspect 33, wherein said probiotic composition comprises the addition of digestive enzymes to said probiotics
[0043] Aspect 37. The method of aspect 36, wherein said digestive enzymes are selected from a group comprising of: a) amylase; b) glucoamylase; c) lipase; d) bromelain; e) maltase; f) lactase; g) hemicellulose; h) xylanase; i) papain, and j) invertase. [0044] Aspect 38. The method of aspect 33, wherein said cancer therapy is selected from a group comprising of: a) radiotherapy; b) chemotherapy; c) immunotherapy; d) hyperthermia; and e) surgery.
[0045] Aspect 39. The method of aspect 33, wherein said hematopoietic toxicity is neutropenia.
[0046] Aspect 40. The method of aspect 33, wherein said hematopoietic toxicity is anemia.
[0047] Aspect 41. The method of aspect 33, wherein said hematopoietic toxicity is thrombocytopenia.
[0048] Aspect 42. The method of aspect 33, wherein said hematopoietic toxicity is pancytopenia.
[0049] Aspect 43. The method of aspect 33, wherein said hematopoietic toxicity is lymphopenia.
[0050] Aspect 44. The method of aspect 1 and 33, wherein said probiotic composition is a pharmaceutical composition comprising: a formulation of probiotics comprising: Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei, and Lactobacillus paracasei; and, a formulation of digestive enzymes comprising: amylase, glucoamylase, lipase, bromelain, maltase, lactase, hemicellulase, xylanase, papain, and invertase.
[0051] Aspect 45. The method of aspect 44, wherein the form of composition is selected from the group consisting of a pharmaceutically acceptable: pill, a tablet, a caplet, a capsule, powder, a suspension, a gel, and a liquid.
[0052] Aspect 46. The method of aspect 45, wherein said probiotics are present in a total weight of 116.20 mg.
[0053] Aspect 47. The method of aspect 45, wherein said digestive enzymes are present in a total weight of 272.65 mg.
[0054] Aspect 48. The method of aspect 44, wherein said formulations of probiotics and digestive enzymes are contained in a plurality of capsules.
[0055] Aspect 49. The method of aspect 48, wherein said formulations of probiotics and digestive enzymes are present in the same, single, capsule.
[0056] Aspect 50. The method of aspect 44, wherein said probiotics are present in the following amounts: Bifidobacterium Infantis - between 5-7 billion cfu,
Bifidobacterium Longum -between 750 million and 2 billion cfu, Bifidobacterium Bifidum -between 3-5 billion cfu, Lactobacillus Rhamnosus - between 5-7 billion cfu, Lactobacillus Acidophilus -between 1-3 billion cfu, Lactobacillus salivarius -between 1- 3 billion cfu, Lactobacillus plantarum - between 1-3 billion cfu, Lactobacillus Casei - between 750,000 million-2 billion cfu, Lactobacillus paracasei - between 1-3 billion cfu.
[0057] Aspect 51. The method of aspect 50, wherein said probiotics are present in the following amounts: Bifidobacterium Infantis - 6 billion cfu, Bifidobacterium Longum
- 1 billion cfu, Bifidobacterium Bifidum - 4 billion cfu, Lactobacillus Rhamnosus - 6 billion cfu, Lactobacillus Acidophilus - 2 billion cfu, Lactobacillus salivarius - 2 billion cfu, Lactobacillus plantarum - 2 billion cfu, Lactobacillus Casei - 1 billion cfu,
Lactobacillus paracasei - 2 billion cfu.
[0058] Aspect 52. The method of Aspect 44, wherein said enzymes are present in the following amounts: hemicellulase - between 5-11 mg, xylanase - between 2-6 mg, amylase - between .5 - 2.5 mg, glucoamylase - between 30 - 70 mg, maltase - between 8
- 12 mg, papain - between .5 - 3mg, portease, such as bromelain - between 13-23 mg, lipase - between 18- 32 mg, invertase - between .5-3 mg, lactase - between 8.0-11 mg.
[0059] Aspect 53. The method of Aspect 52, wherein said enzymes are present in the following amounts: hemicellulase - 8 mg, xylanase - 3.9 mg, amylase - 1.33 mg, glucoamylase - 50 mg, maltase - 10 mg, papain - 1.7 mg, bromelain - 18 mg, lipase - 25 mg, invertase - 1.5 mg, and lactase - 9.5 mg.
[0060] Aspect 54. The method of aspects 1 and 33, wherein said probiotic composition is a pharmaceutical composition comprising: (a) probiotic formulation comprising Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus rhamnosus GG,
Bifidobacterium longum, Lactobacillus casei, Lactobacillus paracasei, and (b) a formulation of digestive enzymes.
[0061] Aspect 55. The method of aspect 54, wherein said digestive enzymes are selected from the group consisting of: a) proteases; b) Carbohydrate-digesting enzymes; c) Fiber-digesting enzymes; and, d) lipases.
[0062] Aspect 56. The method of aspect 55, wherein said carbohydrate-digesting enzymes are selected from the group consisting of: a) Amylase; b) Glucoamylase; c) Lactase; d) Invertase; and, e) Maltase.
[0063] Aspect 57. The method of aspect 55, wherein said fiber-digesting enzymes are selected from the group consisting of: a) Xylanase; and b) Hemicellulase. [0064] Aspect 58. The method of aspect 54, wherein the form of pharmaceutical composition is selected from the group consisting of: a pill, a tablet, a caplet, a capsule, powder, a suspension, a gel, and, a liquid.
[0065] Aspect 59. The method of aspect 54, wherein said formulations of probiotics and digestive enzymes are formulated in the same single formulation.
[0066] Aspect 60. The method of aspect 59, wherein said single formulation is a capsule.
[0067] Aspect 61. The method of aspect 55, wherein said formulation of digestive enzymes comprises at least two proteases.
[0068] Aspect 62. A probiotic formulation having: Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius,
Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum,
Lactobacillus casei, Lactobacillus paracasei; in conjunction with a formulation of digestive enzymes having: amylase, glucoamylase, lipase, bromelain, maltase, lactase, hemicellulase, xylanase, papain, and invertase that is useful in the preparation of a medicament for lowering cancer therapy associated toxicities in a subject in need.
[0069] Aspect 63. The probiotic formulation of aspect 62, wherein said formulation of probiotics and digestive enzymes are combined in capsules.
[0070] Aspect 64. The probiotic formulation of aspect 62, wherein said probiotics and digestive enzymes are formulated to be administered orally.
[0071] Aspect 65. The probiotic formulation of Aspect 62, wherein said probiotics are present in the following amounts: Bifidobacterium Infantis - between 5-7 billion cfu, Bifidobacterium Longum -between 750 million and 2 billion cfu, Bifidobacterium Bifidum -between 3-5 billion cfu, Lactobacillus Rhamnosus - between 5-7 billion cfu, Lactobacillus Acidophilus -between 1-3 billion cfu, Lactobacillus salivarius -between 1- 3 billion cfu, Lactobacillus plantarum - between 1-3 billion cfu, Lactobacillus Casei - between 750,000 million-2 billion cfu, Lactobacillus paracasei - between 1-3 billion cfu.
[0072] Aspect 66. The probiotic formulation of Aspect 65, wherein said probiotics are present in the following amounts: Bifidobacterium Infantis - 6 billion cfu, Bifidobacterium Longum - 1 billion cfu, Bifidobacterium Bifidum - 4 billion cfu, Lactobacillus Rhamnosus - 6 billion cfu, Lactobacillus Acidophilus - 2 billion cfu, Lactobacillus salivarius - 2 billion cfu, Lactobacillus plantarum - 2 billion cfu,
Lactobacillus Casei - 1 billion cfu, Lactobacillus paracasei - 2 billion cfu. [0073] Aspect 67. The probiotic formulation of Aspect 66, wherein said enzymes are present in the following amounts: hemicellulase - between 5-11 mg, xylanase - between 2-6 mg, amylase - between .5 - 2.5 mg, glucoamylase - between 30 - 70 mg, maltase - between 8 - 12 mg, papain - between .5 - 3mg, portease, such as bromelain - between 13-23 mg, lipase - between 18- 32 mg, invertase - between .5-3 mg, lactase - between 8.0-11 mg.
[0074] Aspect 68. The probiotic formulation of Aspect 67, wherein said enzymes are present in the following amounts: hemicellulase - 8 mg, xylanase - 3.9 mg, amylase - 1.33 mg, glucoamylase - 50 mg, maltase - 10 mg, papain - 1.7 mg, bromelain - 18 mg, lipase - 25 mg, invertase - 1.5 mg, and lactase - 9.5 mg.
[0075] Aspect 69. The probiotic formulation of aspect 62, wherein said formulation is administered together with a chemotherapeutic agent.
[0076] Aspect 70. The probiotic formulation of aspect 69, wherein said
chemotherapeutic agent is capable of augmenting expression of immunogenic molecules on a cancer cell or plurality of cancer cells.
[0077] Aspect 71. The probiotic formulation of aspect 70, wherein said
chemotherapeutic agents are selected from a group comprising of: a) alkylating agents; b) plant alkaloids; c) antitumor antibiotics; d) antimetabolites; e) topoisomerase inhibitors; and f) ribonucleotide reductase inhibitors.
[0078] Aspect 72. The probiotic formulation of aspect 71, wherein said
chemotherapeutic agent is vinblastine at a concentration and duration sufficient to augment HLA I expression.
[0079] Aspect 73. The probiotic formulation of aspect 71, wherein said
chemotherapeutic agent is cyclophosphamide at a concentration and duration sufficient to augment HLA I expression.
[0080] Aspect 74. The probiotic formulation of aspect 71, wherein said
chemotherapeutic agent is topotecan at a concentration and duration sufficient to augment HLA I expression.
[0081] Aspect 75. The probiotic formulation of aspect 71, wherein said
chemotherapeutic agent is paclitaxel at a concentration and duration sufficient to augment HLA I expression.
[0082] Aspect 76. The probiotic formulation of aspect 62 wherein said formulation is administered to protect from toxicity effects of chemotherapy. [0083] Aspect 77. The probiotic formulation of aspect 76, wherein said toxicity effect of chemotherapy is gastrointestinal toxicity.
[0084] Aspect 78. The probiotic formulation of aspect 76, wherein said toxicity effect of chemotherapy is hematological toxicity.
[0085] Aspect 79. The probiotic formulation of aspect 76, wherein said toxicity effect of chemotherapy is cardiac toxicity.
[0086] Aspect 80. The probiotic formulation of aspect 76, wherein said toxicity effect of chemotherapy is gastrointestinal toxicity.
[0087] Aspect 81. The probiotic formulation of aspect 76, wherein said toxicity effect of chemotherapy is vascular toxicity.
[0088] Aspect 82. The probiotic formulation of aspect 76, wherein said toxicity effect of chemotherapy is nephrotoxicity toxicity.
[0089] Aspect 83. The probiotic formulation of aspect 76, wherein said toxicity effect of chemotherapy is neurological toxicity.
[0090] Aspect 84. The probiotic formulation of aspect 76, wherein said toxicity effect of chemotherapy is alopecia.
[0091] Aspect 85. The probiotic formulation of aspect 76, wherein said toxicity is severity of grade 2 or higher diarrhea resulting from an anti-cancer chemotherapy in a subject in need thereof.
[0092] Aspect 86. The probiotic formulation of aspect 76, wherein said
chemotherapy comprises antibody therapy with or without small-molecule chemotherapy.
[0093] Aspect 87. The probiotic formulation of aspect 76, wherein said
chemotherapy comprises bevacizumab, cetuximab or panitumumab antibody therapy with or without small-molecule chemotherapy.
[0094] Aspect 88. The probiotic formulation of aspect 76, wherein said probiotic formulation is administered in a regimen comprising daily administration of said formulation for 2 to 10 days, and said chemotherapy cycle is 8 to 24 days.
[0095] Aspect 89. The probiotic formulation of aspect 76, wherein said
chemotherapy comprises administration of one or more compounds selected from the group consisting of antimetabolites, alkylating agents, anticancer antibiotics, microtubule targeting agents, topoisomerase inhibitors, alkaloids, antibodies, pyrimidine analogs, purine analogs, folate antagonists, epidipodophyllotoxins, DNA damaging agents, antiplatelet agents, platinum coordination complexes, hormones, hormone analogs, aromatase inhibitors, anti-angiogenic compounds, growth factor inhibitors, angiotensin receptor blockers, nitric oxide donors, antisense oligonucleotides, cell cycle inhibitors, differentiation inducers, mTOR inhibitors, mitochondrial dysfunction inducers, chromatin disruptors.
[0096] Aspect 90. The probiotic formulation of aspect 76, wherein said
chemotherapy comprises administration of one or more compounds selected from the group consisting of 5-fluorouracil (5-FU), floxuridine, capecitabine, gemcitabine, cytarabine, irinotecan, doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, mitoxantrone, topotecan, lapatinib, oxaliplatin, cisplatin, carboplatin, folinic acid, methothrexate, erlotinib, sorafenib, and lapatinib.
[0097] Aspect 91. The probiotic formulation of aspect 76, wherein said
chemotherapy comprises administration of oxaliplatin or irinotecan.
[0098] Aspect 92. The probiotic formulation of aspect 76, wherein said
chemotherapy comprises administration of oxaliplatin or irinotecan in combination with cetuximab, bevacizumab, and/or panitumumab.
[0099] Aspect 93. The probiotic formulation of aspect 76, wherein said
chemotherapeutic agent(s) is administered at least during the first two consecutive days from the beginning of each chemotherapy cycle.
[00100] Aspect 94. The probiotic formulation of aspect 76, wherein said
chemotherapy is administered as FOLFOX or FOLFIRI chemotherapy regimen.
[00101] Aspect 95. The probiotic formulation of aspect 65, wherein said formulation is administered in 6 to 8 doses per day.
[00102] Aspect 96. The probiotic formulation of aspect 66, wherein said formulation is administered in 6 to 8 doses per day.
[00103] Aspect 97. The probiotic formulation of aspect 67, wherein said formulation is administered in 6 to 8 doses per day.
[00104] Aspect 98. Use of a probiotic formulation in the preparation of a medicament for improving the efficacy of a cancer therapy comprising in a subject with cancer scheduled to undergo or is undergoing treatment for cancer by administering to said subject said probiotic formulation in an amount sufficient to improve the efficacy of the cancer therapy.
[00105] Aspect 99. Use of a probiotic formulation in the preparation of a
medicament for alleviating cancer treatment-related adverse events in a subject with cancer who is scheduled to undergo, is undergoing, or has undergone treatment for cancer by identifying symptoms of cancer treatment-related adverse events or risks for experiencing cancer treatment-related adverse events in said subject; and administering to said subject said probiotic formulation in an amount sufficient to alleviate said cancer treatment-related adverse events.
Brief Description of the Drawings
[00106] Fig. 1A is a bar graph showing that as the reactors acidify during changes in microbial activity, base is added.
[00107] Fig. 1B is a bar graph showing that as the reactors acidify during changes in microbial activity, base is added.
[00108] Figure 2 depicts bar graphs showing analysis of acetate, propionate, and butyrate concentrations in the SHIME.
[00109] Figure 3 depicts bar graphs showing the effects of a curative (CUR) and preventive (PREV) administration of the probiotic with digestive enzymes supplement as compared to a control SHIME (CTRL) on luminal Lactobacillus and Bifidobacterium levels in the proximal (PC) and distal colon (DC).
[00110] Figure 4 depicts bar graphs showing the effect of a curative (CUR) and preventive (PREV) administration of the probiotic with digestive enzymes supplement as compared to a control SHIME (CTRL) on luminal Bacteroidetes, and Finnicutes levels in the proximal (PC) and distal colon (DC).
[00111] Figure 5 depicts a bar graph showing the differences in microbial compositions at the phylum level in the proximal and distal colons caused by the probiotic with digestive enzymes supplement.
[00112] Figure 6A depicts a bar graph showing modulation of IL-6 in an in vitro model of intestinal inflammation by metabolites from SHIME reactors treated with the probiotic with digestive enzymes supplement.
[00113] Figure 6B depicts a bar graph showing modulation of IL-10 in an in vitro model of intestinal inflammation by metabolites from SHIME reactors treated with the probiotic with digestive enzymes supplement.
Description of the Invention
[00114] This invention teaches methods and compositions for augmenting the efficacy of cancer therapeutic drugs using formulations comprising probiotic compositions combined with digestive enzymes. This invention also teaches methods and compositions for preventing, improving or alleviating adverse events that are associated with cancer therapeutic drugs in a subject in need of treatment.
[00115] In the context of the present invention, the terms “probiotic (based) formulation” and“probiotic (based) composition” may refer to a formulation comprising probiotics or to a formulation containing probiotics combined together with digestive enzymes. Specifically, the terms “formulation” and “composition” encompass said probiotics or said probiotics with digestive enzyme ingredients that are mixed together into a delivery vehicle for administration to or consumption by a subject in need thereof. In the context of the present invention, administration or consumption of a probiotic based formulation by said subject may occur orally via swallowing of a capsule, tablet, or soft gel, orally via swallowing of a powder that may be dissolved or distributed in food or water, orally via gavage, sublingually via a troche, or rectally as a suppository.
[00116] In the context of the present invention, each probiotic microorganism in said probiotic based formulation is measured by activity per gram(g), which is expressed as colony forming units (CFU).
[00117] In the context of the present invention, each digestive enzymes in said probiotic based formulation is measured by enzyme activity per gram (g). The activities of each digestive enzyme are measured using methods known in the art, as follows: Hemicellulase units (HU) for hemicellulase, Xylanase unit (XU) for xylanase, Alpha - amylase Dextrinizing units (DU) for amylase, amyloglucosidase (AGU) for glucoamylase, degrees Diastatic power (DP) for maltase, enzyme activity or units (TP) for papain, Gelatin Digestion units (GDU) for bromelain, Fungi Lipase-Intemational FIP standard (FIP) for lipase, Summer units (SU) for invertase, lactase units (ALU) for lactase.
[00118] In the context of the present invention,“cancer therapeutic drug(s)”, “cancer therapeutic agent(s)”,“cancer drug(s)”,“cancer therapy”,“oncology drug(s)”,“treatment agent(s)”, and the like, are used synonymously to refer to the cancer treatment received by a subject in need thereof, as is recognized in the art. Broadly speaking, these cancer treatments may include one or more of the following treatment categories: a) immunotherapy; b) chemotherapy; c) surgery; d) radiation therapy; e) hyperthermia; f) radiation; g) targeted therapy; h) hormone therapy; i) stem cell transplantation. Use of these terms also signifies that one or more of the abovementioned treatment categories may be administered to said subject. [00119] In the context of the present invention,“subject” refers to a human (e.g. a subject with cancer and/or a subject taking one or more cancer therapeutic drugs and/or one or more antibiotics) and, more broadly speaking, may include any mammal. Accordingly, aspects of the present invention may be practiced in human clinical medicine as well as in veterinary medicine.
[00120] In the context of the present invention, the phrase“augmentation of efficacy” or“improved efficacy” of a cancer therapeutic agent includes enhancement of one or more of the therapeutic effect(s) of said cancer drug. In the context of the present invention, the therapeutic effect of a cancer drug can refer to inhibition of tumor growth, induction of tumor regression, suppression of metastasis, inhibition of metastasis, suppression of proliferation, induction of programmed and/or non-programmed cell death. At a clinical level, therapeutic effects of one or more cancer inhibiting drugs can be observed by one or more of the following clinical endpoints: a) Overall survival (OS); b) Progression-free survival (PFS); c) Time to progression (TTP); d) Time to treatment failure (TTF); c) Event- free survival; d) Objective response rate (ORR); e) Duration of response (DR); f) Improvement in immunoscore; and g) Decrease in cancer- associated biomarkers including but not limited to tumor-associated antigens (TAAs).
[00121] In the context of the present invention, said subject may be afflicted with one or more types of cancer including but not limited to the following: brain, melanoma, bladder, breast, cervix, colon, rectal, head and neck, kidney, lung, non- small cell lung, mesothelioma, ovary, prostate, sarcoma, stomach, uterus, as well as carcinomas, including spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, carcinoma villosum, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, and carcinoma scroti, The term "sarcoma" generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar, heterogeneous, or homogeneous substance. Sarcomas include, chondrosarcoma, fibrosarcoma, lymphosarcoma, melano sarcoma, myxosarcoma, osteosarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilns' tumor sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.
[00122] One embodiment of the present invention teaches a method of improving the efficacy of a cancer therapy comprising the following: (a) Identifying a subject with cancer who plans to undergo or is undergoing treatment for cancer; (b) administering to said subject a formulation comprising a probiotic based formulation, which is preferably a formulation comprised of probiotics with digestive enzymes. In one embodiment, said formulation of probiotics with digestive enzymes comprises of one or more probiotics selected from a group comprising of: a) Bifidobacterium inf antis, b) Bifidobacterium bifidum, c) Lactobacillus acidophilus, d) Lactobacillus salivarius; e) Lactobacillus plantarum, f) Lactobacillus rhamnosus, g) Bifidobacterium longum, h) Lactobacillus casei; and i) Lactobacillus paracasei. Said formulation of probiotics with digestive enzymes may also be comprised of one or more digestive enzymes selected from a group comprising: a) amylase; b) glucoamylase; c) lipase; d) bromelain; e) maltase; f) lactase; g) hemicellulose; h) xylanase; i) papain, and j) invertase. In the preferred embodiment of this invention,
[00123] A preferred composition for improving the efficacy of cancer therapy in a subject comprises the following active ingredients: a) Bifidobacterium infantis; b) Bifidobacterium bifidum, c) Lactobacillus acidophilus, d) Lactobacillus salivarius; e) Lactobacillus plantarum; f) Lactobacillus rhamnosus; g) Bifidobacterium longum; h) Lactobacillus casei and i) Lactobacillus paracasei j) amylase; k) glucoamylase; 1) lipase; m) bromelain; n) maltase; o) lactase; p) hemicellulose; q) xylanase; r) papain, and s) invertase. In one embodiment, this compositon comprises between 100-500 mg by weight of digestive enzymes and between 50-250 mg by weight digestive enzymes per dose. In a preferred embodiment, said formulation comprises 272.65 mg by weight of digestive enzymes and 116.20 mg of probiotics per dose. Preferably, this formulation is contained in capsules for oral administration.
[00124] To practice the invention, administration of the probiotic based compositon may begin before or during the initation of cancer therapy, which typically will include numerous treatment cycles for ceratin cancer therapeutic agents (for example, chemotherapy drugs or monoclonal antibodies). The term“treatment cycle" is used herein to refer to a period of time between the initial administration of an anti-cancer agent and its repeat administration. For example, the cycle of the FOLFOX4 chemotherapy includes 14 days, wherein anti-cancer agents are administered only for the first 2 days of the cycle as follows: Day 1: oxaliplatin 85 mg/m.sup.2 IV infusion and leucovorin 200 mg/m.sup.2 IV infusion both given over 120 minutes at the same time in separate bags, followed by 5-FU 400 mg/m.sup.2 IV bolus given over 2-4 minutes, followed by 5-FU 600 mg/m.sup.2 IV infusion as a 22 -hour continuous infusion; Day 2: leucovorin 200 mg/m.sup.2 IV infusion, followed by 5-FU 400 mg/m.sup.2 IV bolus given over 2-4 minutes, followed by 5-FU 600 mg/m.sup.2 IV infusion as a 22-hour continuous infusion. Similarly, the cycle of the FOLFIRI chemotherapy includes 14 days, wherein anti-cancer agents are administered only for the first 2 days of the cycle as follows: irinotecan (180 mg/m.sup.2 IV over 90 minutes) concurrently with folinic acid (400 mg/m.sup.2 [or 2. times.250 mg/m.sup.2] IV over 120 minutes), followed by fluorouracil (400-500 mg/m.sup.2 IV bolus) then fluorouracil (2400-3000 mg/m.sup.2 intravenous infusion over 46 hours). Bevacizumab is usually given intravenously every 14 days, although the frequency can be dose dependent (for example 5 mg/kg by intravenous infusion every two weeks or 7.5 mg/kg by intravenous infusion every three weeks). In colon cancer, it is given in combination with the chemotherapy drug 5-FU (5-fluorouracil), leucovorin, and oxaliplatin or irinotecan. One recommended dose and schedule for cetuximab is 400 mg/m.sup.2 administered intravenously as a l20-minute infusion as an initial dose, followed by 250 mg/m.sup.2 infused over 30 minutes weekly, preferably in combination with FOLFIRI. In the context of the present invention, it may be preferable to iniate daily administration of said probiotic based formulation (for example, consisting of probiotics with digestive enzymes) prior to the initaiton of the first cycle of treatment and then daily for the duration of the all the treatment cycles at a minimum. However, said probiotic based formulation may be given for a duration that surpasses the therapeutic regimen that said subject undergoes. To practice this invention, administration of said probiotic based formulation may also be initiated during any of the treatment cycles with a given cancer therapeutic agent. Preferably, said probiotic based formulation will be administered on a daily basis thereafter.
[00125] To practice the invention, preferable dosing regimens are established. In the context of this invention, the probiotic based formuations may be adminsiterd to a subject in need thereof a doses of 2-10 capsules per day and on a daily basis. Preferably, this formulation is administered in 6-8 doses per day and on a daily basis. In a preferred embodiment, the probiotics based formuation is taken at a dose of 6 capsules per day; specifically, 2 capsules taken in the morning, 2 capsules at midday, and 2 capsules at night on days when cancer treatment agents are not being administered to said subject. Eight capsules may be taken daily on each day that a cancer treatment agent is being administered to said subject; specifically, 2 capsules taken in the morning, 2 capsules at midday, 2 capsules at night, 1 additional capsule taken within 2 hours prior to the cancer treatment agent is administered, and 1 additional capsule taken within two hours after the cancer treatment agent has been administered. This dosing regimen can be continued for the duration of cancer treatment cycles and irrespective of the specific cancer drug(s) being administered to said subject.
[00126] In embodiments of this invention, said probiotic based formulation contains said probiotics having the following activity/gram: between 25-150 billion colony forming units (cfu) for Bifidobacterium inf antis, between 50-150 billion cfu for Bifidobacterium longum, between 350-550 billion cfu for Bifidobacterium bifidum, between 100-300 billion cfu for Lactobacillus rhamnosus, between 100-300 billion cfu for Lactobacillus acidophilus, between 200-400 billion for Lactobacillus salivarius, between 300-500 billion cfu for Lactobacillus plantarum, between 200-400 billion cfu for Lactobacillus casei, and between 300-500 billion cfu Lactobacillus paracasei.
[00127] In embodiments of this invention, said probiotic based formulation comprises digestive enzymes having the following activity/gram: between 300,000-500,000 HCU for hemicellulase, between 100,000-300,000 XU for xylanase, between 100,000-300,000 DU for amylase, between 900-1,100 AGU for glucoamylase, between 10,000-30,000 DP for maltase, between 900,000, 1,100,000 TU for papain, between 2,000-4,000 gdu for bromelain, between 150-300 FIP for lipase, between 100,000-300,000 SU for invertase, between 75,000-150,000 ALU for lactase.
[00128] In other embodiments, reduction of toxic effects or adverse events mediated by therapeutic anti-cancer agents in said subject with cancer is a desired goal of the invention. In some embodiments, the invention provides the ability to augment dosage of chemotherapy or other oncology drugs in order to achieve killing of tumor cells without achieving dose limiting toxicity. In other embodiments, the invention provides means of providing multiple therapies that would otherwise not be possible due to toxicities, in other embodiments the invention provides means of suppressing cytokine release syndrome associated with cancer therapies, particularly immunotherapies, in other embodiments the invention provides means of reducing adverse events associated with administration of checkpoint inhibitors through selectively enhancing tolerance towards self antigens while not suppressing or minimally suppressing antitumor immunity. In the previously mentioned embodiments the invention teaches administration of probiotics and/or combinations of probiotics and digestive enzymes, collectively referred to herein as probiotic based compositions.
[00129] Accordingly, the present invention teaches methods for prevention or management of cancer treatment-related adverse events comprising the following: (a) Identifying a subject with cancer who has plans to undergo, is undergoing or has undergone treatment for cancer; (b) Identifying that said subject also exhibits symptoms of cancer treatment-related adverse events or is at-risk for experiencing cancer treatment-related adverse events (b) administering to said subject a formulation comprising probiotics with digestive enzymes.
[00130] In the context of this invention,“prevention” or“management” of said adverse event(s) may refer to initiation of administration of a probiotic based formulation prior to initiation or re-initiation of treatment with a cancer drug or at some time during the treatment with a cancer drug (i.e. whe treatment with the cancer drug has already commenced).
[00131] In the context of this invention, said cancer treatment-related adverse events may be identified as one or more of the following: a) Nausea; b) Vomiting; c) Diarrhea; d) Abdominal pain; e) Bloating; f) Ulcers in the gastrointestinal tract; g) Mucositis in the gastrointestinal tract; h) Esphagitis; i) Infusion-related reactions; j) Fatigue; k) Malaise; 1) Cytokine release syndrome; m) Serum sickness; n) Alopecia; o) Anemia; p) Leukocytosis; q) Viral infections; r) Bacterial infections; s) Yeast infections t) Acid reflux; u) Neutropenia; v) Lymphopenia; w) Thrombocytopenia; x) Leaky gut; y) Cardiomyopathy; and z) Neuropathy.
[00132] As disclosed herein, combinations of probitoics or probiotics with digestive enzymes may be adminsiterd to a subject with cancer in need thereof. A preferred composition for prevention or management of cancer treatment-related adverse events in a subject comprises the following active ingredients: a) Bifidobacterium infantis b) Bifidobacterium bifidum c) Lactobacillus acidophilus ; d) Lactobacillus salivarius e) Lactobacillus plantarum f) Lactobacillus rhamnosus, g) Bifidobacterium longum h) Lactobacillus cased, and i) Lactobacillus paracasev, j) amylase; k) glucoamylase; 1) lipase; m) bromelain; n) maltase; o) lactase; p) hemicellulose; q) xylanase; r) papain, and s) invertase. In one embodiment, this compositon comprises between 100-500 mg by weight of digestive enzymes and between 50-250 mg by weight digestive enzymes per dose. In a preferred embodiment, said formulation comprises 272.65 mg of digestive enzymes and 116.20 mg of probiotics per dose. Preferably, this formulation is contained in capsules for oral administration.
[00133] To practice this invention, dosing regimens for prevention or management of adverse event(s) in a subject may be similar or identifical to those used to improve the efficacy of cancer therapy in a subject as per the descriptions provided herein. Preferably, the formulations herein administered in 6-8 doses per day and on a daily basis.
[00134] Broadly speaking, said probiotic based formulation(s) described herein can be used for reducing existing inflammation in a subject with cancer, wherein said inflammation may be caused by the cancer disease process and/or treatment agents used to treat cancer or infections.
[00135] Broadly speaking, said probiotic based formulation(s) described herein can be used for modulating the immune system in a subject with cancer, wherein said immune system may require modulation due to the cancer disease process and/or treatment agents used to treat cancer or infections.
[00136] Broadly speaking, said probiotic based formulation(s) described herein can be used for modulating the microbiome in the gut. The microbiome refers to the microorganisms that are present in the gut, which can be assessed based on sequencing tehniques and analytical techniques that are known in the art. The two main approaches for analyzing the microbiome, 16S ribosomal RNA (rRNA) gene amplicons and shotgun metagenomics are widely practiced. For detecting that modulation of the gut microbiome has occurred, these techniques may be performed using stool samples acquired before and after a specific treatment, which, in this case, may be before and after administration of a probiotics-based formulation to a subject in need thereof.
[00137] The probiotic based formulation(s) described herein are intended to treat, reverse, reduce or prevent dysbiosis of the gut microbiome. This may also be referred to generally as“modulating” the microbiome. In the context of the present invention, the term “dysbiosis” can refer to one or more of (i) an increase in the proportion of a first species (bacterial or archaeal species) or strain in the microbiota (eg, gut microbiota) (eg, Bacteriodes fragalis or thetaiotamicron ); (ii) an increase in the relative proportion of first and second species (eg, Bacteriodes fragalis versus Clostridium dificile, or Streptococcus thermophilus verus Escherichia coli or Lactococcus lactis ), first and second strains of the same species, or first and second phyla which are different from each other (eg, Bacteriodetes versus Firmicutes); (iii) an addition of a species or strain that was not comprised by the microbiota prior to the treatment method; (iv) a decrease in the proportion of a first species (bacterial or archaeal species) or strain in the microbiota (eg, Clostridium dificile or Streptococcus thermophilus ); (v) a decrease in the relative proportion of first and second species (eg, Bacteriodes fragalis versus Clostridium dificile, or Streptococcus thermophilus versus Escherichia coli or Lactococcus lactis ), first and second strains of the same species, or first and second phyla which are different from each other (eg, Bacteriodetes versus Firmicutes); and (vi) a removal of a species or strain that was not comprised by the microbiota prior to the treatment method.
[00138] In a preferred embodiment of the present invention, dysbiosis of the gut microbiome is treated, reversed, reduced and/or prevented by administering to a subject in need thereof a probiotic based composition comprising the following: a) Bifidobacterium infantis, b) Bifidobacterium bifiidunv, c) Lactobacillus acidophilus, d) Lactobacillus salivarius; e) Lactobacillus plantarum, f) Lactobacillus rhamnosus, g) Bifidobacterium longum, h) Lactobacillus cased, and i) Lactobacillus paracasev, j) amylase; k) glucoamylase; 1) lipase; m) bromelain; n) maltase; o) lactase; p) hemicellulose; q) xylanase; r) papain, and s) invertase. In a preferred embodiment, said formulation comprises 272.65 mg of digestive enzymes and 116.20 mg of probiotics per dose. In one embodiment, between 2-10 doses are administered daily. In a preferred embodiment, 6-8 doses are administered daily. In a preferred embodiment, said formulation of probiotics with digestive enzymes is contained in capsules for oral administration to a subject in need thereof, wherein one dose is present in each capsule. In the context of the invention, said formulation is administered for a duration of a minimum of three weeks; preferably for at least three months.
[00139] In a preferred embodiment, said probiotic based composition comprising probiotics with digestive enzymes is administered to a subject in need thereof for modulating the ratios of Bacteroidetes to Firmicutes phyla in the gut of said subject. This ratio is known in the art to have significance with regard to the health of the microbiome and the overall wellness of a subject. In the context of the present invention, modification of the presence of phyla can serve as confirmation of the bioactivity of the probiotics present in said formulation. Said modification can be measured by evaluating the composition of the microbiome from stool samples using methods known in the art, for example, 16S rRNA based sequencing methods, that may be performed before and after administration of said probiotic based composition. Within the scope of this invention, changes in the genera and species within the Firmicutes and Bacteroidetes phyla can also be monitored. Examples within the Firmicutes phylum include but are not limited to the following: Clostridium clusters IV and XlVa, the Veillonellaceae family, and Lactobacillus species. Examples within Bacteroidetes phlyum including but are not limited to Bacteroides species. In the context of the present invention, other phyla, species and genera can also be evaluated in order to determine the effects of the probiotic based composition for modulating the gut microbiome; for example, Akkermansia muciniphila (phylum Verrucomicrobia). It is herein implied that modulation of the ratios and quantities of microorganisms belonging to the Firmicutes, Bacteroidetes or other phyla may also modulate the metabolic and fermentation activities in the gut, which may in turn modify the immune status, susceptibility to infections, manifestion of adverse events in response to cancer therapeutics, and/or the efficacy of cancer therapeutics.
[00140] In a preferred embodiment, said probiotic based composition comprising probiotics with digestive enzymes is administered to a subject in need thereof for increasing the presence of Lactobacillus and/or Bifidobacterium genera in the gut of said subject.
[00141] In one embodiment of the invention said probiotic based formulation is administered to a subject in need thereof prophylactically for modulating the gut microbiome. In the context of the present invention, prophylactically refers to initiation administration of said probiotic based formulation at a time prior to administration of one or more cancer therapeutic agents and/or an antibiotics. Administration of said probiotic based formulation may then continue throughout and following the administration of one or more cancer therapeutic agents and/or an antibiotics. Preferably, said probiotic based formulation will be administered for a total duration of at least three or six months. In another embodiment, said probiotic based formulation is administered to a subject in need thereof subsequent to administration of one or more cancer therapeutic agents or antibiotics for the purpose of modulating the gut microbiome. Administration of said probiotic based formulation may then continue throughout and following the administration of one or more cancer therapeutic agents and/or an antibiotics. Preferably, said probiotic based formulation will be administered for at least three or six months.
[00142] To practice the present invention, it should be noted that the probiotic based formulation may require testing for bioactivity of its active ingredients prior to administration to said subject. Methods known in the art can be utilized for determining the activity of probiotic microorganisms. For example, he SHIME® model allows to culture the complex gut microbiota over a longer period under representative conditions of the different intestinal regions. Therefore, the SHIME® does not only allow to obtain detailed information about the fermentation profile of the test products, but importantly also about the localization of the intestinal fermentation activity. The SHIME® allows for performance of mechanistic research as the gut microbiome is fully stable prior to treatment. This is achieved by applying a two- week stabilization period with strict control of the environmental conditions (e.g. nutrients, residence time, pH, temperature, ...) so that the human fecal inoculum evolves to a stable in vitro microbiota that is representative for the different colon regions of interest. This is crucial because a microbiota will inevitably alter after being transferred from an in vivo environment to a laboratory model. Upon reaching a stable composition and activity, one can apply one specific change of interest (= treatment), knowing that all the observed effects are because of this one specific treatment. In the context of the present invention, the probiotic based composition may be added to the SHIME®. The typical reactor setup of the SHIME®, representing the gastrointestinal tract of the adult human, was described previously [10]. It consists of a succession of five reactors simulating the different parts of the human gastrointestinal tract. The first two reactors are of the fill-and-draw principle to simulate different steps in food uptake and digestion, with peristaltic pumps adding a defined amount of SHIME feed (140 mL 3x/day) and pancreatic and bile liquid (60 mL 3x/day), respectively to the stomach and small intestine compartment and emptying the respective reactors after specified intervals. The last three compartments simulate the large intestine. These reactors are continuously stirred, they have a constant volume and pH control. Retention time and pH of the different vessels are chosen in order to resemble in vivo conditions in the different parts of the colon. Upon inoculation with fecal microbiota, these reactors simulate the ascending, transverse and descending colon. Inoculum preparation, retention time, pH, temperature settings and reactor feed composition were previously described by [11].
[00143] In the context of the present invention, the probiotic based composition may be added to the SHIME® together with a chemotherapeutic agent and/or an antibiotic. As control arms, a chemotherapeutic agent and/or antibiotic can be added without the probiotic based composition. As an additional control, the probiotic based composition can be added to the SHIME® reactors prior to addition of a chemotherapeutic agent and/or an antibiotic. Subsequently, activity in the SHIME® reactors can be measured based on acid/base consumption, concentrations of short-chain fatty acids in the reactors, concentration of lactate in the reactors, and the microbial community composition using quantitative PCR methods known in the art. Accordingly, the SHIME® method can be used to assess the bioactivity of a probiotic based composition in the presence of a chemotherapeutic agent or an antibiotic. Additionaly, the SHIME® study can be utilized to gauge the optimum time for administration of the probiotic based composition for its microbiome modulating effects to occur (i.e. before or during chemotherapy and/or antibiotic administration).
[00144] In the context of this invention, the terms“probiotic based compositions” or “probiotic based formulations” may refer to probiotics comprising two or more live microorganisms from the genera Lactobacillus or Bifidobacterium. In one embodiment of the invention, nine probiotic microorganisms from the genera Lactobacillus or Bifidobacterium are present in the composition In a preferred embodiment, the following probiotic species are present in the composition: Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei, Lactobacillus paracasei
[00145] In the context of this invention, the terms“probiotic(s) based compositions” or“probiotic(s) based formulations” may also refer to two or more probiotics with two or more digestive enzymes. In preferred embodiments of the invention, said mixtures or blends of probiotics with digestive enzymes are prepared or manufactured in a manner that they are co-adminisetered to a subject in need thereof.
[00146] In a preferred embodiment of the present invention, said digestive enzymes present in said formulation include enzymes that are have activity for digesting carbohydrates, proteins, and fats. In one embodiment, 10 digestive enzymes are present in the formulation. In a preferred embodiment, the following digestive enzymes are present in the composition: a) amylase; b) glucoamylase; c) lipase; d) bromelain; e) maltase; f) lactase; g) hemicellulose; h) xylanase; i) papain, and j) invertase.
[00147] In a preferred embodiment of the present invention, said probiotic based composition is contained in a capsule. In a preferred embodiment, said capsule contains probiotics and digestive enzymes as active ingredients and inactive ingredients that include one or more of the following substances: a) Inulin; b) Rice extract; c) Hydroxypropyl methylcellulose. In a preferred embodiment of the present invention, said probiotic based composition is administered to a subject in need thereof by the oral route of administration. In another embodiment of the invention, said capsule may be broken open and the contents of said capsules may be mixed into applesauce for subjects who have difficulty swallowing intact capsules.
[00148] In one embodiment of the invention, said probitioc based composition is adminsered to a subject with cancer who is taking antibotics or who may need antibiotics before, during or aftr the course of treatment with cancer drugs. In the context of the present invention, said subject may have an infection or be at-risk for an infection. It is known in the art, that infections in cancer patients often occr due to their immune suppressed status, which is due to their disease and/or secondary to certain cancer drug treatments. Said antibiotics include but are not limited to the following: Aminocoumarins (such as Novobiocin, Albamycin, Coumermycin and Clorobiocin), Aminoglycosides (such as Amikacin, Apramycin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin and Spectinomycin), Ansamycins (such as Geldanamycin, Herbimycin, Rifaximin and Streptomycin), Carbapenems (such as Ertapenem, Daripenem, Cilastatin ('Imipenem') and Meropenem), Cephalosporins (such as Cefadroxil, Cefazolin, Cefalothin ('Cefalotin'), Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil and Ceftobiprole) Glycopeptides (such as Teicoplanin, Vancomycin and Telavancin), Lincosamides (such as Clindamycin and Lincomycin), Lipopeptides (such as Daptomycin), Macrolides (such as Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin and Spiramycin), Monobactams (such as Aztreonam), Nitrofurans (such as Furazolidone and Nitrofurantoin), Oxazolidonones (such as Linezolid, Posizolid, Radezolid and Torezolid), Penicillins (such as Amoxicillin. Ampicillin, Aziocillin, Carbeniallin, Cloxacillin, Didoxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Pencillin G, Penicillin V, Piperacillin, Temocillin and Ticarcillin), Penicillin combinations (such as Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam and Ticarcillin/davulanate), Polyethers (such as Monensin), Polypeptides (such as Sacitracin, Colistin and Polymyxin B), Quinolones (such as Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovalioxacin, Grepafloxacin, Sparfloxacin and Temafloxacin); Sulfonamides (such as Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Sulfamethoxazole (Co-trimexazole, TMP-SMX, 'Trimethoprim') and Sulfonamidochrysoidine), Tetracyclines (such as Demeclocycline, Doxycycline, Minocycline, Oxytetracycline and Tetracycline) and Others (such as Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin ('Rifampin'), Rifabutin, Rifapentine, Streptomycin, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid, Metronidazole, Mupirocin, Platensimycin, Ouinupristin (Dalfopristin), Thiamphenicol, Tigecycline, Tinidazole and Trimethoprim).
[00149] This invention teaches formulations of probiotics with digestive enzymes that can serve as anti-diarrhea agents in subjects with cancer for preventing or improving cancer drug-associated diarrhea.
[00150] Another embodiment of this invention pertains to formulations of probiotics with digestive enzymes that can serve to prevent or allevaite antibiotic-associated diarrhea in a subject with cancer in need thereof.
[00151] Another preferred embodiment of this invention pertains to formulations of probiotics with digestive enzymes that can serve as antiemetic agents in a subject with cancer for the purpose of preventing or improving cancer drug-associated nausea or vomiting.
[00152] Another embodiment of the invention pertains to formulations of probiotics with digestive enzymes that serve to prevent or treat antibiotic-associated nausea or vomiting in a subject with cancer.
[00153] Another embodiment of the invention pertains to formulations of probiotics with digestive enzymes that serve to prevent or treat alopecia in a subject with cancer.
[00154] Another embodiment of the invention pertains to a formulation of probiotics with digestive enzymes that serves to prevent or treat cardiotoxicity in a subject with cancer. Examples of cardiotoxicity in said subject may include clinical syndromes including but not limited to reduced ejection fraction, dilated cardiomyopathy, and congestive heart failure.
[00155] Another embodiment of the invention pertains to a formulation of probiotics with digestive enzumes that serves to prevent or treat neurotoxicity in a subject with cancer. Examples of neurotoxicity in a said subject may include clinical syndromes including but not limited to depression, encephalopathy, cerebellar ataxia, acute neuromuscular syndrome, thrombotic microangiopathy, blurred vision, dizziness, headaches, delayed progressive encephalopathy, optic neuropathy, Parkinsonism, leukocencephalopathy, and acute chemical meningitis.
[00156] Another embodiment of the invention teaches the use of formulations of probiotics with digestive enzymes that serve to prevent or to treat chemotherapy and/or antibiotic associated immune suppression. In some embodiments immune suppression refers to reduction in numbers of immune cells. In other embodiments, immune suppression refers to activity of immune cells. Examples of immune cells include neutrophils, eosinophils, basophils, natural killer cells, natural killer T cells, gamma delta T cells, monocytes, macrophages, dendritic cells, B cells, CD4 T cells, CD8 T cells, and innate lymphoid cells. Examples of immune cell activities include cytokine production, phagocytosis, cytotoxic activity, and antigen presenting activity.
[00157] In another embodiment of the invention, a formulation of probiotics with digestive enzymes is used for treating cytokine release syndrome, serum sickness, or sepsis in a subject with cancer. Said formulation may be used in conjunction with supportive care or other medications used to address said cytokine release syndrome, serum sickness or sepsis. In one embodiment of the invention, a subject receiving treatment with chimeric antigen receptor (CAR) T cells is administered a probiotic based formulation for several days prior to infusion of CAR T cells for the prevention of cytokine release syndrome.
[00158] This invention teaches compositions of probiotics with digestive enzymes for improving efficacy of cancer therapy, for reducing toxicities of cancer therapy, and/or for allowing for higher concentrations of cancer therapeutic agents to be used in a subject with cancer. The invention also teaches compositions of probiotics with digestive enzymes administered to a subject with cancer in order to improve the subject’s tolerability to a cancer treatment for the purpose of allowing continued or extended administration of cancer therapeutic agents to said subject. For example, increased numbers of cycles of chemotherapy may be tolerated or additional cancer therapeutic agents may be administed to said subjects. [00159] The specific probiotic based formulations when used in combination and administered to a subject, can be utilized to improve the health of a subject undergoing cancer therapy; specifically, for subjects afflicted with toxicities associated with cancer therapy including hypotension, cardiovascular disease, disorders related to the vascular system, metabolic syndrome, disorders of the digestive tract, hematological toxicities, hepatotoxicities, and neural toxicities.
[00160] Said probiotics based formulation can also be administered to a subject as a to improve quality of life of said subject and of specific organ systems in said subject in the absence of any specific cancer therapy as a prophylactic. The terms“overall wellness” or “quality of life’ may be used interchangeably in the context of the present invention. Quality of life may be defined subjectively by said subject; for example, based on self- reporting of energy levels, presence or absence of malaise or other measures. Alternatively, quality of life may be defined by a health care professional or clinical study personnel using a validated questionnaire. One non-limiting example of a questionnaire is the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire. In another embodiment of the present invention, efficacy of said probiotic based formulation for improving quality of life in a subject in need thereof is evaluated by a health care professional or clinical study personnel using Kamofsky Performance Status, an assessment tool for functional impairment that is known in the art.
[00161] An object of the invention is to provide a composition that relieves symptoms of chemotherapy induced toxicity. An object of the invention is to provide a composition that relieves symptoms of radiotherapy induced toxicity. An object of the invention is to provide a composition that relieves symptoms of immunotherapy induced toxicity. An object of the invention is to provide a composition that relieves symptoms of hyperthermia induced toxicity. An object of the invention is to provide a composition that relieves symptoms of surgery induced toxicity.
[00162] An object of the invention is to provide a probiotic based formulation that replaces and replenishes the bacteria that are beneficial to the human body in a subject in need thereof, after such bacteria are reduced or destroyed by intervention with a cancer therapy. An object of the invention is to provide a probiotic based formulation to improve digestion and nutrient absorption in a subject with cancer. An object of the invention is to provide the inventive composition in a form that has a long shelf life. An object of the invention is to provide the inventive composition in pill form and to provide a pill that reaches the digestive tract prior to being absorbed. An object of the invention is to provide a probiotic based formulation that allows a subject in need thereof to ingest foods that otherwise result in adverse reactions by the body (for example, any reaction related to food intolerance or allergic responses), subsequent to which the patient was treated with a cancer therapy or a plurality of cancer therapies.
[00163] Chemotherapeutic agents are well known in the art. Specific non-limiting examples of chemotherapeutic agents are provided throughout the specification and include, for example, FOLFOX (a chemotherapy regimen for treatment of colorectal cancer, which comprises administration of folinic acid (leucovorin), fluorouracil (5-FU), and oxaliplatin) and FOLFIRI (a chemotherapy regimen for treatment of colorectal cancer, which comprises administration of folinic acid (leucovorin), fluorouracil (5-FU), and irinotecan), as well as administration of targeted monoclonal antibody therapy (e.g., bevacizumab, cetuximab, or panitumumab) alone or in combination with chemotherapeutic agents.
[00164] During the past few years, attention has turned to using antibodies to target different tumor- associated antigens. These include surface glycoproteins associated with clusters of differentiation, CTLA-A, or pathways regulated by growth factors. The use of immunotherapeutic monoclonal antibodies has had a tremendous impact on cancer treatment, hoaever, some monoclonal antibodies can have severe side effects that are related to the antigens they target. For example, Bevacizumab (AVASTIN®) is a monoclonal antibody that targets a protein called VEGF that affects tumor blood vessel growth. It can cause debilitating side effects such as high blood pressure, bleeding, poor wound healing, blood clots, and kidney damage.
[00165] For the purpose of the invention,“antibody” referes to proteins capable of binding antigens with selectivity. In some embodiments of the invention, the probiotic based composition is used to enhance therapeutic efficacy of passively administered antibodies (such as Herceptin®, Rituximab®, and Cetuximab®), and/or augment cancer killing/inhibitor activity of endogenous antibodies in a subject. Antibodies exist in three formats; monoclonal (mAbs), oligo/polyclonal and antibody-drug conjugates. mAbs represent an effective therapeutic modality and are important to the treatment paradigm of various diseases. Recent insights into the detailed mechanism of mAbs link their strong disease fighting potential to the immune system. Drug manufacturers have leveraged the ability of mAbs to induce an antibody-dependent cell-mediated cytotoxicity (ADCC) effect to develop better treatments that prolong survival and quality of life of patients. In addition, mAbs designed to inhibit specific checkpoints in the immune system have demonstrated strong immune responses and therapeutic benefit in patients. Immune cells express proteins that are immune checkpoints that control and down-regulate the immune response. These are best defined in T lymphocytes and include PD-l, CTLA-4, TIM-3 and LAG3. Tumor cells express the ligands to these receptors. When T cells bind the ligand to these proteins on the tumor cells, the T cell is turned off and does not attempt to attack the tumor cell. These agents, typically mAbs, that block these proteins, are known as checkpoint inhibitor drugs. However, the degree of efficacy of these therapies is heavily reliant on the immune system of patients, many of whom are severely immuno-compromised.
[00166] In the context of the present invention, non-limiting examples of checkpoint inhibitor drugs include the following: a) Inhibitors of Programmed Death 1 (PD-l, CD279), such as nivolumab (OPDIVO.RTM., BMS-936558, MDX1106, or MK-34775), and pembrolizumab (KEYTRODA.RTM., MK-3475, SCH-900475, lambrolizumab, CAS Reg. No. 1374853-91-4), as well as the PD-l blocking agents described in U.S. Pat. Nos. 7,488,802, 7,943,743, 8,008,449, 8,168,757, 8,217, 149, WO 03042402, WO 2008156712, WO 2010089411, WO 2010036959, WO 2011066342, WO 2011159877, WO 2011082400, and WO 2011161699; b) Inhibitors of Programmed Death— Ligand 1 (PD-L1, also known as B7-H1 and CD274), including antibodies such as BMS-936559, MPDL3280A), MEDI4736, MSB0010718C, and MDX1105-01); also including: atezolizumab, durvalumab and avelumab; c) Inhibitors of CTLA-4, such as ipilimumab (YERVOY.RTM., MDX-010, BMS-734016, and MDX-101), tremelimumab, antibody clone BNI3 (Abeam), RNA inhibitors, including those described in WO 1999/032619, WO 2001/029058, U.S. 2003/0051263, U.S. 2003/0055020, U.S. 2003/0056235, U.S. 2004/265839, U.S. 2005/0100913, U.S. 2006/0024798, U.S. 2008/0050342, U.S. 2008/0081373, U.S. 2008/0248576, U.S. 2008/055443, U.S. Pat. Nos. 6,506,559, 7,282,564, 7,538,095 and 7,560,438 (each incorporated herein by reference); d) Inhibitors of PD-L2 (B7-DC, CD273), such as AMP-224 (Amplimune, Inc.) and rHIgMl2B7; and e) Inhibitors of checkpoint proteins, including: LAG3, such as IMP321; TIM3 (HAVCR2); 2B4; A2aR, ID02; B7H1; B7-H3 or B7H3, such as antibody MGA271; B7H4; BTLA; CD2; CD20, such as ibritumomab tiuxetan, ofatumumab, rituximab, obinutuzumab and tositumomab; CD27, such as CDX-1127; CD28; CD30, such as brentuximab vedotin; CD33, such as gemtuzumab ozogamicin; CD40; CD52, such as alemtuzumab; CD70; CD80; CD86; CD112; CD137; CD160; CD226; CD276; DR3; OX-40 (TNFRSF.sub.4 and CD134); GAL9; GITR; such as TRX518; HAVCR2; HVEM; IDOl; ICOS (inducible T cell costimulator; CD278); such as MED 1570 (Medlmmune LLC) and AMG557 (Amgen); KIR; LAIR; LIGHT; MARCO (macrophage receptor with collageneous structure); PS (phosphatidylserine); SLAM; TIGIT; VISTA; and VTCN1; or a combinations thereof. In another variation, the checkpoint inhibitor is an inhibitor of a checkpoint protein selected from the group of PD-l, PD-L1, and CTLA-4. In another variation, the checkpoint inhibitor is selected from the group of an anti-PD-l antibody, and anti-PD-Ll antibody, and an anti- CTLA-4 antibody. In one variation, the anti-PD-l antibody is selected from the group of nivolumab, pembrolizumab, and lambrolizumab. In another variation, the anti-PD-Ll antibody is selected from the group of as BMS-936559, MPDL3280A, MEDI4736, MSB0010718C, and MDX1105-01. In yet other variations, the anti-PD-Ll antibody is selected from the group of durvalumab, atezolizumab, and avelumab. In another variation, the anti-CTLA-4 antibody is selected from the group of ipilimumab and tremelimumab. In one embodiment, the checkpoint inhibitor is selected from the group consisting of nivolumab, pembrolizumab, lambrolizumab, BMS-936559, MPDL3280A, MEDI4736, MSB0010718C, MDX1105-01, durvalumab, atezolizumab, avelumab, ipilimumab, and tremelimumab. In certain embodiment, the checkpoint inhibitor is selected from the group consisting of nivolumab, pembrolizumab, lambrolizumab, durvalumab, atezolizumab, avelumab, ipilimumab, and tremelimumab. In some embodiment, In one embodiment, the checkpoint inhibitor is selected from the group consisting of nivolumab, pembrolizumab, durvalumab, atezolizumab, and avelumab.
[00167] In the context of the present invention, non-limiting examples of mAbs include the following: abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab, ertumaxomab, etaracizumab, farietuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, moxetumomab, narnatumab, naptumomab, necitumumab, nimotuzumab, nofetumomabn, ocaratuzumab, ofatumumab, olaratumab, onartuzumab, oportuzumab, oregovomab, panitumumab, parsatuzumab, patritumab, pemtumomab, pertuzumab, pintumomab, pritumumab, racotumomab, radretumab, rilotumumab, rituximab, robatumumab, satumomab, sibrotuzumab, siltuximab, simtuzumab, solitomab, tacatuzumab, taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tocilizumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab, vorsetuzumab, votumumab, zalutumumab, CC49 and 3F8. In the context of the present invention, the mAbs may be joined to another entity; for example, mAbs joined to a chemotherapy drug or to a radiolabeled particle, or they may be bispecific monoclonal antibodies that are made up of two different mAbs.
[00168] The probiotic based compositions disclosed herein can be administered to a subject in need thereof as a means of enhancing immune stimulatory efficacy of dendritic cell therapy. The dendritic cell approach is designed to indirectly stimulate a patient’s T- cells by leveraging the role of dendritic cells in presenting antigens to T-cells. Cancer vaccines are the most common application of dendritic cells. The only FDA-approved dendritic cell therapy is PROVENGE, which entails collecting monocytes from the patient, maturing them into dendritic cells,“loading” ex vivo with the patient’s cancer antigens, and then re-infusing in the patient. Currently, this process is cumbersome and expensive, and again, relies on an intact and effective immune system of the patient. In one embodiment of this invention, a probiotic based composition is is utilized to increase dendritic cell anticancer activity. Specifically, in one embodiment, said probiotic mix is utilized to increase the number of dendritic cells infiltrating tumors. In fact, several publications show that augmentation of dendritic cells infiltrating tumors is associated with enhanced patient survival.
[00169] Methods of assessing immune infiltrates in tumors and significance to treatment outcome are known in the art and an example is incorporated by reference. In one study 275 patients with cervical cancer who were treated with radiation therapy alone, including 216 patients with Stage III squamous cell carcinomas and 59 with adenocarcinomas of all stages. Langerhans' cell (LC) and T-cell were stained immunohistochemically on the specimens excised from the cervical cancer. In squamous cell carcinoma, 5-year survival rates for patients with LC infiltration were significantly better than those without LC (78% versus 60%; P < 0.01). The 5-year survival rate of patients with T-cell infiltration also was significantly better than that of patients without such infiltration (83% versus 61%; P < 0.05). Similar trends were observed in patients with adenocarcinoma; 5-year survival rates for patients with LC infiltration and those without LC infiltration were 49% and 25%, respectively (P < 0.025). The survival rates for patients with T-cell infiltration and those without were 50% and 33%, respectively (P < 0.1). An analysis of patterns of failure of radiation therapy demonstrated that the favorable prognosis in LC infiltration was attributable mainly to improvement of local control rates, but that in T-cell infiltration was not. T-cells infiltrated into tumor specifically in the patients with LC infiltration in both cell types. The authors suggest that the host anti-cancer immune response of individual patients may be remarkably different at the first step of antigen recognition by LC [12]. The LC may induce T-cell-mediated antitumor response and improve local response in radiation therapy. Accordingly, in some embodiments of the invention, probiotic based compositions are administered to a subject with cancer for enhancing infiltration of immune cells, including antigen presenting cells, into tumors subsequent to radiation and/or chemotherapy.
[00170] In one embodiment, patients treated with cancer therapy are administered a blend of probiotics; specifically, Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum,, Lactobacillus casei, Lactobacillus paracasei, in combination with a blend of digestive enzymes; specifically, amylase, glucoamylase, lipase, bromelain, maltase, lactase, hemicellulase, xylanase, papain, and invertase. Identifying a patient in need can be done by any conventional detection method used in oncology practice, non-exclusively including blood tests, or identifying and assessing risk factors for cardiovascular disease, such as smoking, drinking, lack of exercise, weight of patient, age, family history, etc.
[00171] T cells recognize diseased cells by receptors engaging with antigens that are present on or inside the diseased cells. CAR-T therapy entails genetically engineering T- cells to express synthetic CARs that direct T-cells to antigens on the surface of cancer cells. TCR therapy modifies T-cells to express high-affinity tumor specific TCRs that recognize intra-cellular antigens that must be presented on the surface of target cells. In early clinical trials, CAR-T and TCR therapies have demonstrated impressive anti-tumor activity in a narrow spectrum of hematologic cancers and garnered significant attention by research institutions and biopharmaceutical companies. We believe a key limitation of adaptive autologous immunotherapy is the need to retrieve non-compromised immune cells from a cancer patient which requires a complex and costly manufacturing process to develop the therapy. Phase I clinical trials of CAR-T cell immunotherapy have reported severe adverse toxicities of cytokine release syndrome and neurotoxicity, requiring hospitalization, pre conditioning and, in some instances, intensive care unit admission following side effects associated with cytokine release syndrome. In one preferred embodiment of the present invention, a probiotic based composition is administered to patients prior to CAR-T cell therapy for the purpose of off-setting probable toxicity caused by the treatment. In another embodiment, a probiotic with digestive enzymes formuaiton is administered to patients after CAR T cell therapy, either upon presentation of adverse events or before such adverse events are clinically manifested.
[00172] In some embodiments, the probiotic based composition is used to reduce toxicity and augment efficacy of various cancer therapies, for example chemotherapeutic agents, radiotherapeutic agents, or drugs such as atovaquone, azacitidine, bexarotene, boceprevir, bosentan, bosutinib, brentuximab vedotin, carbidopa-levodopa, carglumic acid, decitabine, eribulin mesylate, foscarnet, metformin, ofatumumab, pomalidomide, prelatrexate solution, ropivacaine, rosiglitazone, sirolimus, temsirolimus, and valganciclovir. In some embodiments, the probiotic mixture and mixtures described in the invention mitigate or prevent the decrease in the production of red blood cells caused by the surgery or second drug.
[00173] In the context of this invention, the“probiotic composition” (which may also exist as a drug or pharmaceutical) may comprise at least two ingredients for administration to a subject with cancer. The two ingredients may include at least one probiotic ingredient. For example, the composition may include at least two different probiotic ingredients or at least one probiotic ingredient and at least one digestive enzyme ingredient, or other ingredients in various combinations. In addition, the composition may be substantially, if not completely, devoid of artificial flavors, colorings or preservatives. Further, the supplement may be developed for human or other animal consumption by swallowing or other ingestion technique. In a situation where the composition is developed for consumption by swallowing, the composition may be enclosed within a capsule or other form known to facilitate swallowing. The terms probiotics, digestive enzymes and dietary supplements have generally accepted definitions. For example, probiotics may be defined as live microorganisms thought to be healthy for the host organism; digestive enzymes may be defined as enzymes that break down polymeric macromolecules into their smaller building blocks in order to facilitate their absorption by the body; dietary supplements may be defined as a preparation intended to supplement the diet and provide nutrients that may be missing or may not be consumed in sufficient quantities in a human's diet. The probiotic ingredients of the composition may be present in an effective dose. For example, at the time of manufacture, the probiotic ingredients may total at least 6 x 1 Q9 colony forming units (cfu) and may include at least 13 x 1Q9 cfu of probiotics or more. In a preferred aspect, the probiotic ingredients total at least 13 x 1Q9 cfu of probiotics. In a more preferred aspect the probiotic ingredients total at least 14 x 1Q9 cfu of probiotics. A colony forming unit (cfu) is generally accepted as a measure of viable bacterial or fungal numbers. Such quantity of probiotic ingredient may facilitate providing a consumer with an effective dose of probiotics at the time of ingestion, as the inventor has realized that probiotics may be destroyed during storage due to undesirable environments (e.g., temperature extremes) and other reasons. The probiotic ingredients may comprise a probiotic blend including one or more of the following: Lactobacillus rhamnosus GG, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus salivarius, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium bifidum. In a preferred aspect the composition includes at least one probiotic from each of the strains listed above. Each probiotic ingredient present in the probiotic based composition may be present in any desired quantity. In one aspect each probiotic ingredient of the composition may be present in an amount up to 1.5 x 109 cfu. In a further aspect each probiotic ingredient of the probiotic based composition may be present in an amount equal to or greater than 1 x 109 cfu, and in a preferred aspect when combined the nine probiotic ingredients may total as much as, or more than, 13 x 109 cfu of probiotic ingredients. Preferably the amounts are equal to or are more than 14 x 109 cfu. In the example, these quantities may be measured at the time of manufacture.
[00174] The inventor appreciates that simply introducing a probiotic based composition into the gastrointestinal (GI) tract as done in prior instances is not effective due to otherwise poor or undesirable placement of the ingredient within the system and / or lack of an effective dose due to the degradation of the living probiotic ingredient and/or lack of the variety and nature of a desired or sufficient strain or strains of probiotic and/or lack of use of the probiotic blend in combination with the digestive enzyme supplement or supplemental blend. Further, as the inventor appreciates that Lactobacillis acidophilis is a prominent strain of probiotic for the small intestine, and Bifidobacterium bifidum is a prominent strain of probiotic for the large intestine, for instance, it is advantageous to have those supplemental ingredients (and other of the respective probiotic ingredients and the digestive enzymes) introduced into the GI tract at the appropriate or preferred locations and in effective amounts. Use of a capsule, such as a vegetable or other capsule that does not immediately release the contents therein (for instance, the capsule delays release beyond the stomach), has a benefit for the positioning of the ingredients within or throughout the GI tract. Use of a blister pack (or other sealing mechanism) for storing the capsule assists in preserving the potency of the ingredients such that the combination of the composition with the capsule in a protected blister package assists with appropriate and effective delivery (location and potency). Further, the particular combination, strains, and sources of thes probiotics and digestive enzymes, and in the various amounts, have been established by the inventor for desired impact and appropriate delivery. Digestive enzymes of the composition may be present in an effective dose to supplement existing quantities of enzymes and improve digestion of ingested food and absorption of the nutrients within the ingested food. The digestive enzyme ingredients may comprise any enzyme that is useful in the digestion of ingested food. For example, inventors has developed a particularly effective probiotic based formulation also containing digestive enzymes comprising some or all of the following: amylase, glucoamylase, lipase, bromelain, maltase, lactase, hemicellulose, xylanase, papain, and invertase.
[00175] A capsule may enclose the probiotic based composition to facilitate increasing the shelf-life of the composition, swallowing of the composition, timing a release of the composition after ingestion and other considerations. The capsule may be a gelatin capsule, vegetable- based (e.g., vegetable cellulose) capsule or other type of capsule. If the capsule is a vegetable-based capsule, the capsule may facilitate releasing the probiotics at a desirable location within the digestive tract. Preferably the capsule is a vegetable-based capsule. An exemplary embodiment of the composition includes a probiotic blend and a digestive enzyme blend enclosed within a vegetable cellulose capsule.
[00176] Protecting the composition after manufacture is particularly important as at least the probiotic ingredients may be sensitive to variations in environmental conditions. To facilitate protection of the composition, capsules comprising the composition may be and are preferably stored in blister packs. That is, the blister packs may seal the capsule from a surrounding environment and thus, extend the life of the effective ingredients of the composition.
[00177] The method of using the composition may be used as desired by the subject in need thereof or as determined by the oncologist or other health practitioner who advises said subject. Preferably, the probiotics based composition is self-administered by said subject on a daily. Continuous daily use of the probiotic based composition may result in greater and sustained benefits for the subject.
[00178] In an aspect of the invention the composition may include at least one of the probiotics of the Lactobacillus species and at least one probiotic of the Bifidobacterium species together with a digestive enzyme and contained in a vegetable-based capsule for at least 2 x 109 colony forming units. Preferably the capsule is stored in a blister pack. More preferably the blend includes at least some additional probiotic ingredients as noted above and at least 6 x 109 colony forming units assuming 1 billion cfu per probiotic at time of manufacture. In a further and preferred aspect, the composition may include, for instance, Lactobacillus acidophilus, Lactobacillus rhamnosus CG, Bifidobacterium infantis, and Bifidobacterium bifidum, together with a digestive enzyme and contained in a capsule. In a further preferred aspect the foregoing composition may include others from the above list of probiotics for at least 9 x 109 colony forming units, and stored in a blister pack or other sealed package. In a further preferred aspect the foregoing composition may include others from the above list of probiotics for at least 13 x 109 colony forming units, and stored in a blister pack or other sealed package. In a further aspect the composition may include a greater amount of Lactobacillus probiotic as compared to Bifidobacterium probiotic.
[00179] According to preferred embodiments, said formulations of probiotics with digestive enzymes can contain said probiotics having the following activity/gram: between 25-150 billion colony forming units (cfu) for Bifidobacterium infantis, between 50-150 billion cfu for Bifidobacterium longum, between 350-550 billion cfu for Bifidobacterium bifidum, between 100-300 billion cfu for Lactobacillus rhamnosus, between 100-300 billion cfu for Lactobacillus acidophilus, between 200-400 billion for Lactobacillus salivarius, between 300-500 billion cfu for Lactobacillus plantarum, between 200-400 billion cfu for Lactobacillus casei, and between 300-500 billion cfu for Lactobacillus paracasei.
[00180] According to preferred embodiments, said formulation of probiotics with digestive enzymes contains digestive enzymes having the following activity/gram: between 300,000-500,000 HCU for hemicellulase, between 100,000-300,000 XU for xylanase, between 100,000-300,000 DU for amylase, between 900-1,100 AGU for glucoamylase, between 10,000-30,000 DP for maltase, between 900,000, 1,100,000 TU for papain, between 2,000-4,000 GDU for bromelain, between 150-300 FIP for lipase, between 100,000-300,000 SU for invertase, between 75,000-150,000 ALU for lactase.
[00181] A formulation comprising probiotics or probiotics with digestive enzymes may be utilized in subjects with cancer who are scheduled to undergo, are currently undergoing, and/or have undergone treatment with one or more types of chemotherapy. Selection of chemotherapy will depend on the subject to be treated, the type of cancer, and the intent of therapy. Selection of a subject for treatment with said probiotic based formulation may be performed prophylactically. Alternatively, the current invention may be practiced by selection of a subject for treatment with said probiotic based formulation during or after the course of chemoetherapy drug treatment.
[00182] The present invention can be practiced with chemotherapeutic agents that are known in the art and include but are not limited to: methotrexate, taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, etoposides, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel, doxorubicin, epirubicin, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrosoureas such as carmustine and lomustine, vinca alkaloids, platinum compounds, mitomycin, gemcitabine, hexamethylmelamine, topotecan, tyrosine kinase inhibitors, tyrphostins, STI-571 or Gleevec.TM. (imatinib mesylate), herbimycin A, genistein, erbstatin, and lavendustin A. taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, etoposides, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel, doxorubicin, epirubicin, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrosoureas such as carmustine and lomustine, vinca alkaloids, platinum compounds, mitomycin, gemcitabine, hexamethylmelamine, topotecan, tyrosine kinase inhibitors, tyrphostinsherbimycin A, genistein, erbstatin, and lavendustin ABCNU, irinotecan, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel. In a preferred embodiment, the anti-cancer agent can be, but is not limited to, a drug listed: Alkylating agents Nitrogen mustards: Cyclophosphamide Ifosfamide Trofosfamide Chlorambucil Nitrosoureas: Carmustine (BCNU) Lomustine (CCNU) Alkylsulphonates: Busulfan Treosulfan Triazenes: Dacarbazine Platinum containing Cisplatin compounds: Carboplatin Aroplatin Oxaliplatin Plant Alkaloids Vinca alkaloids: Vincristine Vinblastine Vindesine Vinorelbine Taxoids: Paclitaxel Docetaxel DNA Topoisomerase Inhibitors Epipodophyllins: Etoposide Teniposide Topotecan 9- aminocamptothecin Camptothecin Crisnatol mitomycins: Mitomycin C Anti-metabolites Anti-folates: DHFR inhibitors: Methotrexate Trimetrexate IMP dehydrogenase Mycophenolic acid Inhibitors: Tiazofurin Ribavirin EICAR Ribonuclotide reductase Hydroxyurea Inhibitors: Deferoxamine Pyrimidine analogs: Uracil analogs: 5-Fluorouracil Floxuridine Doxifluridine Ratitrexed Cytosine analogs: Cytarabine (ara C) Cytosine arabinoside Fludarabine Purine analogs: Mercaptopurine Thioguanine DNA Antimetabolites: 3-HP 2'-deoxy-5-fluorouridine 5-HP alpha-TGDR aphidicolin glycinate ara-C 5-aza-2'-deoxycytidine beta-TGDR cyclocytidine guanazole inosine glycodialdehyde macebecin II pyrazoloimidazole Hormonal therapies: Receptor antagonists: Anti-estrogen: Tamoxifen Raloxifene Megestrol FHRH agonists: Goserelin Feuprolide acetate Anti-androgens: Flutamide Bicalutamide Retinoids/Deltoids Cis- retinoic acid Vitamin A derivative: All-trans retinoic acid (ATRA-IV) Vitamin D3 analogs: EB 1089 CB 1093 KH 1060 Photodynamic therapies: Vertoporfin (BPD-MA) Phthalocyanine Photosensitizer Pc4 Demethoxy-hypocrellin A (2BA-2-DMHA) Cytokines: Interferon-. alpha. Interferon-. gamma. Tumor necrosis factor Angiogenesis Inhibitors: Angiostatin (plasminogen fragment) antiangiogenic antithrombin III Angiozyme ABT-627 Bay 12-9566 Benefin Bevacizumab BMS-275291 cartilage-derived inhibitor (CDI) CAI CD59 complement fragment CEP-7055 Col 3 Combretastatin A-4 Endostatin (collagen XVIII fragment) Fibronectin fragment Gro-beta Halofuginone Heparinases Heparin hexasaccharide fragment HMV833 Human chorionic gonadotropin (hCG) IM-862 Interferon alpha/beta/gamma Interferon inducible protein (IP- 10) Interleukin- 12 Kringle 5 (plasminogen fragment) Marimastat Metalloproteinase inhibitors (TIMPs) 2-Methoxyestradiol MMI 270 (CGS 27023A) MoAb IMC-1C11 Neovastat NM- 3 Panzem PI-88 Placental ribonuclease inhibitor Plasminogen activator inhibitor Platelet factor-4 (PF4) Prinomastat Prolactin 16 kD fragment Proliferin-related protein (PRP) PTK 787/ZK 222594 Retinoids Solimastat Squalamine SS 3304 SU 5416 SU6668 SU11248 Tetrahydrocortisol-S tetrathiomolybdate thalidomide Thrombospondin- 1 (TSP-l) TNP- 470 Transforming growth factor-beta (TGF-b) Vasculostatin Vasostatin (calreticulin fragment) ZD6126 ZD 6474 farnesyl transferase inhibitors (FTI) bisphosphonates Antimitotic agents: allocolchicine Halichondrin B colchicine colchicine derivative dolstatin 10 maytansine rhizoxin thiocolchicine trityl cysteine Others: Isoprenylation inhibitors: Dopaminergic neurotoxins: l-methyl-4-phenylpyridinium ion Cell cycle inhibitors: Staurosporine Actinomycins: Actinomycin D Dactinomycin Bleomycins: Bleomycin A2 Bleomycin B2 Peplomycin Anthracyclines: Daunorubicin Doxorubicin (adriamycin) Idarubicin Epirubicin Pirarubicin Zorubicin Mitoxantrone MDR inhibitors: Verapamil Ca.sup.2+ATPase inhibitors: Thapsigargin [00183]
Additional anti-cancer agents that may be used in the methods of the present invention include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-nl; interferon alfa-n3; interferon beta-I a; interferon gamma-I b; 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; mitocarcin; mitocromin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimu stine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride.
[00184] In one embodiment, the present invention is based on the discovery that the occurrence of chemotherapy-induced diarrhea is prevented or its severity is reduced upon administration of the probiotic with digestive enzyme compositions described herein. In particular, the invention is based on the unexpected finding that the administration of said probiotic compositions provides a protective effect against chemotherapy-induced diarrhea that extends long after the composition is administered. The effect is especially pronounced for diarrhea of Grade II as determined by National Cancer Institute Common Toxicity Criteria for Diarrhea (CTCAE v.4.03). Therefore, in a first embodiment the invention provides a method for preventing or reducing the occurrence of grade 2 or higher diarrhea resulting from an anti-cancer chemotherapy in a subject in need thereof, which method comprises administering to the subject an effective amount of a probiotic based composition. More generally speaking, the invention provides a method for treating gastrointestinal mucositis, or otherwise preventing or reducing gastrointestinal damage and/or dysfunction associated with an anti-cancer chemotherapy in a subject in need thereof, which method comprises administering to the subject a therapeutically effective amount of probiotic based composition for a plurality of consecutive days, preferably commencing at the start of the chemotherapy cycle or prior to the chemotherapy cycle.
[00185] Other anti-cancer drugs that can be used include, but are not limited to: 20- epi-l,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- dorsalizing morphogenetic protein- 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnor spermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflomithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; forme stane; fostriecin; fotemu stine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor- 1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor l-based therapy; mustard anti-cancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum- triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B l; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; 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 system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.
[00186] In some embodiments, the composition(s) of the invention, including probiotics, and probiotics together with enzymes are administered in the form of a nutraceutical. Nutraceuticals, whether in the form of a liquid extract or dry composition, are edible and may be eaten directly by humans or mammals. Said nutraceuticals are preferably provided to humans in the form of additives or nutritional supplements for example they may be administered in the form of tablets of the kind sold in health food stores, or as ingredients in edible solids, more preferably processed food products such as cereals, breads, tofu, cookies, ice cream, cakes, potato chips, pretzels, cheese, and in drinkable liquids such as beverages such as milk, soda, sports drinks, and fruit juices. Thus, in one embodiment a method is provided for enhancing the nutritional value of a food or beverage by intermixing the food or beverage with a nutraceutical in an amount that is effective to enhance the nutritional and probiotic or immune modulatory and/or cancer therapy augmentative value of the food or beverage. In one embodiment, a flavoring agent is added. Preferred flavoring agents include sweeteners such as sugar, com syrup, fructose, dextrose, maltodextrose, cyclamates, saccharin, phenyl-alanine, xylitol, sorbitol, maltitol, and herbal sweeteners such as Stevia. Examples of foods into which probiotics useful for the practice of the invention can be incorporated into include soft drinks, a fruit juice or a beverage comprising whey protein, health teas, cocoa drinks, milk drinks and lactic acid bacteria drinks. Probiotic bacteria may be administered together with agents known to enhance efficacy and retention of probiotics, including In a further embodiment of the present invention various extracts and plant powders are incorporated into our compositions, depending on the desired properties according to the end use of said compositions. These compositions according to the present invention can be characterized in that in addition to the discussed prebiotics and phytosterols and lecithins the said further plant extracts or powders are one or more of those of Panax ginseng (red, Korean ginseng), Panax ginseng (white, Chinese ginseng), Rhodiola rosea (golden root), Panax quinquefolium (American ginseng), Eleutherococcus senticosus (Siberian ginseng), Cynara scolymus (artichoke), Uncaria tomentosa (Cat's claw), Lepidium meyenii (maca, Peruvian ginseng), Paullinia cupana (guarana), Croton lechleri (Sangre de Grado), Whitania somnifera (ashwagandha, Indian ginseng), Panax japonicus (Japanese ginseng), Panax vietnamensis (Vietnamese ginseng), Panax trifolius, Panax pseudoginseng, Panax notoginseng, Malpighia glabra (acerola), Ylex paraguayiensis (Yerba mate), Astragalus membranaceus (astragalus), Stevia rebaudiana (stevia), Pfaffia paniculata (Brazilian ginseng, suma), Ginkgo biloba, Tabebuia impetiginosa (Pau d'arco), Echinacea purpurea, Peumus boldus (boldo), Gynostemma pentaphyllum (Jiaogulan, also known as Southern Ginseng or Xiancao), Sutherlandia frutescens (African ginseng), Aloe vera (aloe), Cistanche salsa, Cistanche deserticola (and other Cistanche sp.), Codonopsis pilosula ("poor man's ginseng."), Nopal opuntia (Prickly pear cactus), Citrus sinensis (Citrus aurantium) and other members of the citrus family (lemon, lime, tangerine, grapefruit), Camelia sinensis (tea), Plantago psyllium (psyllium), Amaranth edulis and other amaranth sp. (amaranth), Commiphora mukul (guggul lipid), Serenoa repens, Serenoa serrulata (saw palmetto), Cordyceps sinensis (Cordycaps), Lentinula edodes (shitake), Ganoderma lucidium (Reishi), Grifola frondosa (maitake), Tremella fuciformis (Silver ear), Poria cocos (Hoelen), Hericium erinaceus (Lion's Mane), Agaricus blazei (Sun mushroom), Phellinus linteus (Mulberry yellow polypore), Trametes versicolo , Coriolus versicolor (Turkey tails), Schizophyllum commune (Split gill), Inonotus obliquus (Cinder conl), oat bran, rice bran, linseed, garlic, Ceratonia siliqua (locust been gum or flour from the seeds of carob tree), Cyanopsis tetragonoloba (guar gum, EU Food additive code E412), Xanthomonas campestris (xanthan gum). These plant extracts and plant powders are capable to potentiate the bioactivity of these compositions based on prebiotics, phytosterols, lecithins, vitamins and minerals. In given cases it also adds other prebiotics to the aforementioned prebiotic mixtures. These can result in more pronounced bioactivities as prebiotics and also in the chosen other bioactivity directions.
[00187] The use of chemotherapy in treatment of cancer is common. Broadly speaking, chemotherapy may be divided into the following classes: 1) Alkylating agents. These drugs kill cells that are not in cell cycle [13, 14], and include mustard gas derivatives such as Mechlorethamine, Cyclophosphamide, Chlorambucil, Melphalan [15], and Ifosfamide. Ethylenimines such as Thiotepa and Hexamethylmelamine, Alkylsulfonates such as Busulfan, Hydrazines. Triazines such as Altretamine, Procarbazine, Dacarbazine and Temozolomide. Nitrosureas such as Carmustine, Lomustine and Streptozocin. Metal salts such as Carboplatin, Cisplatin, and Oxaliplatin [16]. Alkylating agents are one of the original classes of chemotherapies that where historically developed [17, 18]. 2) Plant Alkaloids. These are chemotherapeutic drugs that are extracted from certain types of plants. The plant alkaloids are cell-cycle specific. This means they attack the cells during various phases of division. The vinca alkaloids are made from the periwinkle plant (catharanthus rosea). The taxanes are made from the bark of the Pacific Yew tree (taxus). The vinca alkaloids and taxanes are also known as antimicrotubule agents. The podophyllotoxins are derived from the May apple plant. Camptothecan analogs are derived from the Asian "Happy Tree" (Camptotheca acuminata). Podophyllotoxins and camptothecan analogs are also known as topoisomerase inhibitors, which are used in certain types of chemotherapy. Vinca alkaloids include Vincristine, Vinblastine and Vinorelbine. Taxanes include Paclitaxel and Docetaxel. Podophyllotoxins include Etoposide and Tenisopide. Camptothecan analogs include Irinotecan and Topotecan. 3) Antitumor Antibiotics. This type of chemotherapy is generated from species of the soil fungus Streptomyces. These drugs act during multiple phases of the cell cycle and are considered cell-cycle specific. Antitumor antibiotics include Anthracyclines such as Doxorubicin, Daunorubicin, Epirubicin, Mitoxantrone, and Idarubicin. Anthracyclines are generally a class of compounds that have the structural core of anthracene. They often are highly effective chemotherapeutics and therefore are used for the treatment of many cancers, including leukemias, lymphomas, breast, uterine, ovarian, bladder cancer, and lung cancers and are often used in childhood cancer treatment regimens. Some anthracycline drugs include doxorubicin, daunorubicin, idarubicin, and epirubicin. Although the exact mechanisms may yet to be validated, anthracyclines have been reported to work by inhibiting DNA and RNA synthesis; promoting free radical formation through redox cycling, with iron promoting the conversion of superoxide into hydroxyl radicals; inhibiting topoisomerases (e.g., topoisomerases Il.alpha. and/or Il.beta.); and evicting histones from open chromosomal areas. Chromomycins such as Dactinomycin and Plicamycin. Additional antitumor antibiotics include Mitomycin and Bleomycin. 4) Antimetabolites. This type of chemotherapy resembles normal substances within the cell. When the cells incorporate these substances into the cellular metabolism, they are unable to divide. Antimetabolites are cell-cycle specific. They attack cells at very specific phases in the cycle. Antimetabolites are classified according to the substances with which they interfere. Antimetabolites include the folic acid antagonist Methotrexate, the pyrimidine antagonists 5-Fluorouracil, Foxuridine, Cytarabine, Capecitabine, and Gemcitabine, the purine antagonists 6-Mercaptopurine and 6-Thioguanine, the adenosine deaminase inhibitors Cladribine, Fludarabine, Nelarabine and Pentostatin. 5) Topoisomerase Inhibitors. These chemotherapeutic agents that interfere with the action of topoisomerase enzymes (topoisomerase I and II). During the process of chemo treatments, topoisomerase enzymes control the manipulation of the structure of DNA necessary for replication. Ironotecan and topotecan are considered topoisomerase I inhibitors, whereas amsacrine, etoposide, etoposide phosphate, and teniposide are considered topoisomerase II inhibitors. 6) Alternative types of chemotherapy. Hydroxyurea is considered a ribonucleotide reductase inhibitor. Mitotane is considered an adrenocortical steroid inhibitor. Asparaginase and Pegaspargase are enzymatic types of chemotherapy. Estramustine is considered an antimicrotubule drug and members of the retinoid family of chemotherapies include Bexarotene, Isotretinoin, Tretinoin (ATRA).
[00188] The various probiotic, and probiotic/enzyme mixtures described herein are intended for human consumption and thus the processes for obtaining them are preferably conducted in accordance with Good Manufacturing Practices (GMP) and any applicable government regulations governing such processes. Especially preferred processes utilize only naturally derived solvents. In contrast to nutraceuticals, the so-called "medical foods" are not meant to be used by the general public and are not available in stores or supermarkets. Medical foods are not those foods included within a healthy diet to decrease the risk of disease, such as reduced-fat foods or low-sodium foods, nor are they weight loss products. A physician prescribes a medical food when a patient has special nutrient needs in order to manage a disease or health condition, and the patient is under the physician's ongoing care. The label must clearly state that the product is intended to be used to manage a specific medical disorder or condition. An example of a medical food is nutritionally diverse medical food designed to provide targeted nutritional support for patients with chronic inflammatory conditions. Active compounds of this product are for instance one or more of the compounds described herein. The present invention thus relates to the use of an immuno-modulating properties of probiotics as related to prevention and/treatment of pregnancy complications. Thus said probiotics can be used in the preparation of a medicament, a vaginal suppository, medical food or nutraceutical to induce immune tolerance or immune modulation. [00189] In some embodiments, the compositions according to the present invention comprise prebiotic components selected from fructose polymers GF.sub.n and F.sub.m, either containing a glucose (G) end-group, or without this glucose end-group and one or more component of a group of prebiotics consisting of modified or unmodified starch and partial hydrolysates thereof, partially hydrolysed inulin, natural oligofructoses, fructo- oligosaccharides (FOS), lactulose, galactomannan and suitable partial hydrolysates thereof, indigestible polydextrose, acemannan, various gums, indigestible dextrin and partial hydrolysates thereof, trans-galacto-oligosaccharides (GOS), xylo-oligosaccharides (XOS), beta-glucan and partial hydrolysates thereof, together if desired with phytosterol/phytostanol components and their suitable esters, and if desired other plant extracts, mineral components, vitamins and additives. The fructose polymers of GF.sub.n or F.sub.m structures (G=glucose; F=fructose; n>2; m>2) are linear fructose polymers having either a glucose (G) and -group, or being without this glucose and -group. Oligofructoses are consisted of 3 to 10 carbohydrate units. Above that, chicory inulin contains 10 to 60 carbohydrate units, typically with 27 carbohydrates (fructoses with our without one glucose end-group and a fructose chain). Other plants may produce different fructans. These fructans are capable to increase the number of colonized and planktonic bacteria in the large intestine. This results in a change that those bacteria that are less advantageous or may turn dangerous are suppressed by the higher probiotic colony of bacteria. Depending on the chain length of these fructans or other prebiotics, they can be fermented by probiotic bacteria at different positions in the colon. We have found that the longer inulins are capable to rich the distal colon and sigmoid colon and exert their anticancer actions in the positions where typically most of the cancerous problems occur. The occurrence of these cancers can be the result of various types of carcinogenesis. It has been demonstrated in the literature that directly induced chemical carcinogenesis can be greatly reduced by probiotic bacteria. The prebiotic compositions of our invention can corroborate this effect by considerably increasing the number of Bifidocateria and other beneficial probiotic strains. The local chemical carcinogenesis can also be the result of the formation of secondary bile acids. These secondary bile acids are often formed upon the action of enzymes produced by resident Clostridia. By probiotic suppression of the number of these bacteria according to the invention, the chance of secondary bile acid formation can also be reduced. This can be demonstrated by measuring the faecal primary/secondary bile acid ratio. Other prebiotics can be selected from a group of prebiotics consisting of various gums (guar gum, xanthan gum, locust been gum), carob seed flour, oat bran, rice bran, barley, modified or unmodified starch and suitable partial hydrolysates thereof, partially hydrolysed inulin, natural or synthetic/biosynthetic oligofructoses, fructo- oligosaccharides (FOS), lactulose, galactomannan and suitable hydrolysates thereof, indigestible polydextrose, indigestible dextrin and partial hydrolysates thereof, trans- galacto-oligosaccharides (GOS), xylo-oligosaccharides (XOS), acemannan, lentinan or beta-glucan and partial hydrolysates thereof, polysaccharides P and K (PSP, PSK), tagatose, various fungal oligosaccharides and polysaccharides, together with other components.
[00190] In some embodiments of the invention, probiotic compositions are utilized to enhance efficacy of immunotherapy. Said immunotherapy may be utilized alone, or in combination with other types of cancer therapies such as chemotherapy, surgery, radiation therapy, or hyperthermia. The basis of immunotherapy is activation of a competent T cell response. It is known that T cell responses are controlled in large part by activation of said T cells by antigen presenting cells. In one embodiment of the invention probiotic compositions are utilized to augment efficacy of antigen presentation. In one embodiment said probiotic compositions are utilized as in vivo activators of antigen presentation by stimulation of dendritic cell activity. In another embodiment, said probiotic compositions are administered after a patient is treated with dendritic cell therapy. Numerous animal models have demonstrated that in the context of neoplasia dendritic cells (DCs) can bind to and engulf tumour antigens that are released from tumor cells, either alive or dying, and cross-present these antigens to T cells in tumour-draining lymph nodes. This results in the generation of tumour- specific immune responses that have been demonstrated to inhibit tumor growth or in some cases induced transferrable immunological memory. Mechanistically, DCs recognize tumors using the same molecular means that they would use to recognize apoptotic cells, or cells that are stressed. One set of signals are molecules released from apoptotic cells, which are highly released by tumors, these include the nucleotides UTP and ATP, fractalkine, lipid lysophosphatidylcholine, and sphingosine 1- phosphate [19]. Signals from stressed cells, such as tumor cells include externalization of phosphatidylserine onto the outside of the cell membrane, calreticulin, avB5 integrin, CD36 and lactadherin. There is some evidence that dendritic cells actively promote tumor immunity in that patients with dendritic cell infiltration of tumors generally have a better prognosis [20-23]. The most advanced DC based therapy is the product Provenge (sipuleucel-T), which is approved by the FDA for treatment of androgen resistant prostate cancer. Provenge is a cellular product derived from autologous peripheral blood mononuclear cell (PBMC) derived dendritic cells that have been grown using a chimeric protein comprised of GM-CSF and the prostate specific antigen, prostatic acid phosphatase [24, 25]. In the pivotal trial, this DC based therapeutic resulted in extension of survival by 4.1 months [25]. Prior to approval of Provenge, numerous clinical trials using DC were performed in prostate cancer, which will be discussed below. Tjoa et al reported on 33 participants of a phase I trial in patients with advanced prostate cancer that received autologous DC pulsed HLA-A0201- specific prostate-specific membrane antigen (PSMA) peptides (PSM-P1 or -P2) that were entered into a second trial (Phase II) which involved six infusions of DC pulsed with PSM-P1 and -P2 peptides. The patients were followed up for up to 770 days from the start of the original phase I study. 9 partial responders were identified in the phase II study based on National Prostate Cancer Project (NPCP) criteria, plus 50% reduction of prostate-specific antigen. Four of the partial responders were also responders in the phase I study, with an average response duration of 225 days. Their combined average total response period was over 370 days. Five other responders in the secondary immunizations at the Phase II were nonresponders in the phase I study. Their average partial response period was 196 days. These data support the safety of follow-up infusion of DC that have been pulsed with tumor antigen derived peptide [26]. The same group published a subsequent paper on an additional 33 patients that had not received prior DC immunization in the Phase I. All subjects received six infusions of DC pulsed with PSM-P1 and -P2 at six week intervals without any treatment associated adverse events. Six partial and two complete responders were identified in the phase II study based on NPCP criteria, plus 50% reduction of pro state- specific antigen (PSA), or resolution in previously measurable lesions on ProstaScint scan [27]. The same group analyzed immune response in patients who had clinical remission or relapsed. A strong correlation was found between delayed type hypersensitivity response to the PSM-P1 and PSM-P2 and clinical response [28]. Thus in one embodiment administration of probiotic compositions is performed with the intent of activating efficacy of dendritic cell therapy. The utilization of dendritic cell therapy in various types of cancers has previously been reported and incorporated by reference from the following publications melanoma [29-80], soft tissue sarcoma [81], thyroid [82-84], glioma [85-106], multiple myeloma ,[107-115], lymphoma [116-118], leukemia [119-126], as well as liver [127-132], lung [133-146], ovarian [147-150], and pancreatic cancer [151-153].
[00191] In one embodiment of the invention, the probiotic with digestive enzymes formulation is used to enhance T cell immunity in a subject with cancer. T cells are immune effectors against tumors, possessing ability to directly kill via CD8 cytotoxic cells [154- 156], or indirectly killing tumors by activation of macrophages through interferon gamma production [157-159]. Additionally, T cells have been shown to convert protumor M2 macrophages to Ml [160].
[00192] In one embodiment of the invention, the probiotic with digestive enzymes formulation is used to enhance the functions of macrophages in a subject with cancer Macrophages are key components of the innate immune system which play a principal role in the regulation of inflammation as well as physiological processes such as tissue remodeling [161, 162]. The diverse role of macrophages can be seen in conditions ranging from wound healing [163-166], to myocardial infarction [167-173], to renal failure [174- 177] and liver failure [178].
[00193] A background of macrophage biology is provided to assist one of skill in the art to practice the invention. Differentiated macrophages and their precursors are versatile cells that can adapt to microenvironmental signals by altering their phenotype and function [179]. Macrophages comprise distinct sub-populations, known as classical Ml and alternative M2 [180]. Mirroring the nomenclature of Thl cells, Ml macrophages are described as the pro-inflammatory sub-type of macrophages induced by IFN-. gamma and LPS. They produce effector molecules (e.g., reactive oxygen species) and pro- inflammatory cytokines (e.g., IL-12, TNF-. alpha and IL-6) and they trigger Thl polarized responses [181].
[00194] In one embodiment of the invention, manipulation of macrophages is performed using a probiotic with digestive enzyme formulation in combination with agents and approaches known to modulate macrophage activity. Said macrophage activity may include pro-angiogenic activities or anti-angiogneic and cytotoxic activities. Macrophages can play a tumor inhibitory, as well as a tumor stimulatory role. Initial studies supported the role of macrophages in mediating antibody dependent cellular cytotoxicity in tumors [182-189], and thus being associated with potentiation of antitumor immune responses. Macrophages also possess the ability to directly recognize tumors by virtue of tumor expressed“eat-me” signals, which include the stress associated protein calreticulin [190, 191], which binds to the low-density lipoprotein receptor-related protein (LRP) on macrophages to induce phagocytosis [192]. Tumors protect themselves by expression of CD47, which binds to macrophage SIRP-l and transduces an inhibitory signal [193]. Blockade of CD47 using antibodies results in remission of cancers mediated by macrophage activation [194-198]. Thus on the one hand, macrophages play an important role in induction of antitumor immunity. This can also be exemplified by some studies, involving administration of GM-CSF in order to augment macrophage numbers and activity in cancer patients [ 199-202] . Alternatively, there is also evidence that macrophages support tumor growth. Studies in the osteopetrotic mice strain, which lacks mature macrophages, demonstrate that tumors actually grow slower in animals deficient in macrophages [203]. Several other animal models have elegantly demonstrated that macrophages contribute to tumor growth, in part through stimulating on the angiogenic switch [204-206]. Numerous tumor biopsy studies have shown that there is a negative correlation between macrophage infiltration into tumors and patient survival [207-211]. The duality of macrophages in growth of tumors may be seen in studies of “inverse hormesis” in which low concentrations of antibodies targeting the tumor specific marker sialic acid N-glycolyl-neuraminic acid (Neu5Gc) actually leads to enhanced tumor growth in a macrophage dependent manner [212].
[00195] In one embodiment of the present invention, a probtioic with digestive enzyme formulation is used to generate Ml macrophages in a subject with cancer. The importance of this is that M2 macropahges are typically augmenters of tumor growth whereas Ml usually inhibit tumor growth [213, 214]. In some embodiments of the invention, other agents may be used to modulate M2 to Ml transition of tumor associated macrophages including RRx-OOl [215], the bee venom derived peptide melittin [216], CpG DNA [217, 218], metformin [219], Chinese medicine derivative puerarin [220], rhubarb derivative emodin [221], dietary supplement chlorogenic acid [222], propranolol [223], poly ICLC [224], BCG [225], Agaricus blazei Murill mushroom extract [226], endotoxin [227], olive skin derivative maslinic acid [228], intravenous immunoglobulin [229], phosphotidylserine targeting antibodies [230], dimethyl sulfoxide [231], surfactant protein A [232], Zoledronic acid [233], bacteriophages [234]
[00196] In one embodiment of the present invention, a probtioic with digestive enzyme formulation is used to generate activated and/or tumor-cytotoxic T cells in a subject with cancer. In another embodiment, a probiotic with digestive enzyme formulation is used to reduce the prevalence of regulatory T (Treg) cells in a subject with cancer, as can be measured based on the presence of said cells in the peripheral blood of said subject with cancer using techniques that are known in the art. The importance of T cells in cancer is illustrated by positive correlation between tumor infiltrating lymphocytes and patient survival [235-239]. In addition, positive correlations between responses to various immunotherapies has been made with tumor infiltrating lymphocyte density [240, 241]. Increased T cell activity is associated with reduction in T regulatory (Treg) cells. Studies show that agents that cause suppression of Treg cells correlates with improved tumor control. Agents that inhibit Treg cells include arsenic trioxide [242], cyclophosphamide [243-245], triptolide [244], gemcitabine [246], and artemether [247].
[00197] In some embodiments of the invention, a probiotic with digestive enzyme formulation is utilized to augment the anticancer effects of metformin, and/or metformin together with chemotherapy. The administration of metformin for the treatment of cancer and its activies is known in the art and includes macrophage Ml polarization and activation of CD8 T cells. In one study, Wang et al. showed that by skewing tumor-associated macrophage (TAM) polarization from M2- to Ml -like phenotype, metformin inhibited both tumour growth and angiogenesis. Depletion of TAMs by clodronate liposomes eliminated M2-TAMs-induced angiogenic promotion, while also abrogating Ml-TAMs- mediated anti-angiogenesis, thus promoting angiogenesis in tumours from metformin treatment mice. Further in vitro experiments using TAMs-conditioned medium and a coculture system were performed, which demonstrated an inhibitory effect of metformin on endothelial sprouting and tumour cell proliferation promoted by M2-polarized RAW264.7 macrophages [248] . From the CD8 perspective, Metformin enabled normal but not T-cell-deficient SCID mice to reject solid tumors. In addition, it increased the number of CD8(+) tumor-infiltrating lymphocytes (TILs) and protected them from apoptosis and exhaustion characterized by decreased production of IL-2, TNFa, and IFNy. CD8(+) TILs capable of producing multiple cytokines were mainly PD-l(-)Tim-3(+), an effector memory subset responsible for tumor rejection. Combined use of metformin and cancer vaccine improved CD8(+) TIL multifunctionality. The adoptive transfer of antigen- specific CD8(+) T cells treated with metformin concentrations as low as 10 mM showed efficient migration into tumors while maintaining multifunctionality in a manner sensitive to the AMP-activated protein kinase (AMPK) inhibitor compound C [249].
Examples
[00198] Example 1: Clinical evaluation of a probiotic based formulation in patients with colorectal cancer.
[00199] The following example is a clinical study designed to evaluate the efficacy of a probiotic based formulation comprising a blend of probiotic and digestive enzymes contained in capsules. This example teaches methods of use for a probiotic with digestive enzyme formulation disclosed in the present invention. Specifically, this example also teaches methods of using a probiotic with digestive enzyme formulation for improving the efficacy of a chemotherapy drug and/or for preventing and treating adverse events associated with chemotherapy drug treatment of a cancer patient.
[00200] Study Design:
[00201] A double-blind, randomized, placebo-controlled trial to examine the potential efficacy of a probiotic-based formulation for alleviating cancer treatment-associated adverse events in patients with stage IV colorectal cancer
[00202] Description of Probiotic Based Formulation: the Investisational Product·.
[00203] The Investigation Product is a formulation comprising capsules that contain 9 probiotic microorganisms of the genera Lactobacillus and Bifidobacterium , as well as 10 digestive enzymes, and is being tested for its efficacy as part of a treatment regimen for patients with colon cancer. DBR consists of capsules containing a proprietary blend of probiotics (116.20 mg total weight); specifically, Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei, Lactobacillus paracasei, and digestive enzymes (272.65 mg total weight); specifically, amylase, glucoamylase, lipase, bromelain, maltase, lactase, hemicellulose, xylanase, papain, and invertase. The inactive ingredients are: inulin, rice extract, and hydroxypropyl methycellulose.
[00204] The Invesigational Product is manufactured in the United States under GMP conditions and is NSF certified. Placebo capsules will have the same packaging and labelling as the Investigational Product.
[00205] Daily administration of the Investigational Product will comprise 6 capsules taken at intervals throughout the day (2 in the morning, 2 in the afternoon, and 2 in the evening). Patients will adhere to the 3x per day dosing regimen. On FOLFOX-treatment days, a total of 8 capsules will be given daily as per above and, in addition, one capsule will be given immediately prior to drug treatment and another capsule will be given after drug treatment within a time window of two hours for each. The placebo capsule will be administered in exactly the same daily dosing as DBR on regular days and chemotherapy- treatment days. If the patient can't swallow the capsules, the capsules can be opened, poured into applesauce, or similar food, and consumed immediately. Whenever possible, the patient should take the capsules at approximately the same time every day for each dose. [00206] Study Population :
[00207] Inclusion Criteria
1. Ability to understand and the willingness to provide informed consent.
2. Ability to respond to questionnaire(s) and to fill out a diary alone or with
assistance.
3. Willingness to abstain from ingestion of yogurt products or any product
containing probiotics during the study treatment period.
4. Diagnosis of Stage IV colorectal cancer.
5. Is undergoing or is scheduled to undergo chemotherapy with FOLFOX.
6. Age > 18 years.
7. Kamofsky Performance Status (KPS) > 50%. (APPENDIX C).
[00208] Exclusion Criteria
1. Current or anticipated use of other investigational agents.
2. Known allergy or hypersensitivity to a probiotic preparation.
3. Use of probiotics or prebiotics < 2 weeks prior to registration.
4. Any medical condition that may interfere with the ability to self-administer the Investigational Product.
5. Severe or uncontrolled medical disorder that would, in the investigator’s opinion, impair the ability to receive the study treatment or to be compliant with the study protocol.
[00209] Study Cohorts :
[00210] Cohort 1: Supplementation with the probiotic based formulation for patients with stage IV colorectal cancer that are undergoing chemotherapy with FOLFOX (n=8).
[00211] Administration of capsules will begin at any time during the FOLFOX chemotherapy regimen and will continue for 5 months.
[00212] The capsules will be taken orally at 6 capsules daily (2 capsules in the morning, noon, and night) on days when chemotherapy is not administered. Eight capsules will be taken daily on each day of chemotherapy treatment (one additional capsule to be taken within two hours before chemotherapy and another additional capsule to be taken within two hours after the treatment session has been completed). [00213] Cohort 2: Placebo supplementation for patients with stage IV colorectal cancer that are undergoing chemotherapy with FQLFQX .
[00214] Administration of placebo capsules will begin at any time during the FOLFOX chemotherapy regimen and will continue for 5 months.
[00215] Placebo capsules will be taken orally at 6 capsules daily on days when chemotherapy is not administered and 8 capsules daily on each day of scheduled chemotherapy treatment (one additional capsule to be taken within two hours before therapy and another additional capsule within two hours after the treatment session has been completed).
[00216] Cohort 3: Supplementation with the probiotic based formulation for patients with stage IV colorectal cancer beginning prior to the initiation or re-initiation of chemotherapy with FQLFQX (n=8).
[00217] Administration of capsules will begin prior to initiation or re-initiation of the FOLFOX chemotherapy regimen and will continue for 5 months. This cohort will include patients who have never been treated with FOLFOX or those who have not undergone therapy for at least 30 days prior to commencing the study.
[00218] The capsules will be taken orally at 6 capsules daily on days when chemotherapy is not administered and 8 capsules daily on each day of scheduled chemotherapy treatment (one additional capsule to be taken within two hours before therapy and another additional capsule within two hours after the treatment session has been completed).
[00219] Cohort 4: Placebo supplementation for patients with stage IV colorectal cancer beginning prior to the initiation or re-initiation of chemotherapy with FQLFQX (n=8).
[00220] Administration of placebo capsules will begin prior to initiation or re-initiation of the FOLFOX chemotherapy regimen and will continue for 5 months. This cohort will include patients who have never been treated with FOLFOX or those who have not undergone therapy for at least 30 days prior to commencing the study.
[00221] Placebo capsules will be taken orally at 6 capsules daily on days when chemotherapy is not administered and 8 capsules daily on each day of scheduled chemotherapy treatment (one additional capsule to be taken within two hours before therapy and another additional capsule within two hours after the treatment session has been completed).
[00222] Summary of Study Endpoints'.
[00223] The overall purpose of the analyses will be to determine the efficacy of the probiotic based formuations described herein for reducing the adverse events associated with FOLFOX,
[00224] The following primary endpoints will be evaluated:
• Dose modifications, including delays and discontinuations of cancer drug treatments, summarized as the numbers and percentages of patients having any dose modification.
• Percentage of patients who experience weight loss; percentage changes in body weight and/or body mass index (BMI)
• Percentage of patients who need cancer drug treatment dose reduction or discontinuation due to diarrhea.
• Percentage of patients who need cancer drug treatment dose reduction or discontinuation due to nausea.
• Percentage of patients who require prescribed medications for treatment of diarrhea; duration of time for which the prescription is needed.
• Percentage of patients who require prescribed medications for treatment of nausea; duration of time for which the prescription is needed.
[00225] The following secondary endpoints will also be evaluated:
• Frequencies and severity of adverse events measured using Common Terminology Criteria for Adverse Events (CTCAE) criteria for grading (APPENDIX A).
• Quality of Life patient reporting using EORTC QLQ (APPENDIX B)
• Changes in hematology and blood chemistry after chemotherapy as measured by testing of complete blood counts (CBC) and comprehensive metabolic panel (CMP).
• Changes in plasma C-reactive protein (CRP) levels in plasma as a measure of systemic inflammation.
• Tumor responses assessed by imaging and Response Evaluation Criteria in Solid Tumors (RECIST).
• Changes in composition of the microbiome after DBR intervention vs. placebo, measured by genome sequencing of microorganisms in the gut.
• Changes in immune cell subsets and functions in blood based on immunological phenotyping and cytokine quantification. [00226] Duration of Therapy with the Investigational Product:
[00227] Patients can continue to receive the Investigational Product if they discontinue their standard of care cancer therapy for a total period of 5 months duration unless their physician recommends DBR discontinuation or if the patient chooses to discontinue taking DBR.
[00228] The duration of therapy with DBR for enrolled patients is 5 months.
[00229] Duration of Follow-Uv for the Study:
[00230] Patients removed from the study will continue to be followed-up by their physician/oncologist to receive standard of care treatment for their cancer or other medical conditions.
[00231] The last follow-up for patients remaining in the study will be the 5-month time point of administration of Investigational Product or placebo capsules
[00232] Synopsis of Study Results:
• Patients who receive the Investigational Product (vs. placebo) will have statistically fewer dose modifications of their chemotherapy.
• Patients who receive the Investigational Product experience will exhibit reduced changes in body weight over the course of the study as compared to patients who are randomized to receive placebo capsules.
• Lower percentages of patients who receive the Investigational Product will require dose reduction and/or discontinuation of chemotherapy due to diarrhea as compared to the placebo groups.
• Lower percentages of patients who receive the Investigational Product will require dose reduction and/or discontinuation of chemotherapy due to nausea as compared to the placebo groups.
• Lower percentages of patients will require prescribed medications for treatment of diarrhea in the Investigational Product groups vs. placebo groups.
• Lower percentages of patients will require prescribed medications for treatment of nausea in the Investigational Product groups vs. placebo groups.
• Patients who initiate treatment with the Investigational Product prior to commencing chemotherapy (i.e. Cohort 3) will experience fewer adverse events, including diarrhea and nausea, as compared to patients who initiate treatment with the Investigational Product during their chemotherapy regimens (i.e. Cohort 1). • The frequencies and severity of specific adverse events, as determined using CTCAE grading, will be reduced overall in patients who receive the Investigational Product vs. placebo. This difference is most significant for patients who initiate treatment with the Investigational Product before starting chemotherapy (i.e. Cohort
3).
• Quality of life will be deemed to be improved by the administration of the Investigational Product as compared to the placebo groups as determined using EQRTC QLQ scoring.
• Hematological toxicity will be reduced by administration of the Investigational Product as compared to the placebo groups as determined on the basis of CBC and CMP blood tests.
• CRP levels in plasma will be reduced by administration of the Investigational Product as compared to the placebo groups.
• A proportion of patients given the Investigational Product will exhibit a complete response (CR) or partial response as defined by RECIST.
• Microbiome testing will demonstrate changes in the ratios of Firmicutes to Bacteroidetes that are associated with continued intake of the Investigational Product when analysis is performed at baseline (i.e. prior to Investigational Product administration, 3 months, and 5 months of the study).
• Flow cytometry to identify immune cell subsets present in patients who took the Investigational Product will reveal the presence of reduced percentages of inflammatory monocytes in plasma/serum (identified as CD14+ CD16+ cells having expresson of CD36 and/or toll-like receptor 2) and reduced percentages of immunosuppressive CD4+ CD25+ FoxP3+ T cells, as evaluated by flow cytometry.
[00233] Example 2: Evaluation of the bioactivity of a probiotic based formulation and its impact on a chemotherapeutic agent and an antibiotic
[00234] The following example is an in vitro study that was conducted to evaluate the activity of a probiotic based formulation comprising a blend of probiotic and digestive enzymes. This example validates the specific activity of a formulation disclosed in the present invention.
[00235] Synopsis of the Study :
[00236] The objective of this study was evaluation of the bioactivity of a supplement consisting of capsules with a blend of 9 probiotic organisms of the genera Lactobacillus and Bifidobacterium plus 10 digestive enzymes, in protecting the human gastrointestinal tract from chemotherapy and an antibiotic. We used the Simulator of Human Intestinal Microbial Ecosystem (SHIME) model, an in vitro model of a stable colon microbiota, and introduced 5-fluorouracil (5-FU) and vancomycin as microbiome-disrupting drugs. The probiotic with digestive enzymes supplement, added in capsules at in vivo doses, improved fermentation activity in the colon reactors and accelerated the recovery of microbial populations following 5-FU/vancomycin treatment. The supplement restored the Bacteroidetes to Firmicutes ratios in the colon reactors, increased the diversity of microbiota, and induced the production of microbial metabolites that elicited anti inflammatory cytokines in an in vitro model of intestinal inflammation. In the proximal colon, preventative administration of the supplement resulted in full recovery of the gut microbial community after cessation of 5-FU and vancomycin treatment. These results identify a probiotic with digestive enzymes formulation that protects against drug-induced gut dysbiosis, highlighting its potential utility as a component of routine cancer care.
[00237] Methods:
[00238] Probiotic-Based Formulation:
[00239] The probiotic with digestive enzymes supplement used herein comprises capsules is manufactured using proprietary methods and contains a blends of probiotics (116.20 mg total weight); specifically, Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei, Lactobacillus paracasei, and digestive enzymes (272.65 mg total weight); specifically, amylase, glucoamylase, lipase, bromelain, maltase, lactase, hemicellulose, xylanase, papain, and invertase.
[00240] The capsules used herein are the same formulation that would be administered in vivo. The product was tested at an in vitro dose of 3 capsules/day before chemotherapy/antibiotic treatments, and 4 capsules/day during and after chemotherapy/antibiotic treatment (to be described below). This corresponds to an in vivo dosage of 6 capsules/day and 8 capsules/day before and after chemotherapy/antibiotic treatment, respectively.
[00241] The SHIME Set-un: [00242] Briefly, the SHIME system consists of a series of double-jacketed vessels, simulating the digestive compartments that are initially inoculated with a fecal sample from a healthy adult donor using methods described previously [11].
[00243] The typical reactor setup consists of a succession of five reactors simulating the different parts of the human gastrointestinal tract. The first two reactors simulate different steps in food uptake and digestion, with peristaltic pumps adding a defined amount of SHIME feed (140 mL 3x/day) and pancreatic and bile liquid (60 mL 3x/day), respectively to the stomach (VI) and small intestine (V2) compartment and emptying the respective reactors after specified intervals [11]. The last three compartments simulate the large intestine. These reactors are continuously stirred, and they have a constant volume and pH control. Retention time and pH of the different vessels are chosen in order to resemble in vivo conditions in the different parts of the colon. Upon inoculation with fecal microbiota, these reactors simulate the ascending (V3), transverse (V4) and descending (V5) colon.
[00244] The present experiments employed an adapted SHIME setup to accommodate the following three treatment arms (vs. two arms that are typically compared using this system):
[00245] Control Arm: Chemotherapy and antibiotics; no supplement given.
[00246] Curative Arm: Chemotherapy and antibiotics; probiotic with digestive enzymes supplement added at the same time as the other agents.
[00247] Preventative Arm: Chemotherapy and antibiotics; probiotic with digestive enzymes supplement added prior to the other agents.
[00248] Accordingly, a TripleSHIME set-up was utilized wherein the colon reactors were limited to two instead of three to account for the additional test conditions required; specifically, reactors corresponding to PC-DC units (proximal and descending colon) instead of AC-TC-DC units (ascending, transverse, and descending colon).
[00249] The stages of the experiment are described below: a. Stabilization period (Weeks 1 and 2)
b. After inoculation of the colon reactors with an appropriate fecal sample, a two-week stabilization period allowed the microbial community to differentiate in the different reactors depending on the local environmental conditions. During this period, the basic nutritional matrix was provided to the SHIME to support diversity of the gut microbiota originally present in the fecal inoculum. The third arm of the SHIME setup (preventive arm; PREV), already received 3 capsules/day during the stabilization period (corresponding to an in vivo dose of 6 capsules/day).
c. Control period (Weeks 3 and 4)
d. During this two-week reference period, the standard SHIME nutrient matrix was further dosed to the model. Analysis of samples in this period allows to determine the baseline microbial community composition and activity in the different reactors, which will be used as a reference for evaluating the treatment effects. The third arm of the SHIME setup (PREV), already received 3 capsules/day during the control period (~ in vivo dose of 6 capsules/day).
e. Treatment period (Weeks 5-8; 5-FU plus vancomycin during Week 5) f. During this four-week period, the SHIME reactor was operated under nominal conditions, but each arm was treated as follows. During the first week, all arms received chemotherapy (10 mM of 5-fluorouracil (5-FU)) and vancomycin treatment (62.5 mg/L of vancomycin). During the subsequent weeks, it was considered a“recovery” period from the effects of 5-FU and vancomycin. Arm 1 of the SHIME did not receive any additional treatment (i.e. control arm; CTRL). Arm 2 of the SHIME received the probiotic with digestive enzymes supplement (4 capsules/day ~ in vivo dose of 8 capsules/day) at the start of the chemotherapy administration (i.e. curative arm; CUR), whereas addition of the supplement for arm 3 of the SHIME was continued (at 4 capsules/day ~ in vivo dose of 8 capsules/day) (PREV).
[00250] Analysis of the Stages and Activity of Microbiota in the SHIME:
[00251] The following microbial parameters were monitored throughout the entire SHIME experiment to evaluate the performance of the model and/or to allow monitoring of basic changes in the microbial community composition and activity due to the probiotic with digestive enzymes supplement. The concentrations of SFCA; specifically, acetic acid, propionic acid and butyric acid, were analyzed as by-products of microbial metabolism. Each of these parameters was measured three times/week. Briefly, SCFA were extracted from samples with diethyl ether after the addition of 2-methylhexanoic acid as an internal standard. Extracts were analyzed using gas chromatography as described previously [250]. The concentrations of lactate, the precursor of SCFA, were also monitored using a d- lactate/l-lactate kit (R-Biopharm, Mannheim, Germany), according to the manufacturer's protocols. As part of the SHIME experiments, the following groups were quantified via quantitative PCR (qPCR; once/week): Bacteroidetes phylum, Firmicutes phylum, Lactobacillus spp. and Bifidobacterium spp. as previously reported [251].
[00252] Analysis of the Stages and Activity of Microbiota in the SHIME:
[00253] 16S ribosomal RNA (rRNA) sequencing was used to analyze samples from the SHIME reactors to identify and compare the microorganisms using similar methods as published elsewhere [252]. The Illumina sequencing method was used to amplify microbial sequences until a saturation level was reached. Information on a broad spectrum of OTUs was obtained (>100 different of the most dominant OTUs), however, the results were presented as proportional values versus the total amount of sequences within each sample, thus providing semi-quantitative results. The methodology used primers that span 2 hypervariable regions (V3-V4) of the 16S rDNA. Using a paired sequencing approach, sequencing of 2x250 bp resulted in 424 bp amplicons.
[00254] To provide an ecological interpretation of these data, the Simpson reciprocal index was calculated as a measure of diversity and evenness of the microbiota as described previously [253]. An increase in the Simpson reciprocal index reflects a diversity increase, with 1 being the lowest possible number, and the number of bacterial species/OTUs present in the sample being the maximal number. The index will approach the maximal value when the OTU distribution is more even. The higher the index, the larger the diversity and the larger the evenness.
[00255] In vitro analysis of immune markers:
[00256] Caco-2 cells. The co-culture experiment was performed as previously described [254] H3riefly, Caco-2 cells (HTB-37; American Type Culture Collection) were seeded in 24-well semi-permeable inserts (0.4 pm pore size) at a density of 1 x 105 cells/insert. Caco-2 monolayers were cultured for 14 days, with three medium changes/week, until a functional cell monolayer was obtained. Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 25 mM glucose and 4 mM glutamine and supplemented with 10 mM HEPES and 20% (v/v) heat-inactivated (HI) fetal bovine serum (FBS).
[00257] THP-1 cells. THP1 cells (InvivoGen) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium containing 11 mM glucose and 2 mM glutamine, supplemented with 10 mM HEPES, 1 mM sodium pyruvate and 10% (v/v) HI-FBS. THP1 cells were seeded in 24- well plates at a density of 5 x 105 cells/well and treated with 100 ng/mL of PMA for 48 hours (h). PMA induces the differentiation of the cells into macrophage-like cells.
[00258] Caco-2/THP-l co-cultures: To mimic the interface between host immune cells and the fermentation products of the gut microbiome, in vitro experiments were conducted based on previous studies by Satsu and colleagues [255]. In this setup, the colonic suspensions collected from the SHIME are brought in contact with the apical side of the co-cultures (i.e. Caco-2 cells). The effects observed on the basolateral chamber where the THP-l cells reside are mediated indirectly by signals produced by the Caco-2 cells and/or by the transport of micro- and macro-molecules.
[00259] Briefly, the apical compartment containing Caco-2 cells was filled with sterile- filtered (0.22 pm) colonic SHIME suspensions (diluted 1:5 (v/v) in Caco-2 complete medium). Cells were also treated apically with 12 mM Sodium butyrate (NaB) (Sigma- Aldrich) as a positive control in experiments establishing the system. The basolateral compartment containing THP1 cells was filled with Caco-2 complete medium. Cells were also exposed to Caco-2 complete medium in both chambers in control experiments. Cells were treated for 24 h, at which time the basolateral supernatant was discarded and THP- 1 cells were stimulated with Caco-2 complete medium containing 500 ng/mL of ultrapure LPS (. Escherichia coli K12, InvivoGen). Cells were also stimulated at the basolateral side with LPS in combination with 1 pM hydrocortisone (HC) (Sigma-Aldrich) and medium without LPS (LPS-) in control experiments. Cultures were incubated at 37 degrees Celcius in a humidified atmosphere of air/C02 (95:5, v/v). After 6 h of LPS stimulation, the basolateral supernatants were collected for cytokine measurement (human IL-6 and IL-10) by Luminex. multiplex (Affymetrix-eBioscience) according to the manufacturers’ instructions. All treatments were done in triplicate.
[00260] Results & Conclusions·.
[00261] The probiotic with digestive enzymes formulation increases fermentation activity in the simulated gut microenvironment:
[00262] The consumption of acid and base reflects the overall microbial activity in the SHIME reactors representing the proximal and distal colon. Generally speaking, there are distinct bacterial populations that are native to the proximal and distal colon regions, reflecting the different requirements for digestion in each segment. Ligures 1A and 1B depict the average weekly acid/base consumption during the control and treatment periods (i.e. before and after 5-LU/vancomycin treatment). It should be noted that the treatment period consisted of one week of 5-LU/vancomycin followed by three weeks without these agents during which time recovery from 5-FU/vancomycin could be monitored and compared for the experimental treatment arms, as follows: 1) Control arm: receiving no supplement; 2) Preventative arm that had already been receiving the probiotic with digestive enzymes supplement all through the previous stabilization and control periods; and. 3; The curative arm that commenced supplementation with the probiotic with digestive enzymes supplement only at the start of 5-FU/vancomycin administration.
[00263] Figs 1A and 1B are bar graphs showing that as the reactors acidify during changes in microbial activity, base is added. These results show acid and base consumption during control (C) and chemotherapy/antibiotic treatment (TR) periods for the control (CTRL) curative (CUR), and preventative (PREV) arms with the probiotic with digestive enzymes supplement. Results are shown for the reactors corresponding to the PC (A) and DC (B), and represent the average base/acid consumption over the entire control (n = 6 measurement) and treatment ( n = 12) period (*) represents statistically significant differences between C and TR (i.e. before and after 5-FU/vancomycin addition to the reactors, respectively). The different letters above the bars denote statistical comparison between the indicated groups where uppercase or lowercase letters define distinct comparator groups; p < 0.05 was considered significant.
[00264] As per Figures 1A and 1B, 5-FU/vancomycin treatment caused significant changes in reducing base consumption the proximal colon but not the distal colon. In the proximal colon, the addition of the probiotics with digestive enzymes in both the curative and preventative treatment arms significantly increased base consumption in the proximal colon, countering the effects of 5-FU/vancomycin. It should be noted that, in vivo, more bacterial fermentation activity also occurs in the proximal colon where substrate availability is higher [256]. In the distal colon, the probiotic with digestive enzymes supplement administered in the preventative arm also increased base consumption, however, the 5-FU/vancomycin itself did not significantly diminish this overall marker of metabolic activity in the distal colon reactors.
[00265] Increased short chain fatty acid production is indued b the probiotic with digestive enzymes formulation:
[00266] The abundant SCFA, acetate, propionate and butyrate, are generated by fermentation of dietary fibers by gut microbiota. SCFA have a plethora of health-promoting effects through their interactions with metabolite- sensing G protein-coupled receptors on the gut epithelium and on immune cells. In these experiments, we monitored the production of these three SCFA in the proximal and distal colon reactors, comparing pre- and post-5- FU/vancomycin treatments in the control (non-supplemented), curative, and preventative arms that were treated with the probiotic with digestive enzymes supplement.
[00267] Figure 2 shows the results for acetate, which is produced by a range of gut microbes including Bacteroides and Bifidobacteria , and exerts anti-inflammatory effects [257]. More specifically, Figure 2 shows average acetate, propionate, and butyrate production, as indicated, over the control (C) (n = 6) and treatment (TR) (n = 12) periods for the control (CTRL), curative (CUR), and preventative (PREV) arms given the probiotic with digestive enzymes supplement is presented. (*) represents statistically significant difference relative to the preceding period. The different letters above the bars denote statistical comparisons between the indicated groups where uppercase or lowercase letters are used to delineate the distinct comparator groups, and p < 0.05 was interpreted as significant.
[00268] Chemotherapy treatment resulted in a decrease in acetate levels in both the proximal and distal colon reactors. Although the recovery of acetate did not occur in the proximal colon, preventative treatment with the probiotic with digestive enzymes supplement increased acetate in both control and 5-FU/vancomycin treatment periods in the distal colon, suggesting that the pretreatment with the supplement offsets the adverse impact of the drugs on microbial metabolism.
[00269] In Figure 2, analysis of propionate concentrations in the SHIME is also provided, a product of a diverse group of metabolically active gut microbes, which exerts anti-inflammatory effects in the colon as well as systemically [257]. Once again, the expected reduction in propionate in the SHIME reactors was observed in the 5- FU/vancomycin treatment period vs. the control period in both the proximal and distal colon reactors. Here, both the curative and preventative treatment arms with the probiotic with digestive enzymes supplement resulted in recovery of propionate levels relative to the control arm.
[00270] Figure 2 also shows the results for butyrate production in the SHIME reactors, a primary product of Clostridium clusters IV and XlVa (phylum Firmicutes). In vivo , butyrate is largely metabolized by intestinal epithelial cells where it serves as an energy source as well as a homeostatic factor for normal colonic cell turnover and repair processes. In the SHIME, the expected result was obtained whereby 5-FU/vancomycin treatment strongly decreased butyrate levels in both the proximal and distal colon reactors. Supplementation of the reactors with curative and preventative administration of the probiotic with digestive enzymes supplement resulted in improved butyrate production in the proximal and distal colon. Notably, the differences between the curative and preventative arms were not statistically different in either of the reactors.
[00271] Changes in gut microbiota are mediated by the probiotic with digestive enzymes formulation:
[00272] To further evaluate the impact of the probiotic with digestive enzymes supplement, the next series of experiments utilized qPCR to identify changes the microbial composition in the SHIME reactors representing the proximal and distal colon. These analyses focused on looking at the rates of recovery of healthy microbiota following the administration of 5-FU/vancomycin during treatment week 1 (TR1; refer to Figures 3 and 4), where 5-FU/vancomycin was discontinued during the recovery weeks spanning TR2- TR4. Comparisons were also conducted relative to the control period of the SHIME where no 5-FU/vancomycin had been added but the preventative arm was already receiving the probiotic with digestive enzymes supplement, allowing its influence on a healthy microbiome to be evaluated.
[00273] Specific strains of Lactobacillus and Bifidobacterium strains exert beneficial influences on maintenance of integrity of intestinal tissue in inflammatory circumstances and actions against toxicity of chemotherapeutic agents including intestinal mucositis [258- 262]. In addition to being permanent genera of the human intestinal microbiota, these strains are highly enriched in the probiotic plus digestive enzymes supplement. We evaluated the SHIME reactors for the presence of these lactate -producing bacteria, the Lactobacillus and Bifidobacterium strains. In Figure 3, data are shown for control and 5- FU/vancomycin treatments among the different experimental arms that received no supplement or either curative or preventative administration of the probiotic with digestive enzymes supplement. Figure 3 show the effects of a curative (CUR) and preventive (PREV) administration of the probiotic with digestive enzymes supplement as compared to a control SHIME (CTRF) on luminal Factobacillus (left panels) and Bifidobacterium (right panels) levels (16S rDNA copies/mF) in the proximal (PC; top panels) and distal colon (DC; bottom panels). The data are represented for the control weeks (Cl, C2)andtreatment weeks (TR1, TR2, TR3, and TR4. It should be noted that 5-FU/vancomycin was administered to the system in TR1 and discontinued in TR2- TR4. Preventative administration of the probiotic with digestive enzymes supplement was being administered throughout the control periods (Cl and C2), while curative supplementation of the probiotic with digestive enzymes formulation was initiated and maintained at TR1-TR4. (*) indicatesstatistically significant differences relative to the preceding period, while different letters indicate a statistical difference between different treatments; p < 0.05. First, as we expected, the results showed that 5-FU/vancomycin reduced the populations of these bacteria in the SHIME. For Lactobacillus, after the 5-FU/vancomycin treatment period during TR1 (Figure 3), Lactobacillus levels recovered after administration of the probiotic with digestive enzymes supplement, but also for the negative control in both colon regions. Significantly however, treatment with the supplement resulted in a faster recovery as compared to the negative control that lacked the supplement (Figure 3). For Bifidobacteria, after cessation of 5-FU/vancomycin, the levels of these microbes dropped drastically for the negative control in both colon regions, but especially in the PC where Bifidobacteria levels dropped below detection limit at the end of the treatment period (Figure 3). Administration of the probiotic with digestive enzymes supplement resulted in a slight recovery of Bifidobacteria levels, with no differences observed between the curative and the preventive arms of supplementation. It should be noted that, during the control periods (Cl and C2) prior to 5-FU/vancomycin treatment, overall Lactobacilli and Bifidobacteria did not increase in the PC or the DC.
[00274] We also assessed the composition of Bacteroidetes and Firmicutes, the predominant phyla in the healthy human gut, to determine how they are influenced by the probiotic with digestive enzymes supplement in the SHIME.
[00275] Figure 4 shows the effect of a curative (CUR) and preventive (PREV) administration of the probiotic with digestive enzymes supplement as compared to a control SHIME (CTRL) on luminal Bacteroidetes, and Finnicutes levels (16S rDNA copies/mL) in the proximal (PC; top panels) and distal colon (DC; bottom panels). The data are represented for the control weeks (Cl, C2) and treatment weeks (TRI, TR2, TR3, and TR4. It should be noted that 5-FU/vancomycin was administered to the system in TRI and discontinued in TR2-TR4. Preventative administration of the probiotic with digestive enzymes supplement was being administered throughout the control periods (Cl and C2), while curative supplementation of the probiotic with digestive enzymes formulation was initiated and maintained at TR1-TR4. () indicates statistically significant differences relative to the preceding period, while different letters indicate a statistical difference between different treatments; p < 0.05.
[00276] The results showed that Bacteroidetes levels were particularly depleted during the 5-FU/vancomycin treatment period. Significantly, we observed that administration of the probiotic with digestive enzymes supplement also resulted in faster recovery of Bacteroidetes after 5-FU/vancomycin (after TR1) as compared to the control arm, an effect that was the greatest for the preventative arm of supplementation over the curative arm (Figure 4). Remarkably, the Bacteroidetes levels were restored to control (pre-5- FU/vancomycin) levels during treatment weeks 3 and 4. This finding supports the capacity of the supplement to hasten the recovery of the gut microbiota following 5-FU/vancomycin treatment with the lowest levels observed in the colon reactors on treatment week 2 (TR2; Figure 4). Analysis of Firmicutes showed a more modest depletion in response to 5- FU/vancomycin that was evident at treatment week 1 (Figure 4), however, all three arms (control, preventative, and curative) recovered in the colon reactors after the treatment was discontinued. Interestingly, as can be appreciated in the proximal colon reactors, there was a faster recovery of Firmicutes in the preventative and curative arms vs. the control arm. This recovery was noted during TR2 and TR3 where the probiotic with digestive enzymes supplement promoted the rapid return to the Firmicutes levels in the control period. Overall, these results show that the probiotic and digestive enzymes supplement restores the Bacteroidetes to Firmicutes ratios following 5-FU/vancomycin.
[00277] Increased diversity of the gut microbiome by the probiotic with digestive enzymes formulation:
[00278] l6S-targeted Illumina sequencing was used whereby amplified 16S rRNA marker gene sequences are clustered into taxonomic units of bacteria. When the data had been processed at the phylum and family levels, and the Simpson diversity index was calculated. The lowest possible value of the index is 1, representing a community consisting of only one Operational Taxonomic Unit (OTU). The highest possible value is the total number of OTUs, and the higher the index, the larger the diversity and the larger the evenness.
[00279] Table 1
Changes in the diversity of microbiota resulting from curative or
preventative administration of the probiotic with digestive enzymes
supplement
Figure imgf000070_0001
[00280] Reciprocal Simpson Diversity Index in the lumen of the proximal and distal colon of the SHIME upon a curative (CUR) and preventive (PREY) administration of the probiotic with digestive enzymes supplement was compared to a control SHIME (CTRL) at the end of the control [control week 2 (C)] and treatment [treatment week 4 (TR4)] periods. The intensity of the shading
indicates the absolute diversity index, normalized for each of the environments (i.e. within each row').
[00281] Table 1 shows the Simpson Diversity Index results to evaluate the impact of the probiotic with digestive enzymes supplement on diversity changes in the microbiota in the SHIME that are caused by 5-FU and vancomycin. In the distal colon, the supplement given preventatively had the most significant impact on increasing the microbial diversity. In the proximal colon, the highest diversity was observed in the negative control (i.e. no supplement) following recovery from 5-FU and vancomycin (TR4). Curative treatment with the supplement also increased the microbial diversity in both the proximal and distal colons.
[00282] Lastly, l6S-targeted Illumina sequencing was used to evaluate the differences in microbial compositions at the phylum level in the proximal and distal colons caused by the probiotic with digestive enzymes supplement. Figure 5 shows the abundance(%) of the dominant phyla in the lumen of the proximal (PC) and distal colon (DC) of the SHIME upon a curative (CUR) and preventive (PREV) administration of the probiotic with digestive enzymes supplement as compared to control (CTRL) the end of the control week 2 (C) and treatment week 4 (TR) period. In Figure 5, the results show that the supplement did indeed cause changes in the microbial communities. The preventative treatment prior to 5-FU/vancomycin treatment did not cause any major changes at the phylum level in the proximal colon. However, in the distal colon, preventative supplementation resulted in increases in abundance of Actinobacteria and reductions in Bacteroidetes and Proteobacteria.
[00283] Following 5-FU/vancomycin treatment, the microbiome in the distal colon did not differ at the phylum level for the control, curative or preventative arms (Figure 5). In contrast, in the proximal colon, only preventive administration of the probiotic with digestive enzymes supplement resulted in full recovery of the gut microbiota after cessation of 5-FU/vancomycin treatment, mainly increasing the abundance of Bacteroidetes at the expense of Proteobacteria as compared to the other experimental arms. Collectively, these findings support the most significant changes in beneficial microbial communities as occurring in response to preventative administration with the probiotic with digestive enzymes supplement. However, it is plausible that a longer duration of curative treatment with the supplement would afford similar results as the preventative treatment.
[00284] The probiotic with digestive enzymes formulation modulates cytokine production in a model of intestinal inflammation:
[00285] We next asked how the probiotic with digestive enzymes supplement alters the composition of bacterial-derived metabolites having the potential to impact immune function as a means of offsetting the negative effects of 5-FU/vancomycin. To this end, we leveraged an in vitro model system that has been described as an‘inflammatory bowel disease-like’ model that uses Caco-2 (intestinal epithelial like cells) and THP-l macrophages [254]. Caco-2 cells originate from a human colon adenocarcinoma cell line that can differentiate into mature, enterocyte-like cells that are characterized by the formation of villi, presence of tight junctions, and expression of apical brush border enzymes, thereby recapitulating the colon [263]. THP-l cells, derived from acute leukemia, differentiate into macrophages upon culture with phorbol l2-myristate l3-acetate (PMA), and can then be activated toward a highly pro-inflammatory phenotype upon treatment with lipopolysaccharide (LPS). In the setup used here, Caco-2 cells were placed on top of PMA- treated THP-l cells, on the apical and basolateral sides of culture chambers, respectively. Colonic suspensions collected from the SHIME reactors, treated with the same regimens of 5-FU/vancomycin and probiotic with digestive enzymes supplement as in the previous experiments, were added to the apical side of the culture chambers containing Caco-2 cells. After 24 h of the apical pre-treatment of the Caco-2/THP-l co-cultures with the SHIME samples, the basolateral supernatant was discarded and the THP cells were treated with LPS to provide inflammatory signals. Subsequently, after 6 hours of stimulation, cytokines were measured from the basolateral side of the chamber containing the THP-l cells, which will have been affected indirectly by signals from the Caco-2 cells or directly by the transport of metabolites and molecules. In this manner, the interactions between cells of the gut and the immune system can be recapitulated in vitro. These experiments allow examination of the influence of the microbial fermentations-derived products following preventive and curative administration of the probiotic with digestive enzymes supplement in the face of 5-FU/vancomycin treatment.
[00286] The results of the Caco-2/THP-l co-culture experiments are provided in Figures 6A and 6B. Samples of SHIME suspensions were taken from reactors with curative (CUR) and preventive (PREY) administration of the probiotic with digestive enzymes supplement as compared to a control SHIME (CTRL; no supplement) from the control period (baseline microbial community prior to 5-FU/vancomycin) and treatment period with 5-FU/vancomycin. Cytokine levels ofl L-6 (A) and IL-10 (B) were measured 6 h after LPS treatment on the basolateral side of the Caco2/THP- 1 co-cultures after pre-treatment of the apical side for 24 h with the SHIME samples. The red dotted line corresponds to the experimental control consisting of LPS. Data are plotted as the mean ± SEM.
[00287] Measurement of IL-6 was performed as known pro-inflammatory signature of LPS-stimulated macrophages, and also of colonic mucosal cells [264, 265]. The results showed that preventative treatment with the probiotic with digestive enzymes supplement generated microbial metabolites in the proximal colon reactors that decreased IL-6 concentrations in the co-culture model as compared to the control reactors that were not supplemented but were treated with 5-FU/vancomycin. Thus, LPS-induced inflammation was modulated by fermentation products derived from reactors to which the supplement was administered prior to treatment with 5-FU/vancomycin. From the distal colon reactors, the SHIME suspensions taken from the control period, during which baseline microbial activities were monitored, and the treatment period with 5-FU/vancomycin did not differ with respect to the concentrations of IL-6 in the co-culture. Secondly, we also monitored IL-10 production induced in the Caco-2/THP-l cultures as a representative anti inflammatory cytokine produced by macrophages during LPS stimulation (Figures 6A and 6B). One point of interest was that that the metabolites from SHIME reactors following treatment with 5-FU/vancomycin induced greater IL-10 production than those from the control (baseline) period in the PC. However, administration of the probiotic with digestive enzymes supplement prior to 5-FU/vancomycin (i.e the control period) generated metabolites in the PC with the highest IL-10 inducing capabilities in the co-cultures. In the DC, the results showed statistically significant increases in IL-10 were generated in co cultures given SHIME suspensions from the preventative arm from both the control period and 5-FU/vancomycin treatment period. Collectively, the reductions in IL-6 and increases in IL-10 observed in the Caco-2/THP-l model of intestinal inflammation containing SHIME suspensions suggest that the probiotics with digestive enzymes supplement affects the gut microbiome toward controlling inflammation. [00288] Table 2
Figure imgf000074_0001
[00289] Table 2 outlines the corresponding statistical analyses performed using two- way ANOVA with Dunnett's multiple comparisons test. Significance is depicted where (*), (**), (***) and (****) represent p < 0.05, p < 0.01, p< 0.001 and p < 0.0001, respectively. Ns = non- specific; PC: proximal colon; DC: distal colon.
[00290] Example 3: Prevention of Chemotherapy Associated Ovarian Failure
[00291] A murine model will be implemented to evaluate the effect of a probiotic with digestive enzymes formulation on ovarian failure caused by chemotherapy.
[00292] C57BL6 mice Weight: 20-25 g Age: 4-6 weeks will be allotted randomly to three different groups, and each had 6 mice; group 1; the control group (no chemotherapy; placebo supplement), group 2; the chemotherapy group (chemotherapy; placebo supplement), group 3 (treatment arm; the mice will be given chemotherapy and a probiotic with digestive enzymes supplement). The supplement will be a formulation containing a proprietary blends of probiotic; specifically, Bifidobacterium infantis, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium longum, Lactobacillus casei, Lactobacillus paracasei, and digestive enzymes; specifically, amylase, glucoamylase, lipase, bromelain, maltase, lactase, hemicellulose, xylanase, papain, and invertase. The probiotics with digestive enzymes formulation will be diluted at a concentration of (65 pg/mL) in the drinking water of the mice. Alternatively, a placebo capsule will be diluted into the drinking water. A combination chemotherapy (CTX) of busulfan (12 mg/kg) and cyclophosphamide (70 mg/kg) (Sigma Aldrich., St. Louis, MO, USA) will beadministered intra-peritoneal to mice in groups 2 and 3.
[00293] Mice receiving chemotherapy will become infertile and posses enhanced levels of follicle stimulating hormone (FSH). Mice that received the probiotic with digestive enzymes supplement will be compared in terms of the percentages of animals who developed ovarian failure vs. controls, as well as with respect to the blood concentrations of FSH. The results will show that fewer of the chemotherapy-treated mice that receive the probiotic with digestive enzyme formulation demonstrate an elevation of FSH levels and the FSH levels will be statistically lower in this group vs. the placebo group that will receive chemotherapy.
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Claims

CLAIMS:
1. A method of improving the efficacy of a cancer therapy comprising: (a) identifying a subject with cancer scheduled to undergo or is undergoing treatment for cancer; (b) administering to said subject a formulation comprising probiotics in an amount sufficient to improve the efficacy of the cancer therapy.
2. The method of claim 1, wherein said formulation of probiotics comprises of one or more probiotics selected from the group consisting of: a) Bifidobacterium infantis b) Bifidobacterium bifiulunv, c) Lactobacillus acidophilus ; d) Lactobacillus salivarius e) Lactobacillus plantarum, f) Lactobacillus rhamnosus, g) Bifidobacterium longum h) Lactobacillus cased, and i) Lactobacillus paracasei.
3. The method of claim 1, wherein said formulation of probiotics further comprises a digestive enzyme selected from the group consisting of: a) amylase; b) glucoamylase; c) lipase; d) bromelain; e) maltase; f) lactase; g) hemicellulose; h) xylanase; i) papain, and j) invertase.
4. The method of claim 1, wherein said formulation comprises 272.65 mg by weight of digestive enzymes and 116.20 mg of probiotics per dose.
5. The method of claim 4, wherein said formulation of probiotics with digestive enzymes is administered to said subject in 6-8 doses per day.
6. The method of claim 1, wherein said formulation of probiotics comprises probiotics having the following activity/gram: between 25-150 billion colony forming units (cfu) for Bifidobacterium infantis, between 50-150 billion cfu for Bifidobacterium longum, between 350-550 billion cfu for Bifidobacterium bifidum, between 100- 300 billion cfu for Lactobacillus rhamnosus, between 100-300 billion cfu for Lactobacillus acidophilus, between 200-400 billion for Lactobacillus salivarius, between 300-500 billion cfu for Lactobacillus plantarum, between 200-400 billion cfu for Lactobacillus casei, and between 300-500 billion cfu for Lactobacillus paracasei.
7. The method of Claim 1, wherein said formulation further comprises digestive enzymes having the following activity/gram: between 300,000-500,000 HCU for hemicellulase, between 100,000-300,000 XU for xylanase, between 100,000- 300,000 DU for amylase, between 900-1,100 AGU for glucoamylase, between 10,000-30,000 DP for maltase, between 900,000, 1,100,000 TU for papain, between 2,000-4,000 GDU for bromelain, between 150-300 FIP for lipase, between 100,000-300,000 SU for invertase, between 75,000-150,000 ALU for lactase.
8. The method of claim 1, wherein said cancer therapy involves the administration of a therapeutic agent selected from the group consisting of: a) immunotherapy, b) chemotherapy, c) surgery, d) radiation therapy, e) hyperthermia, f) radiation, g) targeted therapy, h) hormone therapy, i) stem cell transplantation, and j) steroids.
9. The method of claim 8, wherein said chemotherapy comprises administration of a drug selected from the group consisting of: a) folinic acid (leucovorin), b) fluorouracil (5-FU), c) oxaliplatin, d) irinotecan, e) anthracyclins, f) cyclophosphamide, g) carboplatin, h) docetaxel, and i) paclitaxel.
10. The method of claim 1, wherein the efficacy of a cancer therapy in said subject in response to said probiotic with digestive enzymes formuation is measured by a clinical endpoint selected from the group consisting of: a) Overall survival (OS), b) Progression-free survival (PFS), c) Time to progression (TTP), d) Time to treatment failure (TTF), e) Event-free survival, f) Objective response rate (ORR), and g) Duration of response (DR).
11. The method of claim 1, wherein said subject undergoing cancer therapy is further treated with antibiotics or is scheduled to be treated with antibiotics.
12. A method for alleviating or preventing a cancer treatment-related adverse event comprising: (a) identifying a subject with cancer who is scheduled to undergo, is undergoing, or has undergone treatment for cancer; (b) identifying symptoms of a cancer treatment-related adverse event or risks for experiencing a cancer treatment- related adverse event in said subject; and (c) administering to said subject a formulation comprising probiotics in an amount sufficient to alleviate or prevent said cancer treatment-related adverse event.
13. The method of claim 12, wherein said cancer treatment-related adverse events are selected from the group consisting of: a) nausea, b) vomiting, c) diarrhea, d) abdominal pain, e) bloating, f) ulcers in the gastrointestinal tract, g) mucositis in the gastrointestinal tract, h) esophagitis, i) infusion-related reactions, j) fatigue, k) malaise, 1) cytokine release syndrome, m) serum sickness, n) alopecia, o) anemia, p) leukocytosis, q) viral infections, r) bacterial infections, s) yeast infections, t) acid reflux, u) neutropenia, v) lymphopenia, w) thrombocytopenia, x) leaky gut, y) cardiomyopathy, and z) neuropathy.
14. The method of claim 12, wherein said formulation of probiotics comprises a probiotic selected from the group consisting of: a) Bifidobacterium infantis, b) Bifidobacterium bifidum, c) Lactobacillus acidophilus, d) Lactobacillus salivarius, e) Lactobacillus plantarum, f) Lactobacillus rhamnosus, g) Bifidobacterium longum, h) Lactobacillus casei, and i) Lactobacillus paracasei.
15. The method of claim 12, wherein said formulation of probiotics further comprises a digestive enzyme selected from the group consisting of: a) amylase, b) glucoamylase, c) lipase, d) bromelain, e) maltase, f) lactase, g) hemicellulose, h) xylanase, i) papain, and j) invertase.
16. The method of claim 12, wherein said cancer treatment-related adverse events are identified based on a symptom selelcted from the group consisting of: a) nausea, b) vomiting, c) diarrhea, d) abdominal pain, e) bloating, f) ulcers in the gastrointestinal tract, g) mucositis in the gastrointestinal tract, h) esophagitis, i) infusion-related reactions, j) fatigue, k) malaise, 1) cytokine release syndrome, m) serum sickness, n) alopecia, o) anemia, p) leukocytosis, q) viral infections, r) bacterial infections, s) yeast infections, t) acid reflux, u) neutropenia, v) lymphopenia, w) thrombocytopenia, x) leaky gut, y) cardiomyopathy, and z) neuropathy.
17. The method of claim 12, wherein said formulation comprises 272.65 mg by weight of digestive enzymes and 116.20 mg of probiotics per dose.
18. The method of claim 12, wherein said subject undergoing cancer therapy is further treated with antibiotics or is scheduled to be treated with antibiotics.
19. The method of claim 12, wherein said cancer therapy involves the administration of therapeutic agents selected from the group consisting of: a) immunotherapy, b) chemotherapy, c) surgery, d) radiation therapy, e) hyperthermia, f) radiation, g) targeted therapy, h) hormone therapy, and i) stem cell transplantation.
20. A composition for modifying the gut microbiome in a subject comprising the following active ingredients: a) Bifidobacterium inf antis, b) Bifidobacterium bifidum, c) Lactobacillus acidophilus, d) Lactobacillus salivarius, e) Lactobacillus plantarum, f) Lactobacillus rhamnosus, g) Bifidobacterium longum, h) Lactobacillus casei, and i) Lactobacillus paracasei, j) amylase, k) glucoamylase, 1) lipase, m) bromelain, n) maltase, o) lactase, p) hemicellulose, q) xylanase, r) papain, and s) invertase.
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