EP2480242A1 - Lipopolysaccharide isolé de tissu de pyrularia et/ou bactérie associée à pyrularia et leurs utilisations - Google Patents

Lipopolysaccharide isolé de tissu de pyrularia et/ou bactérie associée à pyrularia et leurs utilisations

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Publication number
EP2480242A1
EP2480242A1 EP10819505A EP10819505A EP2480242A1 EP 2480242 A1 EP2480242 A1 EP 2480242A1 EP 10819505 A EP10819505 A EP 10819505A EP 10819505 A EP10819505 A EP 10819505A EP 2480242 A1 EP2480242 A1 EP 2480242A1
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EP
European Patent Office
Prior art keywords
pyrularia
extract
lps
immunostimulatory
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP10819505A
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German (de)
English (en)
Other versions
EP2480242A4 (fr
Inventor
Steven G. Wood
Katie Southwick
L. Dee Nord
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
TheraPro Technologies Inc
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TheraPro Technologies Inc
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Filing date
Publication date
Application filed by TheraPro Technologies Inc filed Critical TheraPro Technologies Inc
Publication of EP2480242A1 publication Critical patent/EP2480242A1/fr
Publication of EP2480242A4 publication Critical patent/EP2480242A4/fr
Pending legal-status Critical Current

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Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • This disclosure relates to immunologically active components derived from bacteria associated with Pyrularia and methods of use.
  • Natural protection against host invasion by, for example, bacteria, viruses, or parasites occurs in all higher organisms including insects, plants, and animals. Protection is mediated by biological sensors within the host, which are triggered by binding to specific molecules associated with the invading organism. Recognition then can lead to sequestration of the organisms, or to initiation of processes that lead to the elimination of the invading organism.
  • LPS-binding protein LPS-binding protein
  • TLR-4 toll-like receptor 4
  • Recognition initiates, via various signal transduction pathways, the synthesis and release of immune mediators (e.g. , cytokines and chemokines), which attract, localize, and/or activate specific cell types in a concerted immune response.
  • immune mediators e.g. , cytokines and chemokines
  • LPS has a profound effect upon the mammalian immune system and LPS in high amounts may over-stimulate the host immune system with lethal effect, as in the case of bacteremia leading to septic shock.
  • LPS is recognized as a potent immune stimulator and has been given systemically without the appearance of clinical symptoms.
  • LPS lipopolysaccharide
  • compositions including LPS isolated from Pyrularia fruit extracts, or LPS isolated from a Pantoea species associated with Pyrularia fruit.
  • the LPS is isolated from Pantoea agglomerans or Pantoea ananatis.
  • compositions comprising the Pyrularia fruit extracts, or LPS isolated from a Pantoea species, and a pharmaceutically acceptable carrier.
  • a method of stimulating immunity comprising administering to a subject in need thereof a composition that includes LPS isolated from Pyrularia fruit extracts or a Pantoea species, such as Pantoea agglomerans or Pantoea ananatis.
  • the method further comprises administering to the subject a therapeutically effective amount of a second immunostimulatory composition, such as a composition comprising a cytokine.
  • the subject has, or is at risk of developing a tumor.
  • Treatment of the subject with a composition comprising LPS isolated from Pyrularia fruit extracts or a Pantoea species can inhibit tumor development in the subject, such as inhibit metastasis of the tumor.
  • the extracts are immunostimulatory. In other embodiments, the extracts are
  • FIG. 1 is a flow cytometry scan that illustrates PyEx-2 treatment simulates murine bone marrow cells.
  • Balb/C bone marrow cells (1.5 x 10 6 cells/ml) were cultured alone (FIG. 1A) or in the presence of 2 ⁇ g/mL PyEx-2 (FIG. IB).
  • 200 ⁇ of cell suspension was stained with anti-Gr-l-FITC and anti-Mac-1- PE and analyzed by means of an EPICS -XLTM flow cytometer as described herein. Those cells staining more intensely along the Y-axis (Mac-l +hi ) are activated with PyEx treatment, as seen in FIG. IB.
  • FIG. 2 is a graph that shows PyEx reverses cyclophosphamide (CP)-induced white blood cell depletion in Balb/C mice.
  • CP cyclophosphamide
  • FIG. 3 is a flow cytometry scan showing that PyEx treatment restores Balb/C blood granulocytes and re-colonization of bone marrow, after injection of a sub-lethal dose of CP.
  • the normal abundance of blood granulocytes (FIG. 3 A) and bone marrow cells (FIG. 3B) was severely depleted following CP treatment, as shown in FIG. 3C and FIG. 3D, respectively.
  • Treatment with PyEx led to reversal of this immunosuppression, as shown by restoration of granulocytes (FIG. 3E) and recolonization of bone marrow (FIG. 3F). All cell samples were labeled with Gr-1- FITC and Mac-l-PE and analyzed by flow cytometry.
  • FIG. 4 is a graph showing that PyEx-2 stimulates human bone marrow cells.
  • Fresh marrow from a normal, 40-year old donor were incubated without treatment (FIG. 4A), with PyEx-2 (2 ⁇ g/mL; FIG. 4B) or with rhGM-CSF (5 ng/mL; FIG. 4C) for 48 hours prior to analysis by flow cytometry.
  • Cells were labeled with Gr-1- FITC and Mac-l-PE.
  • FIG. 5 is a graph showing the MALDI mass spectrum of a mixture of oligosaccharides following deamination.
  • the oligosaccharides were obtained from purification of LPS core derived from Pyrularia extracts.
  • FIG. 6 is a graph showing the reconstructed ESI mass spectrum of O- deacylated LPS obtained from Pyrularia extracts.
  • nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • sequence Listing is submitted as an ASCII text file, created on September 16, 2010 (-1.45 KB), which is incorporated by reference herein. In the accompanying sequence listing:
  • SEQ ID NO: 1 is the nucleotide sequence of a 16S forward PCR primer.
  • SEQ ID NO: 2 is the nucleotide sequence of a 16S reverse PCR primer.
  • SEQ ID NO: 3 is the nucleotide sequence of a PCR amplification product of genomic DNA isolated from Pyre/ana-associated bacteria.
  • rhGM-CSF Recombinant human granulocyte macrophage-colony stimulating factor
  • composition The introduction of a composition into a subject by a chosen route.
  • the composition is administered by introducing the composition into a vein of the subject.
  • Analog A molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure, such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by one or more functional groups, or a change in ionization.
  • Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in Remington: The Science and Practice of Pharmacology, 19 th Edition (1995), chapter 28.
  • a derivative is a biologically active molecule derived from the base structure.
  • a mimetic is a biomolecule that mimics the activity of another biologically active molecule.
  • Biologically active molecules can include both chemical structures and peptides of protein entities that mimic the biological activities of the Pyrularia extracts of the present invention.
  • Animal Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • mammal includes both human and non-human mammals.
  • subject includes both human and veterinary subjects.
  • mammal includes both human and non- human mammals.
  • Bioactive compound A compound that displays a measurable activity in a biological system is considered to be biologically active (bioactive).
  • a cancer is a biological condition in which a neoplasm has undergone characteristic anaplasia with loss of differentiation, increased rate of growth, in some cases invasion of surrounding tissue, and which is capable of metastasis.
  • the resultant neoplasm is also known as a malignant tumor.
  • cancer includes breast carcinomas ⁇ e.g. lobular and duct carcinomas), and other solid tumors, sarcomas, and carcinomas of the lung such as small cell carcinoma, large cell carcinoma, squamous carcinoma, and
  • adenocarcinoma mesothelioma of the lung, colorectal adenocarcinoma, stomach carcinoma, prostatic adenocarcinoma, ovarian carcinoma such as serous
  • cystadenocarcinoma and mucinous cystadenocarcinoma ovarian germ cell tumors, testicular carcinomas, and germ cell tumors, pancreatic adenocarcinoma, biliary adenocarcinoma, hepatocellular carcinoma, bladder carcinoma including transitional cell carcinoma, adenocarcinoma, and squamous carcinoma, renal cell adenocarcinoma, endometrial carcinomas including adenocarcinomas and mixed Mullerian tumors (carcinosarcomas), carcinomas of the endocervix, ectocervix, and vagina such as adenocarcinoma and squamous carcinoma, tumors of the skin like squamous cell carcinoma, basal cell carcinoma, melanoma, and skin appendage tumors, esophageal carcinoma, carcinomas of the nasopharynx and oropharynx including squamous carcinoma and adenocarcinomas, saliva
  • CD1 lb is the a subunit of the heterodimeric integrin Mac-1 (also known as 0 ⁇ 2 or complement receptor 3). Mac-1, which is comprised of CDl lb and CD18, is expressed on monocytes, macrophages, granulocytes and natural killer cells.
  • Cytokine A type of protein secreted by cells of the immune system.
  • Cytokines regulate the immune system through specific effects on cell-cell interaction, communication and behavior of other cells.
  • Cytotoxic refers to a substance that is toxic to cells.
  • Differentiation Process by which cells become more specialized to perform biological functions. Differentiation is a property that is completely or partially lost by cells that have undergone malignant transformation.
  • Gr-1 A GPI- linked protein expressed on the surface of mature granulocytes in the bone marrow and on peripheral neutrophils. Gr-1 is also known as Ly6G.
  • Granulocyte A white blood cell having a multi-lobed nucleus and cytoplasmic granules.
  • Granulocytes also known as polymorphonuclear leukocytes, include neutrophils, eosinophils and basophils. In some embodiments herein, granulocytes are characterized by expression of Gr- 1.
  • Immunostimulatory refers to the activation or stimulation of any aspect of the immune system.
  • a compound that causes stimulation of any aspect of the immune system is said to be “immunostimulatory” or to have an
  • immunostimulatory activity One specific type of immunostimulatory activity is granulocyte-stimulatory activity. This includes any treatment that causes the activation or stimulation of granulocytes, for instance granulocytes derived from peripheral blood or from bone marrow. Activation or stimulation of granulocytes can be measured, for instance, by examining an increase in the number of cells isolated from peripheral blood or bone marrow after a treatment compared with the number isolated from a control (e.g. , untreated or pre-treated) sample, or as seen by more intense staining with markers (i.e. , fluorescent-labeled monoclonal antibodies) specific to this cell type (for example, Gr-1).
  • markers i.e. , fluorescent-labeled monoclonal antibodies
  • a compound or composition that causes such granulocyte stimulation, or is capable of activating granulocytes, is said to have a granulocyte- stimulatory activity.
  • a compound or composition that is capable of activating macrophages is said to have macrophage-stimulating activity.
  • compositions that comprise at least one biologically active compound (e.g. , a lipopoly saccharide) as described herein as an active ingredient will normally be formulated with an appropriate solid or liquid carrier, depending upon the particular mode of administration chosen.
  • biologically active compound e.g. , a lipopoly saccharide
  • parenteral formulations usually comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like.
  • injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like.
  • Excipients that can be included are, for instance, other proteins, such as human serum albumin or plasma preparations. If desired, the
  • composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • the dosage form of the pharmaceutical composition will be determined by the mode of administration chosen.
  • topical and oral formulations can be employed.
  • Topical preparations can include eye drops, ointments, sprays and the like.
  • Oral formulations may be liquid (e.g. , syrups, solutions or suspensions), or solid (e.g. , powders, pills, tablets, or capsules).
  • solid compositions conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.
  • compositions that comprise biologically active compounds as described herein may be formulated in unit dosage form, suitable for individual administration of precise dosages.
  • One possible unit dosage contains approximately 3 mg/m 2 of active extract (or preparation) containing lipopolysaccharide.
  • the amount of active compound administered will be dependent on the subject being treated, the severity of the affliction, and the manner of administration, and is best left to the judgment of the prescribing clinician.
  • the formulation to be administered will contain a quantity of the active component(s) in an amount effective to achieve the desired effect in the subject being treated.
  • a pharmaceutically acceptable fluid composition comprising at least one active ingredient, e.g. , a mitogenic active component of a Pyrularia fruit extract.
  • the active ingredient is usually dissolved or suspended in a physiologically acceptable carrier, and the composition can additionally comprise minor amounts of one or more non-toxic auxiliary substances, such as emulsifying agents, preservatives, and pH buffering agents and the like.
  • non-toxic auxiliary substances such as emulsifying agents, preservatives, and pH buffering agents and the like.
  • Isolated An "isolated" biological component (such as a nucleic acid molecule, lipid, protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, for example other proteins in a naturally occurring mixture, or other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles.
  • Nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • LPS Lipopolysaccharide
  • Macrophage A cell that originates from specific white blood cells called monocytes. Monocytes and macrophages are phagocytes, acting in nonspecific defense (or innate immunity) as well as specific defense (or cell-mediated immunity) of vertebrate animals. Their role is to phagocytize (engulf and then digest) cellular debris and pathogens either as stationary or mobile cells, and to stimulate lymphocytes and other immune cells to respond to the pathogen.
  • Macrophages are derived from bone marrow precursor cells and are found in most tissues of the body.
  • Mimetic A biological compound (such as a peptide) that mimics the effect of a pharmaceutical, for example a peptide that mimics the effect of a biologically active component of a mitogenic and/or cytogenic Pyrularia extract by stimulating the immune system.
  • a biological compound such as a peptide
  • the activity of such mimetic compound(s) can readily be tested by one or more of the mitogen activity assays described herein, or by other such assays known in the art.
  • Myelocyte A type of cell typically found in the bone marrow that gives rise granulocytes and macrophages.
  • Neoplasm A new and abnormal growth, particularly a new growth of tissue or cells in which the growth is uncontrolled and progressive.
  • a tumor is an example of a neoplasm.
  • Non-native sequence or structure refers to the modification to a natural compound, e.g. , a protein, glycoprotein, or oligo- or polysaccharide, lipid, phospholipid, glycolipid, or lipopolysaccharide.
  • compounds that have a non-native sequence or structure maintain the mitogenic and/or cytotoxic activities described herein.
  • Activity-preserved, non-native saccharide chains may contain one or more saccharide (e.g. , monosaccharide) substitutions, deletions or additions.
  • activity-preserved proteins may contain one or more deletions, additions, or conservative amino acid substitutions.
  • Pantoea A genus of Gram-negative bacteria of the family
  • Pantoea genus includes seven species (P. ananatis, P. agglomerans, P. citrea, P. dispersa, P. punctata, P. stewartii and P. terrea).
  • Pantoea species are usually isolated from soil, fruit and vegetables.
  • a Pantoea species "associated with" Pyrularia fruit is any Pantoea species that grows, or is isolated from, the surface of Pyrularia fruit.
  • the Pantoea species is P. ananatis or P. agglomerans.
  • parenteral Administered outside of the intestine, e.g. , not via the alimentary tract.
  • parenteral formulations are those that will be administered through any possible mode except ingestion. This term especially refers to injections, whether administered intravenously, intrathecally,
  • peptide drugs intramuscularly, intraperitoneally, or subcutaneously, and various surface applications including intranasal, intradermal, and topical application, for instance.
  • Parenteral administration is usually preferred for peptide drugs, to avoid gastric degradation of the peptide.
  • compositions and formulations suitable for pharmaceutical delivery of therapeutic agents are conventional. Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of therapeutic agents.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions e.g., powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Pharmaceutical agent or drug A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject.
  • purified, homogeneous compounds The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified preparation is one in which the desired compound ⁇ e.g. , a
  • a compound ⁇ e.g., a biologically active compound of a Pyrularia extract as described herein
  • a compound is "purified” if it has been substantially separated from contaminants, e.g. , cellular components (nucleic acids, lipids, carbohydrates, and other polypeptides) that naturally accompany it.
  • contaminants e.g. , cellular components (nucleic acids, lipids, carbohydrates, and other polypeptides) that naturally accompany it.
  • Such a compound can also be referred to as “pure” or “homogeneous” or “substantially” pure or homogeneous. Purified is intended to be a relative term, and a compound is purified when at least 50-90% by weight of a sample is composed of the compound.
  • the subject compound will be at least 95% or more of the sample, even as much as 99% or more. Purity or homogeneity is indicated, for example, by polyacrylamide gel electrophoresis; high pressure liquid chromatography; or other conventional methods.
  • Pyrularia extract A concentrated preparation of Pyrularia pubera fruit or root that contains immunostimulatory activity.
  • Pyrularia pubera A terrestrial parasitic plant that grows on the roots of various broadleaf tree and shrubs (such as oak trees). Pyrularia pubera is commonly referred to as "buffalo nut.”
  • a recombinant nucleic acid or protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g. , by genetic engineering techniques.
  • a recombinant protein is one encoded by a recombinant nucleic acid molecule.
  • Subject Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals.
  • Therapeutic agent Used in a generic sense, it includes treating agents, prophylactic agents, and replacement agents.
  • Therapeutically effective amount A quantity of a specified compound or extract preparation sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount of a Pyrularia fruit extract (or a biologically active component thereof) necessary to stimulate an immune cell population in a subject (e.g. , to stimulate myelocytes in a subject). Specific examples would be a therapeutically effective amount of a Pyrularia fruit extract (e.g. , PyEx-1 or PyEx- 2), or a mitogenic active component thereof (e.g. , a lipopolysaccharide purified from a Pyrularia fruit extract, and having mitogenic activity).
  • a Pyrularia fruit extract e.g. , PyEx-1 or PyEx- 2
  • mitogenic active component thereof e.g. , a lipopolysaccharide purified from a Pyrularia fruit extract, and having mitogenic activity.
  • this can be the amount of Pyrularia fruit extract necessary to stimulate the immune system of a subject (also referred to as an immunostimulatory effective amount of the extract).
  • an immunostimulatory amount of an extract or purified component is an amount sufficient to stimulate an immune response (for instance, any of the stimulatory responses discussed herein) without causing a substantial cytotoxic effect (e.g. , without killing more than 10% of cells in a sample).
  • the effective amount of Pyrularia fruit extract, or active component thereof will be dependent on the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic composition
  • an effective amount of an immunostimulatory compound may be administered in a single dose, or in several doses, for example daily, during a course of treatment.
  • the effective amount of the compound will be dependent on the preparation applied, the subject being treated, the severity and type of the affliction, and the manner of administration of the compound.
  • a therapeutically effective amount of Pyrularia extract can vary from about 0.01 mg/kg body weight to about 1 g/kg body weight in some embodiments, or from about 0.01 mg/kg to about 60 mg/kg of body weight, based on efficacy and potential toxicity.
  • the mitogenic and/or cytotoxic Pyrularia extracts disclosed in the present invention have equal applications in medical and veterinary settings. Therefore, the general terms “subject” and “subject being treated” are understood to include all animals, including humans or other simians, dogs, cats, horses, and cows.
  • Tumor A neoplasm that may be either malignant or non-malignant and includes both solid and non-solid tumors (such as hematologic malignancies).
  • Tumors of the same tissue type refers to primary tumors originating in a particular organ (such as breast, prostate, bladder, or lung). Tumors of the same tissue type may be divided into tumor of different sub-types (a classic example being bronchogenic carcinomas (lung tumors) which can be an adenocarcinoma, small cell, squamous cell, or large cell tumor).
  • lung tumors can be divided into tumor of different sub-types (a classic example being bronchogenic carcinomas (lung tumors) which can be an adenocarcinoma, small cell, squamous cell, or large cell tumor).
  • Breast cancers can be divided
  • inhibiting tumor development or growth refers to slowing or preventing the initiation of a tumor, or decreasing the rate of growth or size of the tumor.
  • inhibiting tumor metastasis includes preventing metastasis, slowing the progression of metastasis or delaying the formation of metastases.
  • LPS isolated from the Pyrularia plant or LPS isolated from the plant-associated bacteria (such as Pantoea agglomerans or Pantoea ananatis), possesses potent immunostimulatory activity.
  • This immunostimulation activity increases the number of murine or human granulocytes, promotes restoration of white blood cell counts, and decreases the time required for bone marrow re-colonization and restoration of blood granulocytes following sub-lethal chemotherapy.
  • immunostimulatory compositions comprising LPS isolated from Pyrularia fruit extracts. Also provided are immunostimulatory compositions comprising LPS isolated from a Pantoea species associated with Pyrularia fruit. In some embodiments, the LPS is isolated from Pantoea ananatis or Pantoea agglomerans.
  • the immunostimulatory compositions disclosed herein are capable of activating granulocytes and/or macrophages.
  • the LPS is substantially stable at 100°C and/or the
  • compositions further comprise a pharmaceutically acceptable carrier.
  • the method further includes administering to the subject a therapeutically effective amount of a second immunostimulatory composition.
  • the second immunostimulatory composition comprises a cytokine.
  • stimulating immunity comprises inhibiting tumor development or growth in the subject, and the method comprises administering the agent to a subject having, or at risk of developing, a tumor.
  • inhibiting tumor development comprises inhibiting tumor metastasis.
  • stimulating immunity comprises activating granulocytes and/or macrophages. In some embodiments, stimulating immunity comprises inducing mitosis in an immune cell in the subject.
  • the method includes extracting Pyrularia tissue in a fluid to form a primary extract; precipitating the primary extract with 15% ammonium sulfate to form a secondary extract; and dialyzing the secondary extract using 12-14 kD MW cutoff dialysis tubing to produce the immunostimulatory and/or cytotoxic Pyrularia extract.
  • Pyrularia plants harbor bacteria, including Pantoea agglomerans or Pantoea ananatis, which provide the immunostimulatory and/or cytotoxic component of the extract.
  • the method further includes producing a more highly purified Pyrularia extract by passing the immunostimulatory and/or cytotoxic Pyrularia extract over a size exclusion column; and collecting high molecular weight fractions to yield the more highly purified Pyrularia extract.
  • the method includes passing the more highly purified Pyrularia extract disclosed herein over an anion exchange column; and collecting low salt (for example, 10 mM ammonium carbonate) eluate from the column, to form the immunostimulatory Pyrularia extract.
  • the method further includes heating the immunostimulatory Pyrularia extract to at least 60°C for at least 10 minutes.
  • Also provided is a method of preparing a cytotoxic Pyrularia extract that includes passing the more highly purified Pyrularia extract disclosed herein over an anion exchange column; and collecting high salt (for example, 1M NaCl) eluate from the column, to form the cytotoxic Pyrularia extract.
  • the immunostimulatory and/or cytotoxic Pyrularia extract In some embodiments of the methods of preparing an immunostimulatory and/or cytotoxic Pyrularia extract, the immunostimulatory and/or cytotoxic
  • Pyrularia extract comprises LPS from a bacterium associated with the Pyrularia tissue.
  • the immunostimulatory and/or cytotoxic Pyrularia extract comprises LPS from a Pantoea bacterium associated with the Pyrularia tissue.
  • the Pantoea bacterium associated with the Pyrularia tissue is Pantoea agglomerans or Pantoea ananatis.
  • a Pyrularia extract with immunostimulatory and/or cytotoxic activity prepared by any one of the methods disclosed herein.
  • the extract has granulocyte- stimulatory activity.
  • stimulating immunity comprises inhibiting tumor development or growth in the subject, and the method comprises administering the agent to a subject having, or at risk of developing, a tumor.
  • inhibiting tumor development comprises inhibiting tumor metastasis.
  • stimulating immunity comprises activating granulocytes and/or macrophages, and/or comprises inducing mitosis in an immune cell in the subject.
  • CP cyclophosphamide
  • a crude fraction of the Pyrularia pubera fruit is obtained from a basic fraction (see Example 2), such as ammonium sulfate extraction.
  • This crude fraction was found to have both cytotoxic and mitogenic (immunostimulatory) activity. These combined activities are particularly useful in the treatment of tumors, because the cytotoxic effect can directly damage the rapidly dividing tumor cells while the immunostimulatory effect offsets toxic effects on hematopoiesis. Immune stimulation can also recruit the subject's immune defenses against the tumor.
  • the immunostimulatory but not cytotoxic purified fraction is particularly suited for administration as an immunostimulant, for example to an immunocompromised individual who does not have a tumor, such as a leukopenic individual, for example someone who is granulopenic.
  • the cytotoxic component can be inactivated by heating Pyrularia extract at 70°C for 20 minutes.
  • LPS can also be directly isolated from Pyre/ana-associated bacteria, such as by using the solvent extraction procedure as described in Example 13. LPS obtained from Pyre/ana-associated bacteria is immunostimulatory.
  • LPS isolation procedure can include phenol-water extraction of bacteria (e.g. phenol-water extraction of a bacterial cell paste; see Example 13 below) and/or low or high speed centrifugation to remove debris or separate bacterial cell components. Additional steps can be taken to further purify crude LPS preparations obtained from bacterial samples, such as nuclease treatment (e.g. , RNAse or DNAse treatment), size-exclusion chromatography, anion-exchange chromatography, and/or FPLC anion-exchange chromatography.
  • nuclease treatment e.g. , RNAse or DNAse treatment
  • size-exclusion chromatography e.g. , RNAse or DNAse treatment
  • anion-exchange chromatography e.g., FPLC anion-exchange chromatography
  • PyEx activity was initially identified in a screen for immunologically active materials. It was found that PyEx possesses mitogenic activity toward mouse splenocytes and possesses synergistic activity in combination with certain other mitogenic agents. PyEx also demonstrated a mitogenic response in mouse bone marrow cells. In particular, it was found by flow cytometry to stimulate a subset of these cells that are double positive for labeling with Gr- 1 and Mac- 1 monoclonal antibodies (Gr-1 + , Mac-1 + ), which is indicative of activation of a myelocytic cell lineage in the bone marrow and mature granulocytes (specifically neutrophils) in peripheral blood (Fine et al , Blood, 90:795, 1997).
  • LPS isolated from purified bacteria obtained from Pyrularia fruit is also shown to have immunostimulatory activity. As shown for Pyrularia fruit extracts, LPS from Pyre/ana-associated bacteria is capable of activating bone marrow cells.
  • Pyrularia extracts described herein, and the mitogenic components thereof are potent compositions for treatment of neutropenia in clinical settings.
  • Bone marrow stimulating activity could be extracted from Pyrularia fruit extracts with boiling MeOH. However, little comparable activity was able to be extracted directly from ground Pyrularia root or from fruit prepared in the presence of antibiotics or with incubation at 4°C, suggesting the possibility that in spite of surface sterilization, bacterial products are present in the Pyrularia extracts.
  • the bacteria present in Pyrularia fruit extracts was identified as Pantoea ananatis.
  • Bacteria from Pyrularia extracts were grown on LB plates to isolate single colonies. Isolated bacterial colonies were expanded in LB broth and bacterial genomic DNA was isolated. Genomic DNA was PCR amplified using primers specific for 16S ribosomal nucleic acid. From two individual colonies, the identical nucleotide sequence was obtained. Sequence analysis of the PCR-amplified nucleic acid sequence identified the Pyrularia- associated bacteria as Pantoea ananatis.
  • LPS lipopolysaccharide
  • compositions that include at least one Pyrularia extract (e.g. , PyEx-1 or PyEx-2), or a biologically active component thereof (e.g. , a
  • lipopolysaccharide as described herein as an active ingredient, or that include both one of these compounds/compositions and an additional immunostimulatory factor (e.g. , an immunostimulatory cytokine) as active ingredients, may be formulated with an appropriate solid or liquid carrier, depending upon the particular mode of administration chosen.
  • the pharmaceutically acceptable carriers and excipients useful in this invention are conventional.
  • parenteral formulations usually comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like.
  • Excipients that can be included are, for instance, other proteins, such as human serum albumin or plasma preparations.
  • the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • the dosage form of the pharmaceutical composition will be determined by the mode of administration chosen.
  • topical and oral formulations can be employed.
  • Topical preparations can include eye drops, ointments, sprays and the like.
  • Oral formulations may be liquid (e.g. , syrups, solutions, or suspensions), or solid (e.g. , powders, pills, tablets, or capsules).
  • solid compositions conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. Actual methods of preparing such dosage forms are known, or will be apparent, to those of ordinary skill in the art.
  • compositions that comprise a Pyrularia fruit extract (such as PyEx-1 or PyEx-2), or a biologically active component thereof, in some embodiments of the invention will be formulated in unit dosage form, suitable for individual administration of precise dosages.
  • the amount of active compound(s) administered will be dependent on the subject being treated, the severity of the affliction, and the manner of administration, and is best left to the judgment of the prescribing clinician.
  • the formulation to be administered will contain a quantity of the active component(s) in amounts effective to achieve the desired effect in the subject being treated.
  • immunostimulatory Pyrularia extract such as PyEx-1 or PyEx-2
  • mitogenic components thereof are effective for treatment of conditions or diseases that affect the immune system, for instance conditions (including clinical treatments) that inhibit (or suppress) the immune system.
  • the immunostimulatory effect can be enhanced by co-administering one or more additional immunostimulatory Pyrularia extract (such as PyEx-1 or PyEx-2), or mitogenic components thereof, are effective for treatment of conditions or diseases that affect the immune system, for instance conditions (including clinical treatments) that inhibit (or suppress) the immune system.
  • the immunostimulatory effect can be enhanced by co-administering one or more additional immunostimulatory Pyrularia extract (such as PyEx-1 or PyEx-2), or mitogenic components thereof, are effective for treatment of conditions or diseases that affect the immune system, for instance conditions (including clinical treatments) that inhibit (or suppress) the immune system.
  • the immunostimulatory effect can be enhanced by co-administering one or more additional immunostimulatory Pyrularia extract (such as PyEx-1 or PyEx-2),
  • immunostimulants such as IL-2 or GM-CSF
  • the extract or active component is well known, and can be found for instance in U.S. Patent Nos. 5,632,983; 5,726,156; and 5,861,483.
  • Immune deficiencies e.g. , deficiencies of one or more immune cells, or of one or more immunological factors
  • immune suppressive medical treatment acute and/or chronic infection, and aging
  • aging can be treated using the methods and compositions described herein.
  • a general overview of immunosuppressive conditions and diseases can be found in Harrisons "Principles of Internal Medicine," 14 th Edition, McGraw-Hill, 1998, and particularly in chapter 86 (Principles of Cancer Therapy), chapter 88 (Melanoma and other Skin Cancers), chapter 307 (Primary Immune Deficiency Diseases), and chapter 308 (Human Immunodeficiency Virus Diseases).
  • Immune Suppressive Medical Treatment Many medical treatments can impair the immune system. Corticosteroids, for example, can reduce cell-mediated immunity.
  • cancer therapies e.g. , chemotherapy and radiotherapy
  • proliferating cells such as hematopoietic cells
  • immune suppression and depletion of the immune system is required for bone marrow transplantation, in which all hematopoietic cells are eliminated and subsequently replaced with transplanted cells.
  • Certain known bone marrow stimulants e.g. , erythropoietin and colony stimulating factors such as GM-CSF or G-CSF, which is sometimes marketed under the name "Neupogen," U.S. Patent No. 5,536,495
  • leukocytes e.g. , lymphocytes, monocytes and
  • immunostimulatory compounds and mixtures of the invention can be used to stimulate the immune systems of patients suffering from medical treatment or iatrogenically induced immune suppression, including those who have undergone bone marrow transplants, chemotherapy, and/or radiotherapy.
  • Acute and/or Chronic Infection The ability of Pyrularia fruit extracts, and the immunostimulatory components thereof, to facilitate splenic T cell generation indicates that these extracts and components could be used as a general
  • immunostimulant to activate the immune system against various diseases, both chronic and acute.
  • Subject infections include bacterial and viral infections, as well as infestations caused by eukaryotic pathogens and parasites.
  • immunostimulatory PyEx treatment can be used in the treatment of HIV infection.
  • the ability of extracts and components of Pyrularia fruit extracts, extracted and purified as described herein, to stimulate macrophages and cytotoxic T cells will be beneficial in helping to destroy HIV-infected cells.
  • Age-Linked Immune Deficiency Activation of the immune system (via stimulation of T cell production) by PyEx treatment (with or without an added immunostimulant) is also believed to be beneficial in aging subjects, in whom immune function is often compromised.
  • the need for treatment with one of the methods or compositions of this invention can be determined by examining the immune status of a test subject, and comparing this immune status to a control or average immune state (a hypothetical "normal" subject). For example, bone marrow biopsies or peripheral blood lymphocytes can be sampled to assess immune function. Indications of reduced immune function include leukopenia, for example neutropenia or lymphopenia, or evidence of diminished white blood cell function.
  • the immunostimulatory methods and/or compositions of the invention are available as treatments for the immune suppressed condition.
  • a reduced immune status such as a reduction in a peripheral white blood cell count to below normal, for example 25% below normal
  • the immunostimulatory methods and/or compositions of the invention are available as treatments for the immune suppressed condition.
  • the Pyrularia extracts and purified components thereof e.g. ,
  • lipopolysaccharides may be administered to humans, or other animals on whose cells they are effective, in various manners such as topically, orally, intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, intrathecally, and subcutaneously.
  • the particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (e.g. , the subject, the disease, the disease state involved, and whether the treatment is prophylactic). Treatment may involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years.
  • Site-specific administration of the disclosed compounds may be used, for instance by applying a Pyrularia preparation (or an immunostimulatory active component thereof) to a pre-cancerous region, a region of tissue from which a neoplasm has been removed, or a region suspected of being prone to neoplastic development.
  • the present invention also includes combinations of a Pyrularia extract (e.g. , PyEx-1 or PyEx-2), or a purified bioactive component thereof, with one or more other agents useful in the treatment of an immune-related disorder, condition, or disease, e.g. immune suppression caused by a disease or a therapeutic treatment.
  • a Pyrularia extract e.g. , PyEx-1 or PyEx-2
  • one or more other agents useful in the treatment of an immune-related disorder, condition, or disease e.g. immune suppression caused by a disease or a therapeutic treatment.
  • the extracts and/or extract components of this invention may be administered in combination with effective doses of other immunostimulants, anticancer agents, anti-inflammatories, anti-infectives, and/or vaccines.
  • administration in combination or "co- administration” refers to both concurrent and sequential administration of the active agents.
  • Co-administration of a Pyrularia extract (or purified active component thereof) with immunostimulants in vaccines is also useful in prevention and/or protection against diseases that affect the immune system.
  • immunostimulants or agents to prevent certain side effects may be included.
  • Immunostimulants include levamisole, isoprinosine, muramyl dipeptide, colony stimulating factors such as G-CSF, M-CSF, and GM-CSF, IL-1 , IL-2, and other agents. Effective doses of some of these immunostimulatory agents are provided in Munson, Principles of Pharmacology, 1995, pages 1151-1157. Agents may be used in combination with PyEx to eliminate certain undesirable side effects. These include for example, methyl xanthines (e.g. , pentoxyifyilline), rolipram, quercetin phosphodiesterase inhibitors (e.g. , roflumilast), or agents that elevate cAMP levels or stimulate various cellular protein kinases.
  • methyl xanthines e.g. , pentoxyifyilline
  • rolipram rolipram
  • quercetin phosphodiesterase inhibitors e.g. , roflumilast
  • agents 1) which are known to prevent symptoms leading to septic shock such as a) inhibitors of IL- 1 ⁇ converting enzyme (ICE) or b) IL-Ira (interleukinl receptor antagonist), 2) antiinflammatory cytokines such as IL-10, IL-4, or IL-13, or 3) agents which inhibit the production or action of pro-inflammatory cytokines, including IL-1 , IL-6, TNFa, NO, or IFN may be used in combination chemotherapy.
  • ICE IL- 1 ⁇ converting enzyme
  • IL-Ira interleukinl receptor antagonist
  • the combination therapies are of course not limited to the lists provided in these examples, but include any composition for the treatment of an immune disorder, such as an immunosuppressive disorder.
  • Treatment of a subject using the immunostimulatory compositions of the invention may be indicated after (or while) the subject has received an antiproliferative or other cytotoxic therapeutic treatment.
  • antiproliferatives compounds include the following: ifosamide, cisplatin, methotrexate, Cytoxan, procarizine, etoposide, BCNU, vincristine, vinblastine, cyclophosphamide, taxol, gemcitabine, 5-fluorouraci, paclitaxel, and doxorubicin.
  • a subject is given a cytotoxic treatment, then monitored for a period of time (usually in the range of days to weeks) to determine if the treatment leads to an immunosuppressive effect.
  • monitoring can include monitoring peripheral blood for leukopenia or pancytopenia, and/or monitoring T cell function.
  • a subject that displays an immune suppression will be a candidate for treatment using the compounds (e.g. , a Pyrularia extract such as PyEx-2, or a purified immunostimulatory component of such extract) and therapeutic methods of the disclosed invention.
  • PyEx-1 and PyEx-2 have immunostimulatory activity, there are instances in which it would be more appropriate to administer one or the other of the extracts, or more particularly purified active components thereof.
  • the PyEx-2 extract of the invention having immunostimulatory activity, is beneficially administered to subjects in need of stimulation of their immune system, for instance subjects who are immune suppressed as described herein.
  • PyEx-1 can be administered for the treatment of hyperproliferative conditions, such as tumors, where both cytotoxic and immunostimulatory effects are desired.
  • the Pyrularia extracts for instance PyEx-1 and PyEx-2, and purified active components thereof, can be supplied in kits for use in stimulation of an immune system, or for the prevention and/or treatment of a disorder, condition or diseases
  • kits for the treatment of hyper-proliferative conditions, such as tumors, where both cytotoxic and immunostimulatory effects are desired.
  • a clinically effective amount of one or more of the extracts or bioactive components e.g. , an effective amount of PyEx-2, or a purified immunostimulatory active component thereof
  • the compositions may be provided suspended in an aqueous solution or as a freeze-dried or lyophilized powder, for instance. In certain embodiments, the compositions will be provided in the form of a pharmaceutical composition.
  • Kits according to this invention can also include instructions, usually written instructions, to assist the user in treating a disorder, condition, or disease (e.g. , a tumor or an immunodeficiency) with a Pyrularia extract, such as PyEx-1 and PyEx- 2, or a purified active component thereof.
  • a disorder, condition, or disease e.g. , a tumor or an immunodeficiency
  • a Pyrularia extract such as PyEx-1 and PyEx- 2, or a purified active component thereof.
  • Such instructions can optionally be provided in non-printed format, such as in a computer readable medium.
  • the container(s) in which the composition(s) are supplied can be any conventional container that is capable of holding the supplied form, for instance, microfuge tubes, ampoules, or bottles.
  • the therapeutic composition may be provided in pre-measured single use amounts in individual, typically disposable, tubes or equivalent containers.
  • the total amount of an active extract or compound or combination supplied in the kit can be any appropriate amount, depending for instance on the market to which the product is directed. For instance, if the kit is adapted for research or clinical use, the amount of each Pyrularia extract, such as PyEx- 1 or PyEx-2, or a purified active component thereof, provided in a kit would likely be an amount sufficient for several treatments.
  • kits according to this invention will also include one or more other agents useful in stimulating the immune system, or in inhibiting a tumor, e.g. in treating hyper-proliferation.
  • kits may include one or more effective doses of other anti-proliferative or anti-cancer drugs, or an
  • immunostimulant such as GM-CSF or an anti-inflammatory.
  • Cyclophosphamide, melphalan and colony stimulating factors including recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF), were obtained from Sigma Chemical Company (St. Louis, MO). Isoton II and Zap-Oglobin were obtained from Beckman- Coulter (Fullerton, CA).
  • Red cell lysis buffer consisted of 150 mM NH 4 C1, 1 mM KHC0 3 , and 1 ⁇ Na 2 EDTA » 2 H 2 0, pH 7.2.
  • RPMI medium was composed of RPMI-1640 medium (Sigma Chemical Company) supplemented with 10% heat inactivated fetal calf serum and gentamycin.
  • mice Balb/C or C57B 1 mice were obtained from either Charles River
  • mice were acclimated for a period of two weeks in a temperature - controlled environment with alternating light/dark cycles of 12 hours and were allowed food and water ad libitum.
  • PEC Peritoneal exudate cells
  • a mitogenic assay was used as a measure of immunostimulation.
  • isolated cells were diluted to 1.5 x 10 6 cells/mL. Aliquots of cells (200 ⁇ ⁇ ) were incubated in sterile 96-well plates for 24 to 48 hours prior to the addition of 1 ⁇ of [ 3 H]thymidine.
  • Radiolabeled DNA was collected on glass fiber filters by means of an Inotech cell harvester (Rockville, MD) and associated radioactivity was determined by means of an Inotech radiometric instrument or by scintillation counting of individual filters.
  • TLF-D a proprietary reagent, was utilized at 10 ⁇ g/ml to amplify the mitogenic activity of Pyrularia extract (PyEx) in a synergistic fashion.
  • Bone marrow was flushed from the femurs of Balb/C mice with RPMI medium.
  • Cells were collected by centrifugation and the pellet treated with 1 mL of red cell lysis buffer (as described above) for five minutes at 25 °C, followed by dilution with 12 mL of RPMI media.
  • Cells were washed by two cycles of centrifugation and resuspension in RPMI. On the final wash, cells were counted using a hemocytometer and the final pellet was resuspended to 4 x 10 6 cells/mL with RPMI media for use in activation assays, which were quantitated by flow cytometric analysis (described below).
  • Cytotoxic activity toward adherent cells was determined using the sulforhodamine-B (SRB) method of Skehan and coworkers (/. Nat. Cancer Inst. 82: 1107-12, 1990). Briefly, cells were suspended in RPMI-1640 media and placed into a 96 well-plate (1.5 x 10 3 cells/well) in the presence of test cytotoxic extracts or known cytotoxic compounds. After a period of log-phase growth of 18-24 hours, the media was aspirated and protein fixed to the plate by the addition of 75 ⁇ of 0.4 N perchloric acid.
  • SRB sulforhodamine-B
  • Cells (200 ⁇ L ⁇ , prepared as described above) were collected after gentle trituration to resuspend cells. Each aliquot was diluted with 1 mL PBS, centrifuged to pellet the cells, and the supernatant liquid was removed. The washed cells were sequentially stained in the dark at 4°C for 15 minutes with anti-CD 16/32 to prevent non-specific labeling, then for 45 minutes with either anti-Mac- 1-FITC x anti-Mac - 1-PE or with Gr- 1-FITC x Mac-l-PE. Cells were washed once at 4°C with 1 mL of PBS and then resuspended in 1 mL of PBS.
  • LPS lipopoly saccharide
  • LPS was extracted either from the Pyrularia fruit-fraction extract (LPS-FF) or from the isolated bacteria (ATCC deposit PTA-10285, deposited on August 18, 2009) identified as Pantoea ananatis (LPS-P) by the method of Yi and Hackett (Analyst 125: 651-656, 2000) using Tri-Reagent (Molecular Research Center, Cincinnati, OH). Crude fractions were re-purified by the method of Manthey and Vogel (J Endotoxin Res 1-84-91, 1994) as detailed in Manthey et al. (J Immunol 153:2653, 1994) to remove LPS-associated proteins.
  • LPS-associated endotoxin units were quantitated by this variation of the LAL assay that is based on the enzymatic release of a chromophore in the presence of LPS.
  • Kit QCL-1000 materials were obtained from BioWhittaker (Walkers ville, MD).
  • Endotoxin units (EU) were based on standard endotoxin preparation from E. coli (0111 :B4) which was supplied with the kit.
  • Polymyxin B is an antibiotic, which has strong affinity for LPS. Reversal of bioactivity in the presence of 10 ⁇ g/ml of polymyxin B was used as an indication of LPS -dependent bioactivity.
  • Nitric oxide was estimated by quantitation of nitrite in cell medium by the
  • Pyrularia pubera is a terrestrial parasitic plant, which grows on the roots of oak trees.
  • the material of this investigation was harvested from Cane Creek in North Carolina. Fruit was harvested during the fall season when the fruit was mature. The plant material was shipped on ice in an insulated container and immediately stored at -20°C.
  • Pyrularia roots were washed in water to remove any dirt and debris and then blotted dry.
  • the roots were shaved with a vegetable peeler into small, thin strips until the inner root core is exposed.
  • the pieces were then freeze-dried and ground into a powder using a mechanical grinder. Lyophilized root powder was treated at - 20°C with acetone (5 m/g powder) with agitation for one hour to remove lipid material, then centrifuged at 4°C for 5 minutes to pellet solid material, which was treated again with acetone overnight at -20°C. After collecting the solid by centrifugation, the supernatant liquid was removed and the root powder solid was allowed to air-dry.
  • Root powder was extracted with 0.05 M sodium phosphate, pH 8.5 (10 ml/g root powder) for 3-15 hours at room temperature with stirring. The resulting slurry was centrifuged for 40 minutes at 15,000 rpm. The supernatant was retained and the solids were re-extracted with buffer. The first and second extracts were combined. Activity was then fractionated with ammonium sulfate precipitation as described below.
  • the dialyzed samples were concentrated on a Biichi Rotavapor Rl 10 (Brinkmann Instruments) and lyophilized on a SpeedVac Concentrator (Savant) to obtain a light brown solid.
  • the active material was usually enriched in the 15-25% ammonium sulfate cut. Following ammonium sulfate precipitation, the activity was dialyzed (12-14 LD MN cutoff) and lyophilized. This material is referred to as PyEx- 1. Size-exclusion chromatography
  • Sephadex G75- Active lyophilized PyEx (20 mg/ml) was dissolved in water, adjusted to pH 8.5 with dilute ammonium hydroxide and placed onto a Sephadex G75-120 (Sigma Chemical, St. Lois, MO) column which had been pre-equilibrated with ammonia- water, pH 8.5. Initial elution was with water (pH 10). Fractions (1.5 - 2.0 ml) were collected starting before the elution of the colored material.
  • the PyEx-SEC. l material was applied to an Econ Pac anion exchange cartridge (Bio-Rad, Hercules, CA) and eluted with a step gradient of low salt (10 mM ammonium carbonate) and then high salt (1M NaCl).
  • the immunostimulatory activity was found predominantly in the low salt fraction (designated PyEx-AX. lA) and the cytotoxic activity was found predominately in the high salt fraction
  • the cytotoxic activity was found to be heat labile (60°C, 10 minutes)
  • the immunostimulatory activity found in PyEx-2 was found to be stable to boiling, stable to treatment with detergent (0.5% sodium dodecyl sulfate, SDS) or chaotropic agents (6M guanidinium isothicyanate) and was also stable over pH 3-10 at 25°C. In addition, the activity was resistant to the effects of trypsin and proteinase K. Thus, treatment with proteinase K (50 ⁇ g/ml) in 10 mM Tris (pH 8.0), 10 mM EDTA, 0.5% SDS at 56°C for 30 minutes had little effect on mitogenic activity but was sufficient to completely inactivate a similarly treated sample of concanavalin A.
  • Example 4 In vitro stimulation of mouse granulocytes
  • Mouse myelocytes were prepared as described above (Example 1), and responses of these cells to treatment with Pyrularia extract was examined using flow cytometry.
  • the effect of PyEx-2 on stimulation of murine granulocytes in vitro is shown in FIG 1.
  • the control unstimulated population of Gr-1 + x Mac-1 + (Mac-1 +1 °) population of myelocytes had migrated to a more intensely stained population along the y-axis (Mac-l +hi ).
  • 64% of myelocytes were Mac-1 +1 °, with only 3% recognized in the "hi" region.
  • FIG. 1 shows results of the stimulation of murine bone marrow cells by PyEx-2.
  • Example 5 PyEx prevents chemotherapeutic-drug induced death
  • mice were treated with an LD5 0 dose of melphalan, lethal toxicity occurred on day eight in the melphalan alone group. However, in the group which received both melphalan and PyEx-1 (10 ⁇ g, every other day), no drug-related deaths occurred even at the time of termination of the experiment on day 45.
  • Example 6 In vivo neutropenia model
  • mice were injected on day 0 with a sub-lethal dose of cyclophosphamide (200 mg/kg). Eighteen to twenty-four hours later (day 1), control mice received an injection of saline and the PyEx group received a single dose of PyEx (20 ⁇ g/mouse). Mice were bled sequentially via the retro-orbital sinus or tail vein at various times thereafter. Blood was collected in heparinized capillary tubes.
  • This example demonstrates that the Pyrularia fruit extract can effectively stimulate human bone marrow cells.
  • this example also provides an additional system in which immunostimulatory activity of a Pyrularia extract, or component thereof, can be tested.
  • PyEx-2 was capable of stimulating human myelocytes, as shown in FIG. 4. Fresh human marrow from a normal donor was incubated without drug, or in the presence of PyEx-2 or rhGM-CSF, and the activating effect was quantitated by staining with anti-MAC-l-FITC x anti-CD 14-PE. As shown, both the PyEx-2 and rhGM-CSF treatments produced an activated sub-population of myelocytes, with the PyEx-2 sub-population containing 14% of total cells and the GM-CSF-treated cells containing a new population with 9% of the total cells.
  • polymyxin B is an antibiotic which is effective against gram negative bacteria by being able to bind tightly to the lipid A portion of bacterial LPS and prevent binding to cellular receptors. Reversal of immunostimulatory activity on addition of polymyxin B to an assay is considered to be indicative of the involvement of LPS.
  • Table 3 62% of murine bone marrow cells treated with PyEx were 'activated.' However, in the presence of polymyxin B, only 29% of the cells were activated, suggesting that a substantial portion of this activity was a result of PyEx- associated LPS. A similar result was seen on treatment with LPS extracted from plant-free bacteria, LPS-P.
  • PyEx contained 4.7 ⁇ g KDO/mg of extract which was about one third of the 13.8 ⁇ g/KDO/mg of a commercial preparation, but it was only a little more than twice the amount from LPS-P.
  • Table 4 Quantitation of LPS in PyEx and LPS-P by a functional LAL assay and by direct measurement of 2-keto-3-deoxyoctonate (KDO)
  • Example 9 Generation of TNFoc and NO in bone marrow cells and macrophages
  • the stable metabolite N0 2 was assayed and is used as an indication of the amount of NO formed.
  • High NO levels are normally found only in the presence of INFy.
  • bone marrow cells treated with PyEx were found to generate low levels of NO without added INFy.
  • little TNFot was detected in bone marrow, but higher amounts were found in PEC cultures treated with PyEx or LPS-P.
  • TNFot formation was attenuated by treatment with INFy and completely prevented with 1 mM pentoxifylline, a dose which also diminished the amount of NO formed by PEC.
  • Pentoxifylline may also show a similar effect to reverse the formation of NO in bone marrow cells; however, this has not been demonstrated. A higher dose of pentoxifylline did not prevent bone marrow activation (see Example 10).
  • Table 5 Levels of TNFa and NO generated on treatment of PEC with PyEx,
  • Fresh bone marrow cells were cultured with PyEx or certain commercial preparations of LPS including those from Salmonella minnesota (Sigma Chemical L-6261), Salmonella typhosa (Sigma L-7136), Escherichia coli (Sigma L-3254) or Serratia marcescens (Sigma L-6136). These commercial preparations are either phenol or TCA extracts. PyEx was present at 0.5 ⁇ g/ml. Commercial preparations were each tested separately at 0.1 ⁇ g/ml and then in combination with PyEx at 0.5 ⁇ g/ml.
  • Balb/C mouse bone marrow cells derived from a 7 week old animal was activated with LPS at 1.2 x 10 6 cells/ml in RPMI-1640 containing 10% fetal calf serum, L-glutamine, and Pen/Strep for 18 hours at 37°C and 5% C0 2 .
  • Cells were harvested, blocked with CD16/32 antibody and stained with Rat IgG2b FITC, Rat IgG2b PE, CDl lb (Ml/70), and Gr-1 (RB6-8C5).
  • Example 13 Production and extraction of LPS from Pyra/ana-associated bacteria
  • This example describes the growth of Pyre/ana-associated bacteria and subsequent isolation of LPS from the bacteria for analysis of LPS activity (Example 14) and determination of the LPS core structure (Example 15).
  • Isolated Pyre/ana-associated bacteria were used to inoculate 50 mL of LB broth. The inoculated broth was incubated at 27°C overnight with shaking. Frozen stocks of bacteria were thawed and seeded into four shaker flasks containing LB and placed in a shaker box at 30°C. A 10L fermenter was prepared and ready for media prior to thawing bacteria. Modified media was prepared and placed in the fermenter prior to inoculation to ensure sterility. A total of 9L of FB2 media was prepared according to Liu et al. (Proc. Natl. Sci. Counc. Repub. China B. 24(4): 156-160, 2000). Table 11 provides the components of FB2 media per 1L.
  • Bacteria was harvested at OD 6 oo ⁇ 10.0 and concentrated to approximately 2- 3L with a 0.2 M CellfloTM filter (Spectrum Labs, Collinso Dominguez, CA).
  • Concentrated material was spun down at 5,000 rpm for one hour at 4°C. Wet cell paste was then weighed and frozen at -20°C prior to the extraction process.
  • the wet cell paste was stirred with deionized water and heated to 65-68°C using a water bath. Once heated, an equal volume of pre-warmed 90% phenol was added.
  • the 1 : 1 phenol water mixture was stirred vigorously for 20 minutes at 70- 80°C.
  • the phenol/water/cell paste mixture was then cooled in an ice bath to 2°C and dialyzed against deionized water at 4°C for 24 hours. After 24 hours, the dialyzed emulsion was centrifuged for 15 minutes at 6,000 x g at 4°C.
  • the upper water phase was siphoned off and dialyzed for 72-96 hours against deionized water at 4°C.
  • the dialyzed material was lyophilized, weighted and assayed for purity and quality. In total, 400 mg of LPS was extracted from 57 grams of wet cell paste.
  • LAL assay was performed using standard LPS from Escherichia coli. The results showed that the LPS extracted from Pyre/ana-associated bacteria was pyrogenic. In addition, the extracted LPS preparation contained no detectable protein and little nucleic acid as determined by UV absorption at 256 nm.
  • Example 14 Evaluation of LPS activity at increasing levels of LPS purity This example demonstrates that increasing the purity of LPS isolated from
  • Bone marrow cells (1.5 x 10 6 cells/ml) isolated from 7 week old Balb/c mice were stimulated with the indicated concentration of LPS in RPMI 1640 medium containing 10% FBS, 2 mM L-glutamine, and 1% pen/strep for 20 hours at 37°C and 5% C0 2 .
  • Cells were harvested, Fc receptor was blocked with CD 16/32 antibody and the cells were stained with following antibodies: rat IgG2b F, rat IgG2b PE, CDl lb (Ml/70) PE, and Gr-1 (RB6-8C5) F.
  • MFI mean fluorescence intensity
  • This example describes the structure of the core of LPS isolated from Pyre/ana-associated bacteria.
  • NMR spectra were recorded at 25 °C in D 2 0 on Varian UNITY INOVA 600 instrument, using acetone as a reference for proton (2.225 ppm) and carbon (31.5 ppm) spectra.
  • Varian standard programs COSY, NOESY (mixing time of 200 ms), TOCSY (spinlock time of 120 ms), HSQC, and gHMBC (long-range transfer delay of 100 ms) were used.
  • MALDI Matrix-assisted laser desorption ionization
  • Mass spectra were obtained using Perseptive Biosystems Voyager DE STR spectrometer with 2,4-dihydroxybenzoic acid (DHB) matrix. Electrospray mass spectra were obtained using a Micromass Quattro spectrometer in 50% MeCN with 0.2% HCOOH at flow rate 15 mkl/min with direct injection.
  • DVB 2,4-dihydroxybenzoic acid
  • LPS or core (0.5 mg) was hydrolyzed (0.2 mL of 3M TFA, 120°C, 2 hours), followed by evaporation to dryness under a stream of air. The residue was dissolved in water (0.5 mL), reduced with NaBH 4 (approximately 5 mg, 1 hour), neutralized with AcOH (0.3 mL), dried, and MeOH (1 mL) was added. The mixture was dried twice with the addition of MeOH.
  • the precipitate of the lipid was removed by centrifugation at 12000 rpm, and soluble products were separated by gel chromatography on a Sephadex G50 column.
  • Oligosaccharide (core) fraction was further separated on an anion-exchange column and desalted on Sephadex G-15. Deamination of the core
  • LPS LPS was hydrolyzed by 2% AcOH, soluble products were separated by gel chromatography, and the oligosaccharide fraction was further separated by anion-exchange chromatography to give pure core oligosaccharide.
  • the LPS core was analyzed by NMR spectroscopy.
  • a set of 2D spectra (COSY, TOCSY, NOESY, ! H- 13 C HSQC, HMBC and HMQC-TOCSY) was recorded and interpreted using the Pronto program.
  • Spectra were complex because of the presence of several structural variants, differing by the attachment of glucose residues at the non-reducing end (incomplete presence of Glc P, L and K, and Hep T), and the presence of several variants of KDO degradation products ("normal KDO", 4,7- and 4,8-anhydro-derivatives) at the reducing end.
  • Monosaccharides were identified using characteristic patterns of TOCSY and COSY cross peaks, as well as chemical shift data.
  • the connection between monosaccharides was determined from NOE and HMBC data.
  • NOE correlations were identified: E1C5,7; F1E3; G1F7; H1E4,6; G1F7; Y1F3; X1Y4,5; Z1X4; K1Z6; P1K1,2: L1K6; S1Y1.2; Y1T5; T1S1,2.
  • Corresponding inter-residual HMBC were observed.
  • the core was methylated by the Ciucanu- Kerek procedure (Ciucanu and Kerck, Carbohydr. Res. 131 :209-217, 1984) and hydrolyzed. Monosaccharides were reduced with NaBD 4 , acetylated and analyzed by GC-MS. Derivatives of all neutral monosaccharides, expected from the presented structure, were detected: terminal, 2-, 6-, and 2,6- substituted
  • glucopyranose terminal DD- and LD-heptopyranose, 2-substituted DD- heptopyranose, 3,4- and 3,7- disubstituted heptopyranose.
  • ESI-MS electrospray ionization-mass spectrometry
  • oligosaccharide was deaminated by NaN0 2 -AcOH and the products analyzed by MALDI and ESI-MS.
  • Expected cleavage of GlcN X (which was converted into anhydromannose) led to formation of oligosaccharides with maximal structures 1 and 2; both had smaller variants due to lack of Glc and Hep.
  • Full oligosaccharide 1 with a mass of 1518.5 Da and a minor variant lacking Hep T with a mass of 1326.5 Da were visible in ESI and MALDI spectra.
  • Oligosaccharide 2 had no charged groups and was visible only in MALDI spectrum (FIG. 5) as Na- and K-adducts, giving peaks (Na-adducts) at m/z 833, 671 (loss of one glucose), and 509 (loss of two glucose) in agreement with the proposed structure.
  • LPS was O-deacylated by anhydrous hydrazine treatment and analyzed by ESI-MS (FIG. 6). Maximal structure with a composition
  • the LPS has a standard structure with two KDO residues and does not contain other acid-labile components, which could be lost during core preparation.
  • the analyzed core structure has close resemblance to the Serratia marcescens core (Vinogradov et al, Chemistry 12(25):6692-700, 2006), differing by additions of glucose residues at the non-reducing end, and by replacement of LD- Hep T in Serratia with DD-Hep in the analyzed structure. Ko, replacing side-chain KDO in Serratia, was not detected in the analyzed product.
  • This example describes the identification of the bacteria associated with Pyrularia fruit extracts. Bacteria were grown on LB plates by plating fruit extract and isolating single colonies. Single colonies were further expanded in 1 ml cultures of LB. Bacterial genomic DNA was isolated using the gram negative bacteria protocol of the Qiagen Blood and Tissue DNA Isolation Kit (Valencia, CA).
  • PCR cycling conditions included an initial denaturation at 95°C for 30 seconds, 30 cycles of amplification (95°C for 10 seconds, 55°C for 20 seconds and 72°C for 30 seconds) and a final end polishing at 72°C for 7 minutes.
  • PCR reactions were run using 0.1 ⁇ of isolated genomic DNA, 1 ⁇ of each primer and 100 ⁇ of each dNTP in a final volume of 50 ⁇ containing 5 ⁇ 10X NEB Taq buffer and 0.5 ⁇ NEB Taq polymerase.
  • PCR reactions were run on agarose gels to separate PCR products from unincorporated nucleotides and primers. PCR products were isolated using Qiagen Gel Isolation Kit following the standard protocol for fragments greater than 100 base pairs and greater than 4000 base pairs. PCR products were sequenced in both directions using 16S forward and 16S reverse primers, and a commercial sequencing provider (GeneWiz).
  • the bacterial strain has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. ⁇ 1.14 and 35 U.S.C. ⁇ 122.
  • the deposits represent a substantially pure culture of the deposited strain; it is noted that the strain is designated as Pantoea agglomerans for the deposit, and referred to as either Pantoea agglomerans or Pantoea ananatis herein.
  • the deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed.

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Abstract

Cette invention concerne, dans certains modes de réalisation, des procédés de génération et d'utilisation d'extraits de Pyrularia (par exemple de pyrularia pubera) et/ou d'une bactérie épiphyte/endophyte associée à celle-ci, soit seule ou en combinaison avec un ou plusieurs immunostimulants classiques ou des agents anti-inflammatoires, pour la modulation du système immunitaire d'un sujet. L'invention concerne également des compositions qui comprennent des extraits de Pyrularia spécifiques, des bactéries isolées à partir de tissu de Pyrularia (en particulier l'espèce Pantoea) et des extraits provenant de telles bactéries. La présente invention concerne également des composants purifiés d'extraits de Pyrularia et/ou d'extraits de bactéries épiphytes/endophytes, lesquels extraits présentent des activités mitogènes ou cytotoxiques. Les compositions de l'invention peuvent être utilisées pour le traitement de la neutropénie suite à une chimiothérapie et pour le traitement d'une immunodéficience (par exemple telle que celles provoquées par une chimiothérapie toxique, une maladie ou le vieillissement) ainsi que dans d'autres méthodes immunostimulantes. D'autres modes de réalisation de l'invention concernent des procédés de fabrication des extraits de l'invention ainsi que des composants bioactifs purifiés de ceux-ci comprenant en particulier du LPS bactérien.
EP10819505A 2009-09-24 2010-09-24 Lipopolysaccharide isolé de tissu de pyrularia et/ou bactérie associée à pyrularia et leurs utilisations Pending EP2480242A4 (fr)

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CN105530953A (zh) * 2013-10-03 2016-04-27 日东电工株式会社 注射疫苗组合物
WO2018205010A1 (fr) * 2017-05-11 2018-11-15 National Research Council Of Canada Utilisation de composés d'heptose phosphorylés

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
IWAMOTO ICHIRO ET AL: "Mechanistic analysis of high antitumor effect of intradermal administration of lipopolysaccharide from Pantoea agglomerans", MEDICAL ONCOLOGY, SCIENCE AND TECHNOLOGY LETTERS, NORTHWOOD, GB, vol. 13, no. 2, 1 January 1996 (1996-01-01), pages 103-109, XP008139452, ISSN: 1357-0560 *
KOHCHI C ET AL: "Applications of lipopolysaccharide derived from Pantoea agglomerans (IP-PA1) for health care based on macrophage network theory", JOURNAL OF BIOSCIENCE AND BIOENGINEERING, ELSEVIER, AMSTERDAM, NL, vol. 102, no. 6, 1 December 2006 (2006-12-01), pages 485-496, XP028042366, ISSN: 1389-1723, DOI: 10.1263/JBB.102.485 [retrieved on 2006-12-01] *
See also references of WO2011038186A1 *
TANIGUCHI YOSHIE ET AL: "Utility and Safety of LPS-based Fermented Flour Extract as a Macrophage Activator", ANTICANCER RESEARCH, vol. 29, no. 3, Sp. Iss. SI, March 2009 (2009-03), pages 859-864, XP9166715, ISSN: 0250-7005 *

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