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WO2024097250A1 - Support polymère pour probiotiques - Google Patents

Support polymère pour probiotiques Download PDF

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Publication number
WO2024097250A1
WO2024097250A1 PCT/US2023/036523 US2023036523W WO2024097250A1 WO 2024097250 A1 WO2024097250 A1 WO 2024097250A1 US 2023036523 W US2023036523 W US 2023036523W WO 2024097250 A1 WO2024097250 A1 WO 2024097250A1
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probiotic
copd
respiratory
mesh
probiotics
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Inventor
Helen H. Lu
Ming Chau CHAN
Aaron J. Moment
Zvi Loewy
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TOURO UNIVERSITY
Columbia University in the City of New York
TOURO UNIV
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TOURO UNIVERSITY
Columbia University in the City of New York
TOURO UNIV
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • 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
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/064—Saccharomycetales, e.g. baker's yeast
    • 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
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • 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
    • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/30—Compounds of undetermined constitution extracted from natural sources, e.g. Aloe Vera

Definitions

  • COPD Chronic Obstructive Pulmonary Disease
  • a respiratory tract insertable device comprising a mesh that carries a probiotic composition.
  • a method of treating or preventing chronic obstructive pulmonary disease (COPD) or a respiratory infection comprising having a subject suffering from, or at risk of developing, COPD use the device.
  • COPD chronic obstructive pulmonary disease
  • a method of treating or preventing COPD or a respiratory infection comprising controlled intra-oral release of a probiotic composition, or molecules released from the probiotic composition, to the respiratory tract in an effective amount to treat or prevent the COPD or respiratory infection in a human subject.
  • Fig. 1 Research approach diagram - Inhibition of pathogen establishment and regulation of microbiome composition in the lung using novel probiotic scaffold system applied directly into the oral cavity.
  • Figs. 3A-3C Mechanism of Probiotic Action via Biofilm Disruption. Fig. 3A - Biofilm formation/liftoff. Fig. 3B - P.
  • Figs.4A-4C Probiotic Growth on Nano-Mesh.
  • Fig. 5 Illustration of transport mechanisms and steady state measurement of mass transfer coefficient in flow. An example equation which can be used to determine the transport of peptides and cells released from the scaffold is provided below: [0013] Figs.6A-6B: Flow cell used for the evaluation of release of metabolites and life cycle of the probiotic scaffolds. The flow cell is sealed with a glass slide and tested with dye tracers under laminar flow at 2 mL/min for characterization purposes (Fig. 6A).
  • Figs.7A-7D Dose dependent zone clearing of Haemophilus influenzae (at increasing concentrations from left to right: 25 uL, 50 uL, 100 uL) with membranes containing Bacillus coagulans on both TSA (top) and chocolate agar (bottom) plates (Fig. 7A). A duplicate experimental set shown is shown in Fig. 7B. Results of probiotics (increasing concentration from left to right) on growth of microorganism on chocolate agar (Fig. 7C).
  • Fig. 8 Process development diagram for characterization of bacteriocins from Bacillus coagulans for applications in COPD.
  • Fig.9 OD600 of Bacillus coagulans culture over time. Visible cell number increases over time.
  • Fig.10 pH of Bacillus coagulans over time. pH value decreases over time
  • Fig.11 Standard Nisin LC-MS results.
  • Fig.12 Bacillus coagulans sample cation-exchange results.
  • Fig.13 Bacillus coagulans sample Columbia University LC-MS results.
  • Fig.14 Bacillus coagulans sample Touro School LC-MS results.
  • Fig. 15 Bacillus coagulans sample Columbia University LC-MS results zoom in at 2.31 min.
  • Fig.16 Bacillus coagulans sample Touro School LC-MS results zoom in at 2.31 min.
  • DETAILED DESCRIPTION [0024]
  • respiratory tract insertable device shall mean a device that is able to be inserted and maintained in the respiratory tract of a subject on the order of days to hours.
  • a respiratory tract insertable device includes, but is not limited to, a device that is able to be maintained in the oral cavity, nasal cavity, or trachea of a subject.
  • An example of respiratory tract insertable device is an orally wearable device, such as a mouthguard.
  • probiotics for the treatment of medical conditions including CODP, respiratory infections, and also for the maintenance of both GI and oral health, is driven on the one hand by an interest in understanding and leveraging the role of microbiome regulation in human health, and on the other, by documented clinical benefits.
  • Probiotics are active against respiratory pathogens and inflammation, regulate microbiome composition in vitro and in vivo, and may be formulated and delivered intraorally via controlled release devices.
  • This application discloses a device designed to deliver probiotics and their signaling molecules to the respiratory tract of a subject in order to reduce inflammation in the lung tissue and to prevent respiratory infections and exacerbations related to COPD.
  • the device is an intraoral device and delivers probiotics intraorally.
  • the delivery of probiotics intraorally stands in direct contrast to ingestion into the stomach and gut and opens up a completely different method of delivery as well as mechanisms of action.
  • the selected probiotics are based on their activity against common respiratory pathogens.
  • the premise of one of the novel treatment approaches to COPD is the connection between the oral, nasal, and lung microbiomes, and epidemiological studies that collectively suggest a strong association between oral health and COPD conditions 6-7 .
  • the upper airways comprise a continuous surface that terminates in the lungs through which air continuously passes in and out via the oral and nasal cavities; this air flow combined with both microaspiration and gravity flow of mucus and saliva downwards is the reason the lower airway is seeded with microbes from the oral and nasal cavities 8-9 .
  • the described devices are intended to be worn on the order of hours or days, some level of direct exposure to the lungs will be possible, for both the probiotics as well as their metabolites and signaling molecules.
  • clinical success has been observed based on probiotic use, for example, in human fecal microbiota transplantation 10 , the complete elimination of S.
  • the mesh pore size is such that the microbes can become established inside the mesh as well as on the exterior. Confocal fluorescence microscopy shows that these colonies establish themselves in various morphologies.
  • the PCL is attractive because it has robust mechanical properties, long shelf life, and has the flexibility and character of a fabric that can adopt various shapes and configurations.
  • These matrices are flexible in that nutrients, adjuvants, and also other drugs and agents may be introduced to facilitate the growth of the probiotics.
  • the use of probiotics delivered to the oral cavity to treat respiratory disease has not been well studied, and this disclosure describes developing a scaffold and delivery technology, as well as the selection of appropriate microorganisms, for this purpose.
  • a respiratory tract insertable device comprising a scaffold that carries a probiotic composition
  • the scaffold comprises a mesh, film, and/or a gel.
  • a respiratory tract insertable device comprising a mesh that carries a probiotic composition is described.
  • the mesh comprises polylactide-co-glycolide (PLGA) and/or poly-e-caprolactone (PCL).
  • the mesh comprises electrospun polylactide-co-glycolide (PLGA) and/or poly-e-caprolactone (PCL). [0038] In some embodiments, the mesh comprises 5:1 PLGA / PCL. [0039] In some embodiments, the mesh comprises fibers, wherein each fiber having a diameter between 200-900 nm, more preferably between 300-800 nm, more preferably between 500-600 nm. [0040] In some embodiments, the mesh comprises sodium alginate and or collagen. [0041] In some embodiments, the probiotic composition comprises a probiotic microbe culture.
  • the probiotic microbe culture comprises any one of Lactobacillus acidophilus, Bifidobacterium lactis, Bacillus coagulans and Saccharomyces boulardii, or a mixture of probiotics selected from the group consisting of Lactobacillus acidophilus, Bifidobacterium lactis, Bacillus coagulans and Saccharomyces boulardii.
  • the probiotic composition further comprises a food supply for the probiotic microbe culture.
  • the probiotic composition further comprises a nutrient, adjuvant, drug, and/or agent which facilitates growth of the probiotic microbe culture.
  • the probiotic microbe culture has a live/dead ratio greater than 2, more preferably greater than 3, more preferably greater than 3.5, more preferably greater than 4. [0046] In some embodiments, the probiotic microbe culture has a live/dead ratio of about 4. [0047] In some embodiments, the probiotic composition further comprises lactic acid. [0048] In some embodiments, the probiotic microbe culture is grown or seeded onto the mesh. [0049] In some embodiments, the device is a an orally wearable device, a mouthguard, or a nasally wearable device. In some embodiments, the device may be inserted into the trachea.
  • the mesh is conformed to a 3D-printed human tooth model.
  • a method is described for treating or preventing chronic obstructive pulmonary disease (COPD), the method comprising having a subject suffering from or at risk of developing COPD use any one of the respiratory tract insertable devices described herein.
  • COPD chronic obstructive pulmonary disease
  • a method is described for treating or preventing a respiratory infection, the method comprising having a subject suffering from or at risk of developing a respiratory infection use any one of the respiratory tract insertable devices described herein.
  • the respiratory infection is caused by any one of Pseudomonas aeruginosa, Moraxella catarrhalis, Haemophilus influenzae, and Streptococcus pneumoniae.
  • any one of the respiratory tract insertable devices described herein for use in treating or preventing chronic obstructive pulmonary disease (COPD), or for use in treating or preventing a respiratory infection.
  • COPD chronic obstructive pulmonary disease
  • the respiratory tract infection may be caused by, for example, any one of Pseudomonas aeruginosa, Moraxella catarrhalis, Haemophilus influenzae, and Streptococcus pneumoniae.
  • treatment or prevention of COPD or a respiratory infection utilizing any one of the respiratory tract insertable devices described herein results in a decrease of inflammatory cytokines present in the respiratory tract.
  • the respiratory microbiome composition of a subject is normalized after utilizing any one of the respiratory tract insertable devices described herein.
  • any one of the respiratory tract insertable devices described herein is used for a period of about 2, about 4, about 6, about 8, about 10, about 12, about 18, about 24, about 36, or about 48 hours.
  • the period of using the device is repeated daily, weekly, or monthly.
  • the method comprising controlled intra-oral release of a probiotic composition, or molecules released from the probiotic composition, to the respiratory tract in an effective amount to treat or prevent the COPD or the respiratory infection in a human subject.
  • the probiotic composition comprises one or more of the microbes selected from the group consisting of Lactobacillus acidophilus, Bifidobacterium lactis, Bacillus coagulans, and Saccharomyces boulardii.
  • the respiratory infection is caused by any one of Pseudomonas aeruginosa, Moraxella catarrhalis, Haemophilus influenzae, and Streptococcus pneumoniae.
  • this technology identifies the use of probiotic delivered through a wearable scaffold mesh to treat or prevent infection from respiratory pathogens.
  • Probiotics such as Lactobacillus acidophilus and Bifidobacterium lactis, can be isolated from tablets and colonies can be then established on the scaffold mesh.
  • the flexible matrices have robust mechanical properties, a long shelf life, and can allow nutrients, adjuvants and other drugs to facilitate probiotic growth.
  • probiotics and scaffold suggest an optimal 24-hour culture period of the probiotics on the scaffold.
  • Probiotics may prevent infection and reduce inflammation and therefore, probiotic-carrying scaffolds may be useful wearable delivery devices for COPD treatment and prevention.
  • Various other inventive aspects can be integrated or employed, such as probiotic delivery system to treat other GI tract related diseases in addition to COPD or to improve microbiome composition in a subject.
  • Standard Methods in molecular biology are described Sambrook, Fritsch and Maniatis (1982 & 19892nd Edition, 20013rd Edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3 rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol.217, Academic Press, San Diego, CA). Standard methods also appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols.1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning in bacterial cells and DNA mutagenesis (Vol.
  • the first aim of the research plan is to develop intra-oral devices to deliver probiotics and their signaling molecules, including bacteriocins, to the oral cavity and respiratory tract in order to reduce inflammation in the lung tissue and to prevent respiratory infections and flare- ups in COPD patients.
  • the objective is to create and characterize a prototype system comprised of probiotic organisms, a probiotic carrier, and a formulation, and to test the hypothesis that in the oral cavity, this approach may suppresses respiratory pathogens, regulate respiratory microbiome composition 9, 17 vs. triggers 18 , and ultimately, reduce tissue inflammation for COPD in vitro.
  • the hypothesis is that probiotics are active against respiratory pathogens and inflammation, regulate microbiome composition in vitro and in vivo, and may be formulated and delivered intra-orally via controlled release devices.
  • Fig. 1 shows a graphical representation of this hypothesis with the scaffold as part of a wearable mouthguard.
  • the aims of the research plan are: ⁇ Aim 1A - Screening of probiotic strains: Lactobacillus acidophilus, Bifidobacterium lactis, Bacillus coagulans and Saccharomyces boulardii will be screened against the respiratory pathogens Pseudomonas aeruginosa, Moraxella catarrhalis, Haemophilus influenzae, Streptococcus pneumoniae to evaluate the potential of anti-microbial activity and also with inflammatory models to determine anti-inflammatory activity.
  • the selection criteria are 1) significant zone of clearing as measured by a microbial disc assay, and 2) significant reduction in gene expression levels for pro-inflammatory cytokines as measured by microarrays.
  • ⁇ Aim 1B - Structural chemical characterization For probiotic candidates that show sufficient activity, structural chemical characterization of their metabolites and signaling molecules will be characterized in the presence and absence of pathogens, using high powered chemical characterization techniques (e.g. LC-MS/ NMR) to create a molecular level understanding of the pathways and metabolite releases.
  • ⁇ Aim 2A - Scaffold design Design of controlled-release device elements, e.g. films, gels, or meshes that contain the probiotics, nutrient matrix, and delivery capability enabling these microbes to grow, reproduce, and be established in saliva in a controlled manner, during a time frame on the order of 8 hours.
  • Design criteria include the ability to control the time frame of release, the dose, as well as underlying control of the nutrients and scaffolding on which the probiotics grow and detach.
  • Aim 2B - Transport properties Characterize pharmaceutically relevant transport properties of the active metabolites and the lifecycle of the probiotics identified in Aim 1B in a representative formulation developed in Aim 2A, in order to determine the release rate and appropriate dose regimen.
  • the long term impact of this approach is anticipated to be reduction of chronic inflammation in the lungs, protection against respiratory microbes that may enter the lungs via the oral cavity and nose, and finally restoration of the oral microbiome to a healthy condition in those COPD patients who may have also lost some of their teeth and suffer from poor oral microbiome condition and periodontal disease 6-7 .
  • probiotics for the treatment of medical conditions including burns 12 , asthma 19 , respiratory infections 20 , psoriasis 21 , and also for the maintenance of both GI 22-23 and oral health 24 , is driven on the one hand by an interest in understanding and leveraging the role of microbiome regulation in human health, and on the other, by documented clinical benefits.
  • mechanisms of action are often not well understood, nor are treatments controlled or regulated in a traditional pharmaceutical sense.
  • a sensible direction is to develop more controlled and nuanced ways of using probiotics, with an aim of increasing understanding, as well as advancing technology for medical applications.
  • probiotics used to pretreat the wells there is clear reduction in not only total biofilm, but particularly in dispersed cells (upper ring in Fig.3B).
  • the probiotic strains Lactobacillus acidophilus, Bifidobacterium lactis, Bacillus coagulans, and Saccharomyces boulardii will be screened against the respiratory pathogens Pseudomonas aeruginosa, Moraxella catarrhalis, Haemophilus influenzae, and Streptococcus pneumoniae using traditional techniques of microbiology, and also in a tissue inflammation model.
  • Microbial Growth Probiotic strains will be screened to determine whether probiotics impede the growth of respiratory pathogens. The respiratory pathogens to be screened include those implicated in COPD exacerbation. Membrane discs will be prepared by immersion of discs into probiotic solutions of varying concentrations for 30 minutes at room temperature. After 30 minutes, the discs are air-dried in a sterile petri plate.
  • Biofilm Life Cycle To observe the effect of probiotics on growth and survival of a biofilm the method described by O’Toole 26 will be used. An overnight culture of a respiratory pathogenic organism will be grown in the presence of varying concentrations of probiotics in a 96-well microtiter plate. Each concentration of probiotic will be evaluated in quadruplicates. The 96-well plate will be incubated at 37°C for a 24-hour period.
  • A549 The pulmonary adenocarcinoma derived cell line A549 will be used.
  • A549 cells will be purchased from Sigma-Aldrich (catalog number 86012804) and cultured in Dulbecco’s modified eagle’s medium with 10% fetal calf serum (FCS), 100 units/mL penicillin and 100 mG/mL streptomycin, under a humidified atmosphere 5% CO2 plus 95% air at 37oC.
  • FCS fetal calf serum
  • Agonists To model COPD, cigarette smoke extract (CSE) and lipopolysaccharide (LPS) will be added as described by Nachmias et. al. 27 . Research cigarettes (1RGF) will be obtained from the Kentucky Tobacco Research Center. LPS will be purchased from Sigma- Aldrich Co. St. Louis, MO. Treatments will include 2%, 4% and 10% CSE. LPS will be added to the cultures as described by Victoni et al. to stimulate COPD exacerbation conditions. [0081] Antagonists: We have extensive experience in assaying inflammatory gene expression levels using Affymetrix microarrays.
  • gene expression profiles will be performed as described Offenbacher et al. 28 .
  • the in vitro studies will test the potential anti-inflammatory activity of probiotics. Comparison will be made to cells not treated with probiotics. After treatment with or without antagonists, cells will be harvested for mRNA extraction (Qiagen Inc. Germantown, MD). RNA quality will be assessed with a Bioanalyzer 2100. Gene expression profiles will be performed using Affymetrix recommended procedures (Affymetrix Inc. Santa Clara, CA). Gene chip targets will be synthesized from the RNA using Affymetrix target synthesis procedures. Targets will be hybridized to gene chips and scanned using photoluminescence.
  • Affymetrix GeneChip Microarray software will be used for scanning and initial analysis.
  • Aim 1B - Chemical Structural Characterization This aim is to characterize by structural and chemical analysis the probiotic metabolites and signaling molecules that demonstrated clearing in the membrane disc assay.
  • High powered characterization e.g. LC-MS/ NMR
  • requisite purification e.g. chromatography
  • the objective of this work is to elucidate mechanism of behavior when possible.
  • An example of prior work is the showing that fengycin peptides released by Bacillus are responsible for inhibition of quorum sensing in S. aureus 11 .
  • the strategy is to select lead probiotic strains (ideally both a yeast and a bacteria) identified in Aim 1A, and then grown them on mesh-based carriers (see Figs. 4A-4C) in a saliva model, compare their response to those grown in alginate hydrogel and answer the question which of these carriers are best suited to which strains, and under what conditions.
  • the viability and growth of probiotics, as well as their ability to kill COPD-related pathogens (Aim 1A) will be measured and compared.
  • Our results demonstrate that Bacillus coagulans can be grown onto mesh of electrospun PCL via our customized seeding technique and the probiotic can be successfully migrated from agar to the mesh.
  • probiotics were cultured in Lennox (LB) agar and broth (9mL, Invitrogen, CA) and enumerated for 24 hours.
  • Fig. 4A demonstrates the presence of live probiotics on the polymeric meshes in comparison with agar control after 24 hours and 48 hours under culture.
  • fluorescence was detected at excitation and emission wavelengths of 485 and 535 nm for detection of live bacteria.
  • Excitation and emission wavelengths of 485 and 635 nm were used for detection of dead bacteria with a Tecan microplate reader.
  • Fig. 4B and Fig. 4C show that there is fluorescence for live probiotics is significantly higher than dead probiotics after 24 and 48 hours under culture, further providing evidence that the substrate supports cell viability.
  • Fig. 4C shows that the ratio of live versus dead probiotics peaked after 24 hours in culture in comparison to broth control and 48 hours of culturing. Thus suggesting an optimal culture period of 24 hours.
  • Aim 2B - Transport Properties [0090] The release characteristics, life cycle of the probiotic cells, and transport properties of the secreted molecules elucidated in Aim 1B will be characterized in representative formulations developed in Aim 2A, as illustrated schematically in Fig. 5. Flow cells such as those shown in Figs. 6A-6B will be used in which the probiotic mesh can be placed, and observed microscopically and evaluated in flow. Key characteristics including release rate, cell lifecycle, mass transfer coefficients, diffusion coefficients, as well as susceptibility to proteolytic salivary enzymes can all be estimated and quantified in a simple flow configuration. These studies will help us to better understand the mechanism of action of probiotics on COPD biofilm formation and elimination, and ultimately help us to optimize the probiotic carrier design.
  • Example 2 Effect of Probiotics on Respiratory Pathogens Objectives: [0093] To study if probiotics can inhibit the growth of respiratory pathogens and thereby prevent COPD exacerbations. [0094] To determine whether the effects of probiotics at inhibiting the growth of pathogenic microorganisms are dose dependent. [0095] To find a material that will allow growth of probiotics and have the capacity to release the probiotics in a controlled release manner.
  • Dissolve Probiotics ⁇ Dissolve Bacillus coagulans in sterile water at various concentrations: o (Control) 6 mL sterile water o (A) 4 billion cells BC / 6 mL o (B) 8 billion cells BC / 6 mL o (C) 12 billion cells BC / 6 mL [0099] Incubate: ⁇ Place membranes in each probiotic solution prepared in previous step for 30 minutes then air dry for 30 minutes ⁇ After incubation, place membranes on corresponding sections of the plates ⁇ Incubate plates at 37oC overnight Experimental Procedures [00100] Effect of Probiotic on Growth of H.
  • PCL has attractive features such as robust chemical properties and a long shelf life.
  • B. coagulans is successful in inhibiting the growth of the respiratory pathogen, H. influenzae, a key pathogen implicated in COPD exacerbations.
  • H. influenzae a key pathogen implicated in COPD exacerbations.
  • Based on the measured zones of clearing the effects of probiotic on inhibiting pathogenic microorganisms are dose dependent.
  • Confocal fluorescence microscopy showed that colonies of probiotics establish themselves in various morphologies on fibrous mesh.
  • B. coagulans can successfully be migrated from agar to electro spun PCL mesh. Conclusion [00106] B.
  • coagulans has demonstrated antimicrobial activity and therefore has clinical applications in terms of reducing inflammation in the lung tissue and preventing exacerbations in patients with chronic respiratory disease.
  • Probiotics are effective due to their ability to alter the biofilm lifecycle and thereby impede the growth of respiratory pathogenic microorganisms. The eradication of the growth of the biofilms is dose-dependent and therefore a minimum dosage of probiotic treatment is required to demonstrate beneficial outcomes in COPD patients.
  • B. coagulans can be grown onto mesh of electrospun polycaprolactone (PCL); this scaffold design serves as a site where probiotics can grow, reproduce and detach in a controlled manner.
  • PCL electrospun polycaprolactone
  • Example 3 Characterization of Bacteriocins from Bacillus Coagulans for Applications in COPD Motivation
  • COPD Chronic Obstructive Pulmonary Disease
  • This proposal is to characterize and purify active peptides derived from Bacillus coagulans, a probiotic bacteria, that inhibits pathogens associated with COPD flare ups, specifically Haemophilus influenzae.

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Abstract

La présente divulgation concerne un dispositif pouvant être inséré dans les voies respiratoires comprenant une maille qui porte une composition probiotique. Par exemple, une méthode de traitement ou de prévention d'une bronchopneumopathie chronique obstructive (BPCO) ou d'une infection respiratoire peut comprendre l'utilisation d'un tel dispositif par un sujet souffrant de, ou risquant de développer, une BPCO. En tant qu'autre exemple, une méthode de traitement ou de prévention de la BPCO ou d'une infection respiratoire peut comprendre une libération contrôlée, par exemple, par voie intraorale, d'une composition probiotique, ou de molécules libérées de la composition probiotique, vers les voies respiratoires en une quantité efficace pour traiter ou prévenir la BPCO ou une infection respiratoire chez un sujet humain.
PCT/US2023/036523 2022-10-31 2023-10-31 Support polymère pour probiotiques Ceased WO2024097250A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018188124A1 (fr) * 2017-04-11 2018-10-18 成都益植生物科技有限公司 Préparation probiotique colonisant, ses applications et médicament
US20200016290A1 (en) * 2017-03-16 2020-01-16 Microsintesis Inc. Compositions and methods involving probiotic molecules
WO2021077042A1 (fr) * 2019-10-16 2021-04-22 The Trustees Of Columbia University In The City Of New York Échafaudages à base de fibres pour la migration et la régénération de cellules de tendon
US20220296657A1 (en) * 2019-06-17 2022-09-22 Mayo Foundation For Medical Education And Research Prevotella preparations and treating chronic obstructive pulmonary disease (copd) and other lung conditions

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200016290A1 (en) * 2017-03-16 2020-01-16 Microsintesis Inc. Compositions and methods involving probiotic molecules
WO2018188124A1 (fr) * 2017-04-11 2018-10-18 成都益植生物科技有限公司 Préparation probiotique colonisant, ses applications et médicament
US20220296657A1 (en) * 2019-06-17 2022-09-22 Mayo Foundation For Medical Education And Research Prevotella preparations and treating chronic obstructive pulmonary disease (copd) and other lung conditions
WO2021077042A1 (fr) * 2019-10-16 2021-04-22 The Trustees Of Columbia University In The City Of New York Échafaudages à base de fibres pour la migration et la régénération de cellules de tendon

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