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WO2025019495A2 - Exosomes dérivés de produits laitiers fermentés - Google Patents

Exosomes dérivés de produits laitiers fermentés Download PDF

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Publication number
WO2025019495A2
WO2025019495A2 PCT/US2024/038214 US2024038214W WO2025019495A2 WO 2025019495 A2 WO2025019495 A2 WO 2025019495A2 US 2024038214 W US2024038214 W US 2024038214W WO 2025019495 A2 WO2025019495 A2 WO 2025019495A2
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exosomes
yogurt
centrifugation
isolated
supernatant
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WO2025019495A9 (fr
WO2025019495A3 (fr
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Peixuan Zhu
Javed Siddiqi
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Exom Biopharma Inc
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Exom Biopharma Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/20—Milk; Whey; Colostrum
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5184—Virus capsids or envelopes enclosing drugs

Definitions

  • Exosomes are extracellular nanoparticles (30-150 nm) in size that arc secreted by most cells including normal and cancer cells, bacteria, and plants. They are also present in almost all body fluids such as serum, milk, saliva, urine, and cerebrospinal fluid. Exosomes have several functions such as immune modulation, cell-cell communications, and even tumor metastasis.
  • Exosomes originate from different tissue and possess low immunogenicity, high biocompatibility, differentiation capacity, potent imnmnomodulatory properties. and the ability to be favorably cultured and manipulated. Exosomes transfer functional, cargos like miRNA andmRN'A molecules, peptides, proteins, cytokines, and lipids from derived tissue sources to the recipient cells, which contributes to use in biomedical healing and drug delivery. Exosomes are powerful cellular communication traffickers that are being used to power today’s biomarker discovery and therapeutics applications. Exosame cargo amount and composition depend on the cell type from which they are released, which makes them useful biomarkers and functional characterization of disease states.
  • nanoparticles and viral vectors have been reported as delivery platforms for vaccines and drugs, such as liposomes, lipid nanoparticles, kmtivirus, and AAV.
  • messenger RNA rnRNA
  • Lipid nanoparticle vehicles have been crucial to the success of the Pfizer-BioNTech and Moderns COVID-19 vaccines.
  • one major drawback of these delivery vehicles is their immunogenicity, that is, their tendency to induce an unwanted immune response against themselves. Therefore, the therapeutic agents delivered by these vehicles cannot be repeatedly administrated because the neutralizing antibodies can bind to the active site of drag molecules and inactivate their functional activities.
  • exosomes are naturally occurring bioparticles with intrinsic characteristics, including low immunogenicity, innate stability, and ability to cross blood-brain barriers, offering several advantages as therapeutic vehicles for functional cargo delivery.
  • exosomes share similar lipid bilayer structures to the cell membranes, allowing them to enter recipient cells and deliver therapeutic agents more efficiently.
  • the exosomes can be chemically or genetically modified with selected ligands that enable targeted delivery of therapeutic agents to specific cells or tissues of interest.
  • Eixosomes are present in almost all biological fluids.
  • exosomes have been isolated from various sources, including cell culture media, bacteria culture media, plants, milk, and body fluids.
  • the exosomes are commonly isolated from cell culture media of mesenchymal stem cells (MSCs) that are known as promising sources for cancer therapy and can be utilized as vehicles in cancer gene therapy.
  • MSC-derived exosomes have similar biological functions to the stem cells for tissue regeneration in therapeutic applications, known as the novel cell-free alternatives to MSC-based cell therapy.
  • Exosomes isolated from plants have been used for skin care and wound healing.
  • Exosomes in the body fluids, such as blood, urine, saliva, and CSF are also novel biomarkers for diagnostic applications. Compared to traditional biomarkers of circulating tumor cells and circulating tumor DNA, exosomes provide higher sensitivity and specificity thus they have been recognized as next-generation biomarkers in non-invasive liquid biopsy for early detection of cancers.
  • Exosomes which are considered natural cell-secreted extracellular vesicles, encapsulate endogenous bioactive molecules, and can efficiently deliver these cargoes to recipient cells, to enable cell-to-cell communication and regulate the biological functions of recipient cells.
  • exosomes offer the advantages of high biocompatibility, low toxicity, long- circulating time, and intrinsic ability to encapsulate therapeutic agents, exosomes have been emerging as novel platforms for drug delivery.
  • the clinical application of exosome- based therapeutics in clinical settings remains an ongoing challenge.
  • exosomes isolated from cell culture media often result in a low yield and a high cost, which makes it difficult to scale up the manufacturing production of clinical-grade exosomes.
  • researchers have been continuing to search for an alternative, cost-effective, and scalable source for exosome production.
  • bovine milk has been reported as an alternative source of exosomes (e.g., U.S. Patent Application Publication No. US2016/0000710AI, European Patent Application Publication No. EP3620519A1).
  • Milk- derived exosomes exhibited a similar potential to serve as drug-delivery nanocarriers. Compared to cell culture media, milk is a more affordable and accessible source. It was reported that chemical compounds and nucleic acids encapsulated in milk exosomes could be delivered orally into the bloodstream in bioactive form, but the potential to deliver large biomolecules such as proteins and peptides remained to be further studied.
  • casein and whey protein are the major proteins of milk.
  • casein constitutes approximately 80% (29.5 g/L) of the total protein, and whey protein accounts for about 20% (6.3 g/L), respectively. It is difficult to separate exosomes from these proteins and fat using conventional methods.
  • the isolation of exosomes from milk often results in low yield and high contaminations, limiting manufacturing capability and clinical applications, hydrogen present invention disclosed herein overcomes the shortcomings of previous technologies and provides a novel type of exosomes as well as an effective isolation method that will facilitate the clinical applications of exosomes.
  • the present disclosure provides novel compositions of exosomes derived from fermented dairy products.
  • the embodiments disclosed herein overcome the limitations of previous technologies and provide a cost-effective source for the mass production of high-quality exosomes.
  • the disclosure also provides a method for the Isolation and purification of exosomes from fermented dairy products. Purified exosomes from fermented dairy products can be directly used as therapeutic agents themselves for the treatment of diseases, such as antiinflammation, anti-fungal infections, anti-aging, and skin care.
  • the exosomes from fermented dairy products can also be used as drug delivery vehicles for the delivery of various drugs, such as chemotherapeutic compounds, antibiotics and antifungal drugs, proteins, peptides, small molecules, and nucleic acid therapeutics such as mRNAs, circular RNAs, miRNAs, shRNAs, and various other RNA therapeutics agents or a combination of drugs.
  • drugs such as chemotherapeutic compounds, antibiotics and antifungal drugs, proteins, peptides, small molecules, and nucleic acid therapeutics such as mRNAs, circular RNAs, miRNAs, shRNAs, and various other RNA therapeutics agents or a combination of drugs.
  • the exosomes have been isolated from yogurt samples and characterized by nanoparticle tracking analysis, protein concentration analysis, SDS-PAGE and Western Blot analysis, and transmission electron microscopy analysis.
  • exosomes isolated from a fermented dairy product as herein described are used directly as therapeutic agents, preferably as part of a pharmaceutical composition comprising the exosomes and a pharmaceutically acceptable excipient.
  • exosomes are isolated from a fermented dairy product and used to encapsulate one or more therapeutic agents.
  • an exosome composition is provided that comprises an effective amount of a therapeutic agent encapsulated by a fermented dairy-derived exosome.
  • compositions comprising exosomes isolated from a fermented dairy product are provided.
  • a pharmaceutical composition is provided that comprises a fermented dairy product-derived exosome composition disclosed herein and a pharmaceutical vehicle, carrier, or excipient
  • the pharmaceutical composition is pharmaceutically acceptable in humans.
  • This invention also provides a new type of exosomes as drug delivery vehicles.
  • the exosomes isolated from fermented dairy products are highly biocompatible to humans.
  • the probiotic bacteria in fermented dairy products have been known for many years to help the human body maintain a healthy community of microorganisms. However, these bacteria cells can only stay in the digestive system and are not able to carry drugs to the bloodstream.
  • the exosomes derived from fermented dairy products are smaller nanoparticles compared to bacterial cells. They can cany the drugs, cross intestinal barriers to penetrate the bloodstream circulation, and deliver drugs to target tissues and organs of interest. They even can cross blood-brain barriers and deliver drugs to disease sites in the brain that are difficult to reach by traditional drug delivery systems.
  • Exosomes isolated from fermented dairy products have intrinsic functions as therapeutic agents or can be loaded with selected drugs for the targeted treatment qf diseases. Ibere are myriad examples for the treatment of diseases with exosomes derived from fermented dairy products including different types of cancers, infectious diseases, autoimmune diseases, cardiovascular diseases, neurodegenerative diseases, obesity treatment, skin care, wound healing, and anti-aging treatment.
  • Figure 2 is a comparison of particle diameters from 11 source samples. Y-axis indicates the peak size Of particle nanometers in diameter. In general, exosomes from yogurt are smaller than that from milk while exosomes from kefir are larger except for one sample (KI).
  • Figures 3A-C show the representative histograms of the nanoparticle size distribution of the exosomes isolated from three yogurt samples (Fig. 3 A) Q5-SI, (Fig. 3B) Q5-S2, (Fig. 3C) Q5-S5.
  • the histograms are generated by nanoparticle tracking analysis (NT A) using the ZetaView PMX 130Z instrument.
  • NT A nanoparticle tracking analysis
  • X-axis indicates the particle sizes in the nanometer while Y- axis indicates particle concentrations corresponding to their sizes.
  • Figure 4 is the ratio of particles to protein as a means of comparing exosome purity.
  • X-axis indicates the sample ID numbers.
  • Y-axis indicates the ratio of exosome particle numbers per ⁇ g of protein.
  • Exosome preparations that are pure exhibit a relatively high ratio of particles to protein while introducing contaminating protein to the samples wi ll reduce the value of the ratio.
  • Exosomes isolated from yogurt curds (Q5-S2 and Q5-S.3) show higher purity compared to the exosomes isolated from fresh milk. (Ml and M2).
  • FIG. 5 is the Zeta potential evaluation of exosome stability.
  • Zeta potential was measured in exosomes isolated from yogurt (Q5-S1 , S2, S3) and milk (Ml and M2). Each bar represents mean values of zeta potential and error bars indicate standard deviations.
  • Zeta potential (ZP) is a common method to measure the surface potential of exosomes, while used as an indicator of stability. A large negative value of the zeta potential of exosomes indicates higher stability of exosomes in suspension due to the electrostatic repulsion of individual exosomes.
  • Exosomes isolated from yogurt curds (Q5-S2 and Q5-S3) show higher stability compared to the exosomes isolated from fresh milk (M I and M2),
  • Figure 6 is 4*15% SDS-PAGE analyses of exosomes isolated from yogurt and fresh milk.
  • Li (lane 1 ), Page Ruler Plus Prestained Protein Ladder (ThermoFisher); L2, original milk control; L3, Ml milk-derived exosomes; L4, M2 milk-derived exosomes; L5, Q5-S1 exosomes; L6, Q5-S2 exosomes; L7, Q5-S3 exosomes; L8, exosomes isolated from cell culture media of human breast cancer MCF7 cell line.
  • Figure 7 is 4-20% SDS-PAGE and Western blotting analyses of exosomes isolated from yogurt and fresh milk.
  • Figure 7 A LI (lane 1), Page Ruler Plus Prestained Protein Ladder (ThermoFisher); L2, Q5-S1 exosomes; L3, Q5-S2 exosomes; L4, Q5-S3 exosomes; L5, Ml milk-derived exosomes; L6, M2 milk-derived exosomes.
  • Figure 713 LI, Q5-S1 exosomes; L2, Q5-S2 exosomes; L3, Q5-S3 exosomes; L4, Ml milk-derived exosomes; L5, M2 milk derived exosomes; L6, exosomes isolated from cell culture media of human breast cancer MCF7 cell line.
  • Figure 8 is a transmission electron microscopy (TEM) imaging analysis of yogurt- derived exosomes from sample Q5-S2. Arrows indicate the exosomes with typical cup-shaped structures at a size of -100 nm in diameter.
  • TEM transmission electron microscopy
  • Figures 9A-B show update of DAPl and PK.H26 dyes by MCF-7 cells.
  • Figure 9A shows exosomes prepared by the methods disclosed herein loaded with DAPI (nuclear dye) and incubated with MCF-7 breast cancer cells.
  • Figure 9B shows exosomes loaded with PKH 26 (cytoplasmic dye) and incubated with .MCF-7 breast cancer cells.
  • extracellular vesicles are the vesicles produced inside of cells but released outside of cells through different biogenesis processes. There are three major types of extracellular vesicles, including exosomes, microvesicles, and apoptotic bodies. They are generated through different biogenesis procedures and with various sizes ranging from 30 nm to 1 ,000 nm. As used herein, “Exosomes” are one of the most important types of extracellular vehicles that are smaller in size about 30-150 nm, and have now been recognized as emerging platforms for diagnostic and therapeutic applications.
  • a new source of exosomes involves the discovery of a new source of exosomes that can be used as therapeutic agents or drug-delivery vehicles.
  • exosomes have been isolated from various sources of materials, such as cell culture media, bacterial culture media, body fluids, plants, and milk.
  • 'Hie present invention provides novel types of exosomes isolated from fermented dairy products, particularly yogurt curds. Aspects of the present invention overcome a major dra wback of previous exosomes from milk by providing high yield and purity of final composition and simple methods involving isolation and purification that can promote large-scale manufacturing of exosomes for therapeutic applications.
  • the exosomes can be isolated from different types of fermented dairy products.
  • Fermented dairy products provide numerous potential health benefits to the human diet.
  • the most common fermented dairy products are yogurt, kefir, cultured buttermilk, and sour cream.
  • Other, lesser-known products include koumiss, acidophilus milk, cottage cheese, kimchi, and miso.
  • Fermented dairy products are produced by culturing milk with specific kinds of bacteria, specifically Lactobacillus bulgaricus and Streptococcus thermophilus. During this culturing process, the milk is fermented and partially digested by the bacteria.
  • exosomes are isolated from yogurt (whey and/or curd) or kefir.
  • the fermented dairy products used for exosome isolation may include one type or at least two, three, four, five, six, seven, eight, nine, ten, or even more different types of probiotic bacteria.
  • Fermented dairy products also known as cultured milk products, are very ancient foods that have been produced since around 10,000 BC. Many studies have shown that consuming fermented dairy products, such as yogurt, can enhance normal bacterial flora and improve human health.
  • Yogurt is a daily product made by fermenting milk with lactic acid bacteria culture. Two main lactic acid bacteria, Lactobacillus bulgaricus and Streptococcus thermophilus are used to produce yogurt.
  • lactobacilli and bifidobacteria may be added. Lactobacillus bulgaricus and Streptococcus thermophilus are commonly used together because these two bacterial species are synergistic. During yogurt production, these bacteria work together throughout the fermentation process to produce lactic acid, decreasing pH and causing milk protein to coagulate. In this invention, specific bacterial strains or a combination of strains can be selected for inoculation of milk fermentation. 'Che resulting exosomes will possess unique molecular profiles that have therapeutic potential for various clinical applications. In some aspects, exosomes are derived from a milk product fermented with one or more of the bacterial strains listed in Table I .
  • the yogurt-derived exosomes are concentrated and highly purified Substances of probiotics. Therefore, the composition of this invention carries various biomolecules that have active functions to regulate the immune response and restore a healthy balance to improve human health.
  • the composition can be administered to human subjects by any standard route. Examples include oral, nasal, rectal, vaginal, intravenous intramuscular, and subcutaneous routes of administration to disease sites of interest or by other methods or combinations for delivery.
  • yogurt-derived exosomes may be added as active ingredients into other treatment modalities that are provided to patients.
  • yogurt-derived exosomes as drug delivery vehicles.
  • the method and composition of this invention have important improvements over existing technologies.
  • dairy-derived exosomes Compared to lipid nanpparticles (LNP), adeno-associated viruses (AAV) and lentivirus vectors, and other traditional synthesis delivery systems, yogurt-derived exosomes have their special advantages: (i) exosomes are natural nanoparticles with low toxicity and immunogenicity; (ii) exosomes have high biocompalibility for the delivery of therapeutic payloads due to its lipid bilayer membrane structures, (iii) exosomes have high stability in blood circulation while artificial LNP-based drug particles are more likely be removed by macrophages and other immune cells, (iv) exosomes can cross biological barriers, such as the blood-brain barrier or intestinal barrier, enabling the delivery of preloaded drugs to specific tissues and cells that conventional delivery systems cannot reach.
  • yogurt-derived exosomes leverage the power of the interactions between bacteria and the human host.
  • Bacterial cells communicate with their host and other bacteria through direct contact and secretion of extracellular products, such as metabolites, lipoglycaps, proteins, peptides, and nucleic acids.
  • extracellular vesicles also known as bacterial exosomes, outer membrane vesicles (OMV), or bacterial extracellular vesicles (BEV)
  • OMV outer membrane vesicles
  • BEV bacterial extracellular vesicles
  • yogurt-derived exosomes are more stable in the gastrointestinal tract and particularly suitable for the oral delivery of drugs.
  • exosomes isolated from a fermented dairy product and encapsulating one or more therapeutic agents are provided.
  • the phrase "encapsulated by an exosome,” or grammatical variations thereof is used herein to refer to exosomes whose lipid bilayer surrounds a therapeutic agent.
  • the encapsulation of various therapeutic agents within exosomes can be achieved by mixing one or more therapeutic agents with isolated exosomes as herein described in a suitable solvent, such as ethanol.
  • the exosome/therapeutic agent mixture is then subjected to a low-speed centrifugation (e.g., 10,000 x g) to remove any unbound therapeutic agent and one or more highspeed centrifugation centrifugations to isolate the exosomes encapsulating the therapeutic agents.
  • a low-speed centrifugation e.g., 10,000 x g
  • miRNAs There are three forms of miRNAs existing in vivo, primary miRNAs (pri- miRNAs), premature miRNAs (pre-miRNAs), and mature miRNAs.
  • Primary miRNAs are expressed as stem-loop structured transcripts of about a few hundred bases to over 1 kb.
  • the pri- miRNA transcripts are cleaved in the nucleus by an RNase II endonuclease called Drosha that cleaves both strands of the stem near the base of the stem loop. Drosha cleaves the RNA duplex with staggered cuts, leaving a 5 ! phosphate and 2 nt overhang at the 3* end.
  • the cleavage product, the premature miRNA is about 60 to about 110 nt long with a hairpin structure farmed in a fold-back manner.
  • Pre-miRNA is transported from the nucleus to the cytoplasm by Ran-GTP and Exportin-5.
  • Pre-miRNAs are processed further in the cytoplasm by another RNase II endonuclease called Dicer, Dicer recognizes the 5 * phosphate and 3’ overhang, and cleaves the loop off at the stem-loop junction to form miRNA duplexes.
  • the miRNA duplex binds to the RNA-induced silencing complex (RISC), where the antisense strand is preferentially degraded and the sense strand mature miRNA directs RISC to its target site. It is the mature miRNA that is the biologically active form Of the miRNA and is about 17 to about 25 nt in length.
  • RISC RNA-induced silencing complex
  • the miRNAs encapsulated by the exosomes of the presently-disclosed subject matter are selected from miR-155, which is known to act as a regulator of T- and B-cell maturation and the innate immune response, or miR-223, which is known as a regulator of neutrophil proliferation and activation.
  • an exosome composition as herein described comprises exosomes that encapsulate a messenger RNA (mRNA).
  • the mRNA encodes an antigen useful in the generation of an immune response against the antigen.
  • the mRNA may encode a viral antigen useful in the generation of neutralizing antibodies against a virus (e.g., a COVJD-19 mRNA vaccine).
  • the mRNA may encode a protein useful in generating an antitumor response.
  • an exosome composition as herein described comprises exosomes that encapsulate a therapeutic peptide or protein.
  • therapeutic proteins include catalase.
  • an exosome composition as herein described comprises exosomes that encapsulate a small molecule.
  • therapeutic small molecules include chemotherapeutic drugs such as emeumin, doxorubicin, paclitaxel, antibiotics and antifungal compounds.
  • composition of this invention provides a novel platform for oral drug delivery, a patient-friendly route for the local administration of intestinal therapeutics.
  • oral delivery of drugs has often resulted in low efficiency due to low bioavailability, which is determined by three vital factors called dissolution ⁇ permeability, and solubility. Poor bioavailability of oral delivery may be caused by incomplete absorption, incomplete dissolution of the drug in the gastrointestinal tract, or incomplete absorption resulting from low permeability of the drug through the intestinal epithelia.
  • food, other drugs, and digestive disorders can affect drug absorption and bioavailability.
  • drugs can be incorporated into yogurt- derived exosomes, which considerably contribute to the protection of the drug, increase permeability, and target specific cells or tissues to improve therapeutic efficacy.
  • examples of drugs loaded into yogurt-derived exosomes include chemotherapy drugs, proteins, peptides, small molecules, nucleic acid therapeutics such as mRNAs, circular RNAs, miRNAs, shRNAs, and various other RNA therapeutics agents.
  • yogurt-derived exosomes may be loaded with anti-fungal drugs, such as Amphotericin B.
  • anti-fungal medicines are loaded into exosomes to form exosomal anti-fungal drugs.
  • Fungal infections can be treated with anti-fungal medicines. However, people who have compromised immune systems ate more likely to develop serious fungal infections. These infections are called opportunistic infections. Fungal infections can be life-threatening for people who have compromised immune function which can be caused by conditions such as AIDS, autoimmune diseases like lupus, cancers, organ transplants, or stem cell (bone marrow) transplants.
  • Amphotericin B is one of the most common anti-fungal medicines and has been used for the treatment of serious and potentially life-threatening fungal infections for more than 50 years.
  • Liposomal amphoteric B (AmBisome®; LAmB) is a lipid formulation of amphotericin B that results in reduced toxicity as compared with conventional amphoteric B while retaining the antifungal effect of the active agent. After the amphoteric B is packed into liposomes, LAmB provides a long terminal half-life and retention in tissues, suggesting that single or intermittent dosing regimens are feasible.
  • amphoteric B is loaded into new delivery vehicles exosomes, especially the exosomes isolated from yogurt to Anther reduce toxicity and improve delivery efficiency.
  • a method of treating an inflammatory disorder in a subject need thereof comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising exosomes isolated from a fermented dairy product
  • a pharmaceutical composition comprising exosomes isolated from a fermented dairy product
  • the term "inflammatory disorder” includes diseases or disorders which are caused, at least in part, or exacerbated, by inflammation, which is generally characterized by increased blood flow, edema, activation Of immune cells (e.g., proliferation, cytokine production, or enhanced phagocytosis), heat, redness, swelling, pain and/or loss of function in the affected tissue or organ.
  • the cause of inflammation can be due to physical damage, chemical substances, micro-organisms, tissue necrosis. cancer, or other agents or conditions.
  • Inflammatory disorders include acute inflammatory disorders, chronic inflammatory disorders, and recurrent inflammatory disorders.
  • Acute inflammatory disorders are generally of relatively short duration, and last from about a tew minutes to about one to two days, although they can last several weeks. Characteristics of acute inflammatory disorders include increased blood flow, exudation of fluid and plasma proteins (edema), and emigration of leukocytes, such as neutrophils.
  • Chronic inflammatory disorders generally, are of longer duration, e.g., weeks to months to years or longer, and are associated histologically with the presence of lymphocytes and macrophages and with the proliferation of blood vessels and connective tissue.
  • Recunent inflammatory disorders include disorders which recur after a period of time or which have periodic episodes. Some inflammatory disorders fall within one or more categories. Exemplary inflammatory disorders include, but are not limited to atherosclerosis; arthritis; inflammation-promoted cancers; asthma; autoimmune uveitis; adaptive immune response; dermatitis; multiple sclerosis; diabetic complications; osteoporosis; Alzheimer's disease; cerebral malaria; hemorrhagic fever; autoimmune disorders; and inflammatory bowel disease.
  • exosomes are isolated from a fermented dairy product and used directly for the treatment of an inflammatory disorder.
  • isolated exosomes are encapsulated with one or more therapeutic agents useful in the treatment of an inflammatory disorder (in other words, the exosomes are Used as a drug delivery vehicle).
  • therapeutic agents include anti-inflammatory cytokines such as IL- 10 and IL-2, microRNAs, and steroids such as dexamethasone. See also, Suh et at, Int. J. Mol. Sci. 22(3):I144 (2021 ), doi; 10.3390/ijms22031144, the entire contents of which are incorporated herein by reference.
  • exosomes are isolated from a fermented dairy product and used directly for the treatment of cancer.
  • isolated exosomes are encapsulated with one or more therapeutic agents useful in the treatment of cancer.
  • the cancer is selected from the group consisting of breast cancer, uterine cancer, lung cancer, prostate cancer, ovarian cancer, cervical cancer, and pancreatic cancer.
  • the therapeutic agent is a chemotherapeutic agent or microRNA. See also, Salazar et al., Molecules, 28(9):3816 (2023), doi: 10.3390/molecules28093816, the entire contents of which are incorporated herein by reference.
  • cancer refers to all types of cancer or neoplasm or malignant tumors found in animals, including leukemias, carcinomas, melanoma, and sarcomas.
  • leukemia is meant broadly progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow, including, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, and adult T-cell leukemia.
  • carcinoma refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.
  • exemplary carcinomas include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamuus cell carcinoma, bronchioalveolar Carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebrifoim carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephalpid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic
  • sarcoma generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance.
  • Sarcomas include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft pan sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wiln$* tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma,
  • melanoma is taken to mean a tumor arising from the melanocytic system of the skin and other organs.
  • Melanomas include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma subungal melanoma, and superficial spreading melanoma.
  • Additional cancers include, for example, Hodgkin’s Disease, Mon-Hodgkin's
  • Lymphoma multiple myeloma, neuroblastoma, breast ameer, ovarian Cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, and adrenal cortical cancer.
  • the cancer is selected from the group consisting of breast cancer, uterine cancer, lung cancer, prostate cancer, ovarian cancer, cervical cancer, and pancreatic cancer.
  • Methods for isolation arid purification of exosomes from yogurt 'Che provides a novel method for the effective isolation and purification of exosomes from yogurt.
  • procedures typically consist of the following steps, including cultivation of the cells in a liquid medium, removal of cells and debris by low-speed centrifugation, concentration of the exosomes by polymer-based precipitation or ultrafiltration, and purification of the exosomes by ultracentrifugation or size-exclusion chromatography. See e.g., Yang ct al., Theranoslics, 10(8): 3684-3707 (2020); U.S.
  • a novel isolation method is described herein that overcomes these disadvantages and provides a simple and highly effective method for the isolation and purification of exosomes from yogurt as disclosed herein.
  • the methods for the isolation of exosomes provided herein are simple, fast, robust, and easily scalable to large volumes.
  • the method comprises at least one centrifugation step, at least one microfiltration step, and at least one precipitation step.
  • a method of preparing exosomes isolated from a fermented dairy product comprising: (I) optionally centrifuging a fermented daily product to form a first supernatant; (2) microfiltering the first supernatant to remove residual debris; (3) incubating the microfiltrate with a precipitation agent to form a mixture; and (4) centrifuging the mixture to form a precipitate comprising exosomes.
  • step (1 ) should be performed after the yogurt curd is mixed with a Sterile carrier (such as PBS) and homologized.
  • the initial centrifugation step comprises differential centrifugation of a homologized fermented milk product sample at about 15,000 x g to about 20,000 x g, preferably about 17,700 x g, for about 30 minutes to about 90 minutes, preferably for about 1 hour, to obtain a first supernatant.
  • the obtained first supernatant is filtered through a filter of about 0.1 pm to about 0.7 pm, preferably about 0.45 pm, to remove debris and large particles.
  • Exosomes may be precipitated from the filtration passthrough by any method known in the art, such as those described in U.S. Application Publication Nos. 2013/0337440 and 2013/0273544, foe entire contents of each of which are incorporated herein by reference.
  • exosomes are precipitated from the filtration passthrough with a water-excluding polymer such as polyethylene glycol (PEG) having a molecular weight from 200 to 1 ,000,000 Da, speh: as PEG6.000 or PEG8,000.
  • PEG polyethylene glycol
  • ExoPrepTM reagent commercially available from Exom Biopharma Inc. (Catalog No. El 001) is used.
  • exosomes are precipitated by overnight incubation with PEG at the temperature of about 2°C to about 7°C.
  • the precipitated exosomes are isolated using one or more centrifugation steps.
  • the precipitated exosomes are isolated as the pellet following centrifugation at about 5,000 x g to about 15,000 x g, preferably about 10,000 x g for about 15 minutes to about 60 minutes, preferably about 30 minutes, which may be followed by an additional centrifugation step io remove any remaining liquid, at about 5,000 x g to about 15,000 x g, preferably about 10,000 x g for about 5 to about 15 minutes, preferably about 10 minutes.
  • the method comprises the following steps: (1 ) mixing yogurt with a sterile liquid carrier (e.g., PBS) to create a homologized mixture (e.g., 1 volume of yogurt may be mixed with 0.5 volume of 1 x DPBS buffer and shaken to create a homologized mixture); (2) centrifuging the mixture, at about 17,700 x g for I hour to generate a supernatant (The exosomes are in the supernatant while all insoluble materials are in the pellet); (3) filtering the supernatant through a 0.45 pm filter to remove all debris and large particles; (4) precipitating exosomes by adding a precipitation agent to foe filtrate to generate a mixture comprising precipitated exosomes (e,g., by adding 0.5 volume of ExoPrepTM reagent to the supernatant) and incubating the mixture at 4°C overnight; (5) centrifuging foe mixture at about 10,000 x g for about 30 min to generate a pellet comprising isolated ex
  • foe method further comprises an additional centrifugation step at about 10,000 x g for about 10 min to spin down all liquid residue to foe bottom.
  • the exosomes can be retrieved by removing all residual liquid without disturbing the pellet and resuspending the exoSomes to a select Volume of sterile liquid carrier (e.g., DPBS).
  • DPBS sterile liquid carrier
  • the exosomes can be stored at -80°C for long-term storage until use.
  • the exosomes can be lyophilized and stored at room temperature requiring only foe addition of a sterile liquid carrier immediately prior to use.
  • Several methods can be used to characterize exosomes during the isolation process, including flow cytometry, nanoparticle tracking analysis, dynamic light scattering, western blot, mass spectrometry, and microscopy techniques. Exosomes can also be Characterized and marked based on their protein compositions, with intergrins and tetraspanins being the two most abundant proteins found in exosomes; Other protein markers include I'SOl 01 , ALG-2 interacting protein X (ALIX), flotillin 1, and cell adhesion molecules. Similar to proteins, lipids are major components of exosomes and can be utilized to characterize them.
  • the yield of exosomes may be determined as a ratio of the particle number to the input sample (per gram or mL).
  • the working examples below demonstrate that yields of at least 1x10 11 , at least 1 ,5x10 11 , at least 2.0x10 11 , at least 2.5x 10 11 , at least 3.0x10 11 , at least 3.2x10 11 or at least 3.3x10 11 parlicles/gram of input sample are obtainable from fermented dairy products, preferably from yogurt ctirds.
  • exosomes isolated from milk have relatively low yield as low ratios of 4.25x10 10 to 4.50x10 10 particles/gram of input sample.
  • the purity of exosomes may be determined as the ratio of the particle number to protein concentration. A high ratio indicates high purity of exosomes while a low ratio indicates low purity with nonexosomal protein contaminations.
  • the working examples below illustrate high purity of exosomes isolated from yogurt of 3.64x10 9 particles/pg protein to 4.28x10 9 partici es/pg protein. In comparison, exosomes isolated from milk have relatively low purity pf 3.58x10 8 to 4.11x10 8 particles/ pg protein.
  • compositions comprising exosomes from yogurt.
  • a pharmaceutical composition as described herein comprises exosomcs isolated from a fermented dairy product arid a pharmaceutical carrier such as aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, bactericidal antibiotics, and solutes that render the formulation isotonic with the bodily fluids of the intended recipient; arid aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents.
  • compositions used can take such forms as Suspensions, solutions, or emulsions in oily of aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.
  • tlie formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and can be stored in a frozen or freeze-dried or room temperature (lyophilized) condition requiring only the addition of sterile liquid carrier immediately prior to use.
  • Suitable methods for administering a therapeutic composition in accordance with the methods of the presently-disclosed subject matter include, but are not limited to, systemic administration, parenteral administration (including intravascular, intramuscular, and/or intraarterial administration), oral delivery, buccal delivery, rectal delivery, subcutaneous administration, intraperitoneal administration, inhalation, dermally (e.g., topical application), intratracheal installation, surgical implantation, transdermal delivery, local injection, intranasal delivery, and hyper-velocity injection/bombardment Where applicable, continuous infusion can enhance drug accumulation at a target site (see, e.g,, U.S. Patent No. 6,180,082).
  • the therapeutic compositions are administered orally, intravenously, intranasally, or intraperitoneally to thereby treat a disease or disorder.
  • the therapeutic compositions are administered orally.
  • compositions of the presently-disclosed subject matter typically not only include an effective amount of a therapeutic agent, but are typically administered in an amount effective to achieve the desired respotise.
  • the term "effective amount” is used herein to refer to an amount of the therapeutic composition (e.g., an exosome encapsulating a therapeutic agent, and a pharmaceutical vehicle, carrier, or excipient) sufficient to produce a measurable biological response (e.g., a decrease in inflammation).
  • a measurable biological response e.g., a decrease in inflammation.
  • Actual dosage levels of active ingredients in a therapeutic composition of the present invention can be varied so as to administer an amount of the active compound ⁇ ) that is effective to achieve the desired therapeutic response for a particular subject and/or application.
  • the effective amount in any particular case will depend upon a variety of factors including the activity of the therapeutic composition, formulation, the route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated.
  • a minimal dose is administered, and the dose is escalated in the absence Of dose-limiting toxicity to a minimally effective amount.
  • Exosomes were isolated from four types of samples, fresh milk, yogurt whey (clear liquid), yogurt curd (solid portion), and kefir. Ihese samples were from different vendors. A total of 11 samples were processed (Table 1 ). The methods for isolation and purification of exosomes from these samples are described below.
  • the pellet containing exosomes was resuspended in 2.5 mL of PBS buffer.
  • the exosome resuspension was centrifuged at 10,000 xg for 10 min.
  • the exosomes were present in the supernatant while all indissoluble substances were in the pellet and discarded.
  • the supernatant containing exosomes was transferred to a new tube and used for downstream analyses or stored at ⁇ 80°C for long-term storage.
  • the passthrough was mixed with 0.5 volume of ExptepTM total exosome isolation reagent and incubated at 4°C overnight. After incubation, the mixture was centrifuged at 10,000 xg for 30 min. The supernatant was aspirated and discarded. The tube containing the pellet was centrifuged again at 10,000xg for 10 min. All leftover liquid residue is removed. The pellet containing exosomes was resuspended in 0.5 mL of PBS buffer. The resuspension was centrifuged at 10,000 xg for 10 min to pellet all indissoluble substances. The supernatant containing exosomes was used for downstream analysis or stored at -80°C for long-term storage.
  • the tube containing the pellet was centrifuged again at 10,000*g for 10 minutes. All leftover liquid residue is removed.
  • the pellet containing exosomes was resuspended in 4 mL of PBS buffer. The resuspension was centrifuged at 10,000 xg for 10 minutes to remove all indissoluble substances. The supernatant containing exosomes was used for downstream analysts or stored at -80°C for long-term storage.
  • Kefir sample The method for isolating exosomes from the Kefir sample was the same as that for the yogurt curd sample. The only difference was that the input raw materials required were reduced. 10 mL of kefir sample was processed, and the final exosomes were resuspended in 0.8 mL of PBS.
  • Example 2 Nano-tracking analysis of exosomes derived from yogurt and fresh milk.
  • NanO-tracking analysis was performed for the evaluation of the exosomes isolated from 11 samples using the ZetaView PMX 130Z instrument (Particle Metrix). The particle sizes and concentrations were determined for each purified exosome sample. To compare the y ield of exosomes, the particle concentration was normalized to the input materials as a ratio of particle numbers versus one milliliter of fresh milk, yogurt whey, and kefir samples, or gram of yogurt curd.
  • Figure 1 shows the yield of exosome particles for the 11 samples. The highest yield of exosomes was obtained from the yogurt curd (Q5-S2), while the lowest yield of exosomes was the yogurt whey (Q5-S1).
  • the yield of exosomes is determined as a ratio of the particle number to the input sample (per gram or mL).
  • the exosomes isolated from yogurt curds have higher yield as higher ratios of 3.4E+ 11 particles per gram of input sample (sample Q5-S2) and 1.4E+11 particles per gram of input sample (sample Q5-S3), while the exosomes isolated from the milk have relatively low yield as low ratios of 4.25E-H0 particles per gram of input sample (sample M 1 ) and 4.50E+10 particles per gram of input sample (sample Q5-M2), respectively.
  • yogurt curd samples produced a much high concentration of exosomes compared to the fresh milk samples (3-8 times higher, average > 5 times).
  • the particle sizes are in the range of 91.3 nm to 138 nm ( Figure 2).
  • exosome particles isolated from yogurt samples have relatively smaller sizes (96-103 nm) than those from the milk samples (108-125 nm).
  • the exosome particles from all kefir samples were larger (114- 138 nm) except for one sample (KI) that has smaller sizes at 913 nm.
  • the size distribution of three Q5 exosomes based on NTA measurements is shown in Figure 3.
  • Example s Comparison of purity of the exosomes isolated from the yogurt and milk samples.
  • the ratio of particle numbers to protein concentrations is often used as a Criterion for evaluating exosome purity.
  • the protein concentration of the exosomes isolated from two milk samples and three yogurt samples was determined by standard BCA assay using Micro BCATM Protein Assay Kit (Thermo Scientific).
  • the ratio of particle concentration (particle numbers/mL) to protein concentration (pg/mL) was calculated for each sample and shown in Figure 4.
  • the ratios of the exosome preparations from yogurt curds were determined 3.64E+9 (Q5-S2) and 4.28E+9 (Q5-S3) particles per pg of protein, indicating the high purity of yogurt-derived exosomes.
  • the ratios of the exosome preparations from fresh milk were determined 4.11E+8 (Ml) and 3.58E+8 (M2) particles per pg of protein, indicating relatively low purity of milk- derived exosomes because of more protein contamination in the exosome products.
  • Example 4 Comparison of stability of the exosomes isolated from the yogurt and milk samples.
  • ZetaView PMX 130Z instrument ( Figure 5).
  • Zeta potential is the charge that develops at the interface between an exosome surface and its liquid medium.
  • Hie zeta potential value indicates foe potential stability of the exosomes in the solution. If all the exosomes in suspensi on have a large negative zeta potential, then they will tend to repel each other electrostatically and there will be no tendency to form aggregates.
  • the results show that the exosomes isolated from the yogurt curd samples have larger negative zeta potential values as -29.38 ⁇ 0.75 mV (Q5-S2) and - 38.42 ⁇ 0.75mV (Q5-S3), indicating the exosomes are more stable in solution.
  • Example 5 SDS-PAGE and Western blot analyses of the exosomes isolated from the yogurt and milk samples.
  • the original Ml milk sample shows two heavy bands of casein and whey proteins that constitute the majority of the total protein in the milk. Most proteins with larger molecular weights were cleaned up in Ml exosomes (Lane 3) with small amounts of leftover casein and whey proteins.
  • the M2 exosomes show different protein patterns because the milk input was from different host species.
  • the Q5-S2 (Lane 6) and Q5-S3 exosomes (Lane 7) from the yogurt curds have similar patterns but differ from the Q5-S1 exosomes isolated from yogurt whey.
  • the protein patterns of the exosomes isolated from the human breast cancer MCF-7 cell line (Lane 8) are very different from the exosomes derived from milk and yogurt.
  • FIG. 7 A shows a comparison of protein patterns of exosomes isolated from yogurt and milk. A major band of about 30 kDa was observed in all samples of Q5-exosomes.
  • Figure 7B shows the results of Western blot analysis. Western blot analysis was performed using an anti-human CD9 antibody (Antibodies.com, clone MM2/57). As expected, the anti-human CD9 antibody bound to the exosomes from human MCF-7 cells (positive control).
  • the antibody had no reactivity with Q5- derived exosomes, suggesting that Q5-derived exosomes are a new type of exosomes, arid the tetraspanins markers are different between human and Q5 exosomes.
  • a cross-reactivity with an anti-human CD9 antibody was observed for M2 but not Ml .
  • MCF7 human breast cancer cells MCF7 human breast cancer cells.
  • EMEM media, Fetal bovine serum, L-glutamine, penicillin-streptomycin sulphate (pen-strep), tiypsin-EDTA solution and FBS were obtained from ATCC, PKH26 and DAPI stains were obtained from Millipore-Sigma.
  • FBS depleted with exosome were purchased from Thermo-Fisher.
  • MCF-7 cells were cultured in Eagle’s Minimum Essential Medium (EMEM) with L glutamine, pen-strep, and FBS for several hours till the cells became confluent. Cells were removed by gentle treatment with trypsin-EDTA and centrifuged at 2,000xg for 10 minutes to remove the media. The cells were resuspended in fresh EMEM media with exosome depleted FBS and cultured for 24 hours.
  • EMEM Eagle’s Minimum Essential Medium
  • FBS FBS
  • Q5 exosome prepared according to the methods described herein were labelled with PKH26 (red) using the PKH26 red cell linker kit (Millipore-Sigma), according to the manufacturer’s instructions. Briefly, Q5 EV were resuspended in PBS and 500 pl of Diluent C was added. In another reaction tube 500 ⁇ l of Diluent C was mixed with 2 pl of PKH26 dye ( 1 mM) and incubated for 10 minutes. The reaction was stopped by adding EMEM cultured media with depleted exosomes. The unbound dye was removed for centrifugation using LOOkd amicon membrane filter (Millipore Sigma).
  • PKH26 labelled Q5 exosomes were incubated with MCF-7 cells for 60 minutes and washed with PBS to remove excess exosomes, fixed in 2% paraformaldehyde before further analysis by fluorescent microscope.
  • Q5 exosomes were mixed with nuclear stain DAPI dye and incubated for an hour before unincorporated dye using amicon lOOkd membrane filter.
  • the DAPI labelled exosomes were mixed with MCF-7 cells for 60 minutes and later cells were washed with PBS to remove the excess exosomes, fixed in 2% paraformaldehyde before further analysis by fluorescent microscope.
  • Figs. 9A-B The data as shown in Figs. 9A-B indicates that Q5 exosomes was taken up by the cells and migrated inside the cytoplasm (PKH26; Fig. 9B) and nucleus (DAPI; Fig. 9A). This confirms that exosomes prepared according to the present methods are useful for drug delivery (e.g., anti-cancer agents such as siRNA) as well as nutritional supplements.
  • drug delivery e.g., anti-cancer agents such as siRNA

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Abstract

Sont divulgués des procédés et des utilités pour l'isolement de nouveaux exosomes à partir de micro-organismes présents dans le yaourt et les probiotiques. L'invention propose également un procédé pour que ces exosomes encapsulent des médicaments, des acides nucléiques ou des peptides pour une administration à des sites de maladie. Ces exosomes peuvent aussi être utilisés directement sur des sites de maladie pour leur activité antifongique et anti-inflammatoire intrinsèque.
PCT/US2024/038214 2023-07-17 2024-07-16 Exosomes dérivés de produits laitiers fermentés Pending WO2025019495A2 (fr)

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