JP2023554316A - Formulation for non-parental administration of exosomes - Google Patents

Abstract

Described herein are compositions for oral delivery of mammalian exosomes, the compositions comprising a plurality of mammalian exosomes mixed with a matrix and surrounded by an enteric coating, the matrix comprising about 0 .5% to about 3.0% methylcellulose and about 0.5% to about 3.0% alginate gel. Further described herein are compositions for rectal or vaginal delivery of mammalian exosomes, the compositions comprising a lipophilic or hydrophilic coating surrounding a plurality of mammalian exosomes mixed with a solid matrix. The matrix includes about 0.5% to about 3.0% methylcellulose and about 0.5% to about 3.0% alginate gel. [Selection diagram] Figure 1

Description

CROSS-REFERENCE This application claims the benefit of previously filed U.S. Provisional Patent Application No. 63/124,312, filed December 11, 2020, which is incorporated herein by reference in its entirety.

Described herein are formulations and compositions useful for non-parental administration of exosomes.

In one aspect, a composition for oral delivery of mammalian exosomes is described herein, the composition comprising a plurality of mammalian exosomes mixed with a matrix and surrounded by an enteric coating, the matrix comprising: The matrix includes about 0.1% to about 5.0% methylcellulose and about 0.1% to about 5.0% alginate gel. In certain embodiments, the matrix includes about 0.5% to about 1.0% methylcellulose and about 0.5% to about 1.0% alginate gel. In certain embodiments, the matrix includes about 0.25% to about 0.75% methylcellulose and about 0.25% to about 0.75% alginate gel. In certain embodiments, the matrix includes about 0.5% to about 3.0% methylcellulose and about 0.5% to about 3.0% alginate gel. In certain embodiments, the matrix includes about 1.0% to about 2.0% methylcellulose and about 1.0% to about 2.0% alginate gel. In certain embodiments, the matrix includes about 1.3% to about 1.8% methylcellulose and about 1.3% to about 1.8% alginate gel. In certain embodiments, the matrix includes about 1.5% methylcellulose and about 1.5% alginate gel. In certain embodiments, the matrix comprises a ratio of methylcellulose to alginate gel of about 1:1. In certain embodiments, the exosome is a mesenchymal stem cell exosome or a hematopoietic stem cell exosome. In certain embodiments, the exosome is a hematopoietic stem cell exosome. In certain embodiments, the exosomes are mesenchymal stem cell exosomes. In certain embodiments, the mesenchymal stem cells are type I mesenchymal stem cells. In certain embodiments, the mesenchymal stem cells are type II mesenchymal stem cells. In certain embodiments, the exosome comprises a protein or nucleic acid selected from the list consisting of CD81, CD63, CD9, HSP60, HSP70, HSP90, VEGF, PGE2, and combinations thereof. In certain embodiments, the exosome comprises a nucleic acid selected from the list consisting of miRNA Let7, 155, 146, 122, and combinations thereof. In certain embodiments, mammalian exosomes have a diameter of about 30 nanometers to about 150 nanometers. In certain embodiments, the mammalian exosome is a human exosome. In certain embodiments, the mammalian exosome is a canine, feline, bovine, equine, ovine, or porcine exosome. In certain embodiments, the enteric coating comprises methyl methacrylate-methacrylic acid copolymer (1:1). In certain embodiments, the enteric coating consists essentially of methyl methacrylate-methacrylic acid copolymer (1:1). In certain embodiments, the composition comprises at least 100 billion exosomes. In certain embodiments, the composition comprises at least 100 billion to no more than 2 trillion exosomes.

In yet other aspects, compositions for non-parental and/or rectal or vaginal delivery of mammalian exosomes are described herein, comprising a plurality of mammalian exosomes mixed with a solid matrix. The matrix includes a lipophilic or hydrophilic coating surrounding the exosomes, the matrix comprising about 0.5% to about 3.0% methylcellulose and about 0.5% to about 3.0% alginate gel. . In certain embodiments, the matrix comprises a matrix comprising about 1.0% to about 2.0% methylcellulose and about 1.0% to about 2.0% alginate gel. In certain embodiments, the matrix includes about 1.3% to about 1.8% methylcellulose and about 1.3% to about 1.8% alginate gel. In certain embodiments, the matrix includes about 1.5% methylcellulose and about 1.5% alginate gel. In certain embodiments, the matrix comprises a ratio of methylcellulose to alginate gel of about 1:1. In certain embodiments, the exosome is a mesenchymal stem cell exosome or a hematopoietic stem cell exosome. In certain embodiments, the exosome is a hematopoietic stem cell exosome. In certain embodiments, the exosomes are mesenchymal stem cell exosomes. In certain embodiments, the mesenchymal stem cells are type I mesenchymal stem cells. In certain embodiments, the mesenchymal stem cells are type II mesenchymal stem cells. In certain embodiments, the exosome comprises a protein or nucleic acid selected from the list consisting of CD81, CD63, CD9, HSP60, HSP70, HSP90, VEGF, PGE2, and combinations thereof. In certain embodiments, the exosome comprises a nucleic acid selected from the list consisting of miRNA Let7, 155, 146, 122, and combinations thereof. In certain embodiments, mammalian exosomes have a diameter of about 30 nanometers to about 150 nanometers. In certain embodiments, the mammalian exosome is a human exosome. In certain embodiments, the mammalian exosome is a canine, feline, bovine, equine, ovine, or porcine exosome. In certain embodiments, the composition is formulated for rectal delivery. In certain embodiments, the composition is formulated for vaginal delivery. In certain embodiments, the formulation does not swell upon contact with an aqueous medium. In certain embodiments, the lipophilic or hydrophilic coating melts at normal body temperature or above 36°C. In certain embodiments, the lipophilic or hydrophilic coating consists essentially of cocoa butter, coconut oil, glycerinated gelatin, hydrogenated oils, polyethylene glycol (PEG), fatty acid esters of PEG, or a Gattefosse lipophilic base. Become. In certain embodiments, the lipophilic or hydrophilic coating comprises cocoa butter, coconut oil, glycerinated gelatin, hydrogenated oils, polyethylene glycol (PEG), fatty acid esters of PEG, or Gattefosse lipophilic bases. In certain embodiments, the enteric coating consists essentially of methyl methacrylate-methacrylic acid copolymer (1:1). In certain embodiments, the composition comprises at least 100 billion exosomes. In certain embodiments, the composition comprises at least 100 billion to no more than 2 trillion exosomes. In certain embodiments, 40% or more of intact exosomes are recovered from the composition after 2 hours of incubation at pH 1.2 compared to the initial amount. In certain embodiments, 50% or more intact exosomes are recovered from the composition after 2 hours of incubation at pH 1.2 compared to the initial amount. In certain embodiments, 60% or more intact exosomes are recovered from the composition after 2 hours of incubation at pH 1.2 compared to the initial amount.

In one aspect, the compositions described herein are useful for treating inflammatory disorders in an individual, including administering the composition non-parenterally, orally, vaginally, or rectally to the individual. May be used in methods of treating immune disorders. In certain embodiments, the individual is a human individual. In certain embodiments, the individual is a dog, cow, cat, pig, horse, or sheep. In certain embodiments, the inflammatory or autoimmune disorder is a gastrointestinal inflammatory or autoimmune disorder. In certain embodiments, the gastrointestinal inflammatory disorder or autoimmune disorder comprises inflammatory bowel disease or Crohn's disease. In certain embodiments, the gastrointestinal inflammatory disorder or autoimmune disorder comprises Crohn's disease. Additionally, described herein is a method of reducing neutrophil accumulation or neutrophil activation in the gastrointestinal tract of an individual, which method comprises administering to the individual a composition described herein. including doing.

The novel features described herein are pointed out with particularity in the appended claims. For the features described herein and the advantages of those features, please refer to the following detailed description, which describes illustrative embodiments utilizing the principles of each feature described herein, and the accompanying drawings. It will be better understood by doing so.

A representative diagram of an oral exosome delivery capsule is shown. In the top single-capsule single-phase delivery illustration, therapeutic mammalian exosomes can be appropriately sourced and manufactured, added or mixed into a matrix, packaged into capsules, and finally enteric coated. In another embodiment, the therapeutic mammalian exosomes may be manufactured and added into the matrix or mixed together before being packaged into a single capsule. Subsequently, a second dose of exosomes can be mixed into the matrix and packaged into capsules, which in turn combine with the first dose, as shown in the capsule-in-capsule biphasic delivery diagram below. can be placed within a capsule. Figure 3 shows a representative, non-limiting diagram of oral delivery of exosome-loaded capsules and intended deployment to the ileum and colon. In single-capsule monophasic delivery (left) or capsule-in-capsule biphasic delivery (right), therapeutic mammalian exosomes are ingested orally and pass through the mouth, esophagus, and duodenum. As shown, the enteric protective coating disappears and the exosomes are deployed in the targeted ileum (pH 7.3-8) followed by the colon (pH 5.5-6.5). In. Representative illustrations of transrectal or transvaginal exosome delivery capsules are shown. In the top single-capsule single-phase delivery diagram, therapeutic mammalian exosomes can be manufactured, added or mixed into a matrix, packaged into capsules, and finally enteric coated. In another embodiment, therapeutic mammalian exosomes can be suitably sourced, manufactured, added into a matrix, or mixed as before being packaged into a single capsule. Subsequently, a second dose of exosomes can be mixed into the matrix and packaged into capsules, which in turn combine with the first dose, as shown in the capsule-in-capsule biphasic delivery diagram below. can be placed within a capsule. Figure 3 shows a representative, non-limiting diagram of rectal or vaginal delivery of exosome-loaded capsules and intended deployment to the ileum and colon. In single-capsule monophasic delivery (left) or capsule-in-capsule biphasic delivery (right), therapeutic mammalian exosomes are ingested orally and pass through the mouth, esophagus, and duodenum. As shown, the hydrophilic or lipophilic protective coating disappears and the exosomes are deployed to the targeted vagina (pH 3.8-4.7) or rectum (pH 5.5-4.7). 8.0) and subsequently in the colon (pH 5.5-6.5). 2 shows a manufacturing scheme for the delivery system described herein. Figure 3 shows that exosome particles are released from oral formulation capsules after reaching neutral pH. Exosomes were derived from adult human mesenchymal stem cells and were added to enteric-coated oral formulations and incubated at the indicated pH. Figure 6A shows a heatmap of the data. Figure 6B shows the number of exosomes expressing CD63, CD9, or CD81 recovered from incubation at each pH. Figure 6C shows immunofluorescence microscopy images showing that intact exosomes were recovered. We show that exosome particles are released from orally formulated capsules only after incubation in simulated gastric fluid or simulated intestinal fluid. FIG. 7A shows a heat map of the data. FIG. 7B shows the number of exosomes expressing CD63, CD9, or CD81 recovered from simulated gastric fluid (SIG) or simulated intestinal fluid (SIF). Figure 7C shows immunofluorescence microscopy images showing that intact exosomes were recovered. We show that oral exosome therapy alleviates IBD colitis in a mouse model. Figure 8A shows the conditions of the experiment. Figure 8B shows the scoring of the in vivo model. This shows that the recovered exosomes retain anti-inflammatory ability.

Described herein are compositions for non-parenteral delivery of mammalian exosomes, the compositions comprising a plurality of mammalian exosomes mixed with a matrix, the matrix comprising an alginate gel. In certain embodiments, the composition is for oral delivery. In certain embodiments, the composition releases mammalian exosomes at a pH of about 6.4, 6.6, 6.8, or 7.0 or higher.

Described herein are compositions for non-parenteral delivery of mammalian exosomes, the compositions comprising a plurality of mammalian exosomes mixed with a matrix, the matrix comprising methylcellulose. In certain embodiments, the composition is for oral delivery. In certain embodiments, the composition releases mammalian exosomes at a pH of about 6.4, 6.6, 6.8, or 7.0 or greater.

Described herein are compositions for non-parenteral delivery of mammalian exosomes, the compositions comprising a plurality of mammalian exosomes mixed with a matrix and surrounded by an enteric coating, the matrix being , containing alginate gel. In certain embodiments, the composition is for oral delivery. In certain embodiments, the composition releases mammalian exosomes at a pH of about 6.4, 6.6, 6.8, or 7.0 or higher.

Described herein are compositions for non-parenteral delivery of mammalian exosomes, the compositions comprising a plurality of mammalian exosomes mixed with a matrix and surrounded by an enteric coating, the matrix being , including methylcellulose. In certain embodiments, the composition is for oral delivery. In certain embodiments, the composition releases mammalian exosomes at a pH of about 6.4, 6.6, 6.8, or 7.0 or higher.

Described herein are compositions for non-parenteral delivery of mammalian exosomes, the compositions comprising a plurality of mammalian exosomes mixed with a matrix and surrounded by an enteric coating, the matrix being , containing a matrix containing methylcellulose and alginate gel. In certain embodiments, the composition is for oral delivery. In certain embodiments, the composition releases mammalian exosomes at a pH of about 6.4, 6.6, 6.8, or 7.0 or higher.

Described herein is a composition for oral delivery of mammalian exosomes, the composition comprising a plurality of mammalian exosomes mixed with a matrix and surrounded by an enteric coating, the matrix comprising about 0 .1% to about 5.0% methyl cellulose and about 0.1% to about 5.0% alginate gel.

In the following description, certain specific details are set forth to provide a thorough understanding of the various embodiments. However, one of ordinary skill in the art will understand that the embodiments provided may be practiced without these details. Unless the context otherwise requires, throughout the following specification and claims, the word "comprise" and its variations, such as "comprises" and "comprising", are used as the open term. should be construed in an inclusive sense, ie, "including, but not limited to." As used in this specification and the appended claims, the singular forms "a," "an," and "the" refer to plural referents unless the content clearly dictates otherwise. is included. It should also be noted that the term "or" is generally used to include "and/or" unless the context clearly states otherwise. . Furthermore, the headings provided herein are for convenience only and are not intended for interpretation of the scope or meaning of the claimed embodiments.

In some cases, the compositions described herein "consist essentially of" the specified ingredients, which includes other active ingredients and, to the exclusion of the specified ingredients, the composition Other diluents, carriers, and excipients that do not alter the essential properties of the drug (which are often included to contribute to a measure of dosability or stability) may be included.

"Non-parental" as described herein refers to any route of administration that is enteral, including via the oral, rectal, or nasal routes. Non-parental as described herein also refers to vaginal administration.

The term "about" as used herein refers to an amount that is within 10% of the stated amount.

As used herein, the term "individual," "patient," or "subject" refers to an individual who has been diagnosed with, is suspected of having, or is at risk of developing at least one disease. refers to individuals for whom the described compositions and methods are useful in treating diseases. In certain embodiments, the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. In certain embodiments, the individual is a human.

The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues, and there is no minimum length limitation. The provided antibodies and antibody chains, as well as polypeptides, including other peptides such as linkers and binding peptides, may contain amino acid residues, including natural and/or non-natural amino acid residues. The term also includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In some embodiments, a polypeptide may include modifications to the native or natural sequence so long as the protein maintains the desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or accidental, such as mutations in the host producing the protein or errors due to PCR amplification. good.

"Hematopoietic stem cells" and grammatical equivalents as described herein include red blood cells; platelets; lymphoid cells such as T cells and B cells; monocytes, macrophages, eosinophils, basophils, mast cells, and stem cells that give rise to other blood cells, including myeloid cells such as neutrophils. Hematopoietic stem cells according to the present disclosure are cells that can give rise to at least lymphoid cells and myeloid cells. Common surface markers for hematopoietic stem cells include CD34. Hematopoietic stem cells may also contain surface markers such as EPCR and CD90. Hematopoietic stem cells can be identified by any one or more of CD34, EPCR, and CD90. Such surface positivity can be identified, for example, by immunohistochemistry or flow cytometry. Hematopoietic stem cells can be conveniently isolated from bone marrow, umbilical cord blood, and peripheral blood. Hematopoietic stem cells can be cultured using methods known in the art. For example, Frisch et al. "Hematopoietic Stem Cell Cultures and Assays.", Methods Mol Biol. 2014;1130:315-324.

"Mesenchymal stem cells" and grammatical equivalents as described herein refer to stem cells that are mesenchymal in origin. This term refers to cells capable of differentiating into at least two of the following types: osteoblasts, chondrocytes, adipocytes, or myocytes. MSCs may be isolated from any type of adult tissue. MSCs are typically isolated from bone marrow, adipose tissue, umbilical cord, or peripheral blood. Waterman et al. “A New Mesenchymal Stem Cell (MSC) Paradigm: Polarization into a Pro-Inflammatory MSC1 or an Immunosuppressive MSC2 Phenotype .”, PLoS One. 2010;5(4):e10088, MSCs can be divided into pro-inflammatory MSC type I and anti-inflammatory MSC type II. Methods for deriving various MSC types are described, for example, in US Patent Application Publication No. 20140017787 (A1) and US Patent Application Publication No. 20160097038 (A1).

“Exosome” (abbreviated EV), as described herein, and grammatical equivalents refer to membrane-bound extracellular vesicles produced by many eukaryotic cells. These vesicles may contain proteins and nucleic acids such as DNA, RNA, mRNA, miRNA. Exosomes can be produced from cultured cells in a variety of ways. Such methods include, for example, centrifugation techniques and commercially available kits. Lane et al. "Analysis of exosome purification methods using a model liposome system and tunable-resistive pulse sensing.", Scientific Rep orts volume 5, Article number: 7639 (2015), Li et al. "Progress in exosome isolation techniques.", Theranostics. 2017;7(3):789-804, WO2019035880(A1), and WO2019231562(A1).

In certain embodiments, EVs according to the present disclosure have a diameter of about 25 nm to about 150 nm. In certain embodiments, the EV according to the present disclosure is about 25 nm to about 50 nm, about 25 nm to about 75 nm, about 25 nm to about 100 nm, about 25 nm to about 125 nm, about 25 nm to about 150 nm, about 50 nm to about 75 nm, about 50 nm. ~ about 100 nm, about 50 nm - about 125 nm, about 50 nm - about 150 nm, about 75 nm - about 100 nm, about 75 nm - about 125 nm, about 75 nm - about 150 nm, about 100 nm - about 125 nm, about 100 nm - about 150 nm, or about 125 nm - It has a diameter of approximately 150 nm. In certain embodiments, EVs according to the present disclosure have a diameter of about 25 nm, about 50 nm, about 75 nm, about 100 nm, about 125 nm, or about 150 nm. In certain embodiments, EVs according to the present disclosure have a diameter of at least about 25 nm, about 50 nm, about 75 nm, about 100 nm, or about 125 nm. In certain embodiments, EVs according to the present disclosure have a diameter of at most about 50 nm, about 75 nm, about 100 nm, about 125 nm, or about 150 nm.

Recovery of exosomes from the compositions described herein refers to the recovery rate after a given event, such as incubation, compared to the number of exosomes originally included in the composition. Established techniques for determining the (average) size and concentration of EVs include electron microscopy (EM), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), resistive pulse sensing, and There is fluorescence correlation spectroscopy. For purposes of this disclosure, the number or size of particles is determined by either standard flow cytometry or high resolution flow cytometry, depending on the size of the exosomes. See, for example, van der Vlist EJ, Nolte-'T Hoen EN, Stoorvogel W, et al. "Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry.'', Nat Protoc. 2012/06/23.2012;7(7):1311-1326;van der Vlist EJ, Nolte-'T Hoen EN, Stoorvogel W, et al. "Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry.'', Nat Protoc. Please refer to 2012/06/23.2012;7(7):1311-1326, respectively.

Oral Delivery System As shown in FIG. 1, one embodiment of the delivery system described is a single-phase delivery system. A single-phase delivery system includes multiple exosomes and a pH-sensitive enteric coating. As shown in FIG. 1, another embodiment of the described delivery system is a two-phase delivery system. The two-phase delivery system includes a first plurality of exosomes encapsulated within an enteric coating and a second plurality of exosomes within the enteric coating. The first plurality of exosomes surrounds the second plurality of exosomes, the first plurality of exosomes for release at a first pH and the second plurality of exosomes for release at a second pH. It is supposed to be left behind. As a result, a two-phase delivery system can deliver two different exosomes to two different locations within an individual's gastrointestinal tract, as shown in FIG. 2. In certain embodiments, the first and second plurality of exosomes include the same type of cell. In certain embodiments, the first and second plurality of exosomes include different types of exosomes. In certain embodiments, exosomes are embedded in a matrix and then surrounded by an enteric coating. The matrix protects the exosomes from osmotic stress, pH stress, and mechanical disruption until they are released at the intended delivery site, which may include any one or more of the ileum, jejunum, duodenum, or colon. play a role. Such systems are useful for delivering therapeutic exosomes to individuals by non-parental and oral routes. FIG. 3 provides an overview of the process for making and dispensing the oral exosome compositions described herein.

Transrectal or Vaginal Delivery System As shown in FIG. 3, one embodiment of the described delivery system is a single phase delivery system. Single-phase delivery systems include a preparation of exosomes and a pH-sensitive lipophilic or hydrophilic coating. As shown in FIG. 3, another embodiment of the described delivery system is a two-phase delivery system. The two-phase delivery system includes a first preparation of exosomes encapsulated within a lipophilic or hydrophilic coating and a second preparation of exosomes within the lipophilic or hydrophilic coating. The first preparation of exosomes surrounds the second preparation of exosomes, and the first preparation of exosomes is released at a first pH and the second preparation of exosomes is released at a second pH. is left for release. As a result, a two-phase delivery system can deliver two different multiple exosome preparations to two different locations within an individual's gastrointestinal tract, as shown in FIG. In certain embodiments, the first and second preparations of exosomes include the same type of exosomes. In certain embodiments, the first and second preparations of exosomes include different types of exosomes. In certain embodiments, exosomes are embedded in a matrix and then surrounded by a lipophilic or hydrophilic coating. The matrix is permeable until released at the intended delivery site, which may include any one or more of the colon, large intestine, small intestine, appendix, cecum, colon, rectum, duodenum, jejunum, ileum, or vagina. It plays a role in protecting exosomes from pressure stress, pH stress, and mechanical destruction. Such systems are useful for delivering therapeutic exosomes to individuals by the rectal or vaginal route. FIG. 3 provides an overview of the process for making and dispensing the rectal or vaginal exosome preparation compositions described herein.

Additionally, the formulation can include pharmaceutically acceptable organic or inorganic carrier materials that do not adversely react with the components of the composition and are suitable for non-parental administration. Suitable pharmaceutically acceptable carriers include water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscoparaffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like. Examples include, but are not limited to, arbitrary combinations. The formulations can be sterilized and, if desired, contain adjuvants, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that influence osmotic pressure, buffers, colorants, flavorings, aromatic substances, or Can be mixed with any combination of them.

Compositions of the invention may be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension, such as, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

The compositions of the invention may be prepared and formulated as or containing emulsions. The emulsion may contain further ingredients, which may be present in solution in either the aqueous or oily phase, or themselves as a separate phase. . If desired, further pharmaceutical excipients such as emulsifiers, stabilizers, dyes, antioxidants, etc. may be present in the emulsion. Pharmaceutical emulsions may also be multiple emulsions consisting of more than two phases, as is the case, for example, in oil-in-water-in-oil emulsions and water-in-oil-in-water emulsions. Emulsions according to the invention include oil-in-water emulsions, water-in-oil emulsions, oil-in-water-in-oil emulsions, and water-in-oil-in-water emulsions. Emulsions may be characterized by little or no thermodynamic stability. Often the dispersed or discontinuous phase of an emulsion is well dispersed in the external or continuous phase and maintained in this form by the emulsifier or the viscosity of the formulation. Either phase of the emulsion may be semi-solid or solid. Other means of stabilizing emulsions include, of course, the use of emulsifiers, which may be incorporated into either phase of the emulsion.

Natural emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin, and acacia. Absorption bases such as dehydrated lanolin and hydrophilic petrolatum have hydrophilic properties that allow them to retain their semisolid consistency while absorbing water and forming water-in-oil emulsions. has. Finely divided solids are also used as excellent emulsifiers, especially in combination with surfactants or in viscous preparations. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate, and colloidal magnesium aluminum silicate, pigments, and carbon or non-polar solids such as glyceryl tristearate.

Various non-emulsifying substances are also included in emulsion formulations and contribute to the properties of the emulsion. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, wetting agents, hydrophilic colloids, preservatives, antioxidants, or any combination thereof. Hydrophilic colloids or hydrocolloids include natural gums and synthetic polymers, such as polysaccharides (e.g. acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g. carboxymethyl cellulose and carboxy propylcellulose), and synthetic polymers (eg, carbomers, cellulose ethers, carboxyvinyl polymers).

Commonly used preservatives included in emulsion formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. The antioxidants used are free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, as well as citric acid, tartaric acid, lecithin, etc. It may also be an antioxidant synergist.

Compositions of the invention may be prepared and formulated as or containing microemulsions. Microemulsions can improve drug solubilization, protect drugs from enzymatic hydrolysis, and have the potential to enhance drug absorption due to changes in membrane fluidity and permeability by surfactants. ease of oral administration over solid dosage forms, may improve clinical efficacy, and may reduce toxicity. In many cases, microemulsions can form spontaneously at ambient temperature. Microemulsion compositions and formulations of the present invention can facilitate systemic absorption of compositions from the gastrointestinal tract, as well as delivered exosome preparations within the gastrointestinal tract, vagina, and other areas of non-parental administration. can improve local cellular uptake of substances.

The microemulsion of the present invention may also contain sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and enhance the absorption of the oligonucleotides and nucleic acids of the present invention. It may further contain additional ingredients and additives. The penetration enhancers used in the microemulsions of the invention fall into five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. It can be classified as belonging to one.

The compositions of the present invention can be prepared in capsules, coated capsules, tablets, coated tablets, pills, etc. in a known manner using inert, non-toxic pharmaceutically suitable excipients or solvents. It can be processed into common formulations such as agents, pellets, granules, aerosols, syrups, emulsions, suspensions, gels, lotions, pastes, creams, balms, and solutions, or any combination thereof. In each case, the therapeutically active compound should be present in an amount calculated to ensure the desired therapeutic effect. The compositions may be formulated in a conventional manner using additional pharmaceutically acceptable carriers or excipients as required. The compositions thus include binders (e.g. pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose), fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate), lubricants (e.g. magnesium stearate, They may be prepared by conventional means using carriers or excipients such as talc or silica), disintegrants (eg, starch or sodium starch glycolate), or wetting agents (eg, sodium lauryl sulfate).

In some embodiments, the compositions of the present invention may further include hydroxypropyl methylcellulose (HPMC).

In some embodiments, the compositions of the present invention may further include about 0.63% to about 1.5% hydroxypropyl methylcellulose (HPMC).

In certain embodiments, the pharmaceutical compositions of the invention contain penetration enhancers such as fatty acids, bile salts, chelating agents, surfactants, and non-surfactants such as unsaturated cyclic ureas, 1-alkyl - an alkanone, 1-alkenylazacyclo-alakanone, or a steroidal anti-inflammatory agent.

In some embodiments, the composition is contained in a capsule, which may be coated by methods well known in the art. Capsules used for non-parental delivery can include formulations well known in the art.

In some embodiments, the capsule is a single-piece capsule, a two-piece capsule, a transparent capsule, a non-transparent capsule, an opaque capsule, a sustained release capsule, an extended release capsule, a standard release capsule, a rapid release capsule, an immediate release capsule, a hard shell capsule. , soft gel capsule, gel capsule, hard gelatin capsule, soft gelatin capsule, animal capsule, vegetarian capsule, polysaccharide capsule, cellulose capsule, mucopolysaccharide capsule, tapioca capsule, hydroxypropyl methylcellulose (HPMC) capsule, pullulan capsule, intestine Dissolved capsules, uncoated capsules, coated capsules, capsules containing titanium dioxide, fatty acids, waxes, shellac, plastics, plasticizers, glycerin, sorbitol, vegetable fibers, additives, preservatives, colorants, or any combination thereof may be included.

Furthermore, multi-compartment capsules, lipid-coated capsules, water permeable capsules, capsules with controlled release properties, capsules with multi-stage drug delivery systems, or any combination thereof, may also be used to formulate the compositions of the present invention. may be used for.

Formulations of the invention may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solution may also contain buffers, diluents, and other suitable additives. Additionally, pharmaceutically acceptable organic or inorganic carrier materials that do not adversely react with the components of the mammalian exosome composition and are suitable for non-parental administration can be included in the formulation. Suitable pharmaceutically acceptable carriers include water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like. , but not limited to. The formulations can be sterilized and, if desired, contain adjuvants, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that influence osmotic pressure, buffers, colorants, flavorings, aromatic substances, or Can be mixed with any combination of them. Aqueous suspensions may contain substances which increase the viscosity of the suspension, such as, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In some embodiments, solutions according to the invention consist essentially of nucleoside moieties. In some embodiments, the invention provides pharmaceutical compositions that include at least one nucleoside moiety, such as a nucleoside, nucleotide, or nucleic acid, in a solution or emulsion. The nucleic acid may be a ribozyme, PNA, or aptamer, but is preferably an oligonucleotide. In some embodiments, a solution according to the invention consists essentially of a nucleoside moiety and a solvent comprising, for example, saline or cocoa butter.

In some embodiments, a solution according to the invention consists essentially of a nucleoside moiety and a solvent comprising, for example, saline or cocoa butter.

The formulations may conveniently be presented in unit dosage form and may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredient with a pharmaceutical carrier or excipient. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Non-parental administration of the compositions of the invention may involve suppositories. Suppositories are well known in the art. They are usually formulated to be solid at room temperature, with a melting temperature below the human normal body temperature of 36 or 37°C. Therefore, it is common to formulate suppositories using a fatty base, such as cocoa butter, that meets the melting point criteria described above. In some embodiments, suppository forms may contain additional excipients such as, but not limited to, binders and adhesives, lubricants, disintegrants, colorants, and fillers. . In some embodiments, the suppository includes a combination of any of the above base materials.

Non-parental administration of compositions of the invention may include coating materials that can alter delivery of the therapeutic agent. Coating materials can facilitate administration without interfering with the pharmacological activity of the product.

In some embodiments, suppositories containing one or more fatty acids include fatty or oily bases, such as cocoa butter, hard fats and hydrogenated vegetable oils, waxes, water-soluble or water-miscible bases, such as Coatings of polyethylene glycol, glycol-surfactant combinations and polyoxyethylene sorbitan fatty acid esters, and combinations thereof are applied. Additionally, coated suppositories may be in the form of rectal capsules in which a core containing free fatty acids is coated with a water-soluble compound such as gelatin.

In some embodiments, the composition may further include cocoa butter, coconut oil, glycerinated gelatin, hydrogenated oils, polyethylene glycol (PEG), or fatty acid esters of PEG.

In order to reduce discomfort during the action of the drug and during defecation, it may be convenient to include excipient oil components, such as triacylglyceride oils, in the suppository forms of the present invention and to reduce the overall composition of the dosage form. It is constructed so that it has a suitable melting point.

Compositions of the invention include any pharmaceutically acceptable compound that is capable of providing (directly or indirectly) its biologically active metabolite or residue when administered to an individual. Thus, for example, this disclosure also relates to "prodrugs" and "pharmaceutically acceptable salts" of the compounds of the invention, as well as other biological equivalents.

Also provided are methods of treating an animal comprising administering to the animal a therapeutically effective amount of a pharmaceutical composition according to the invention. The compositions can be administered via non-parental routes, such as buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, or urethral routes. In a preferred embodiment, the compositions of the invention are administered vaginally or rectally by enema or suppository.

Uses Described herein are non-parental oral, rectal, or vaginal delivery of exosomes to the gastrointestinal tract, skin, eyes, pulmonary tract, urethra, vagina, or circulatory system of a subject. It is a useful system for This system can be used to deliver exosomes across the barrier into the blood or lymphatic system by the rectal or vaginal route. The systems described herein can be used to deliver therapeutic doses of mammalian exosomes to a subject. In certain embodiments, the system is used to deliver therapeutic doses of human exosomes to a human subject. In certain embodiments, the system is used to deliver a therapeutic dose of human exosomes to a human subject who has been diagnosed with, is suspected of having, or is at risk of having a disease or disorder. Ru.

In certain embodiments, the system and mammalian exosome compositions deliver therapeutic doses to the colon (e.g., large intestine) of an individual diagnosed with, suspected of having, or at risk of having a disease or disorder. Deliver exosomes. In certain embodiments, the system delivers a therapeutic dose of exosomes to the small intestine of an individual diagnosed with, suspected of having, or at risk of having a disease or disorder. In certain embodiments, the system delivers a therapeutic dose of exosomes to the vagina of an individual who has been diagnosed with, is suspected of having, or is at risk of having a disease or disorder. In certain embodiments, the system delivers a therapeutic dose of exosomes to the ileum of an individual diagnosed with, suspected of having, or at risk of having a disease or disorder. In certain embodiments, the system delivers a therapeutic dose of exosomes to the duodenum of an individual diagnosed with, suspected of having, or at risk of having a disease or disorder. In certain embodiments, the system delivers a therapeutic dose of exosomes to the jejunum of an individual diagnosed with, suspected of having, or at risk of having a disease or disorder. In certain embodiments, the system delivers a therapeutic dose of exosomes to the vagina of an individual who has been diagnosed with, is suspected of having, or is at risk of having a disease or disorder. In certain embodiments, the individual is a human individual and the exosome is a human exosome.

The non-parental, oral, rectal or vaginal systems used herein are useful for delivering therapeutic doses of mammalian exosomes to domestic animals for veterinary applications. In certain embodiments, the system delivers a therapeutic dose of canine exosomes to a dog. In certain embodiments, the system delivers a therapeutic dose of canine exosomes to a dog suffering from inflammatory bowel disease. In certain embodiments, the system delivers a therapeutic dose of feline exosomes to a cat. In certain embodiments, the system delivers therapeutic doses of bovine exosomes to bovine species. The transrectal or vaginal systems described herein may also be combined with other treatments administered rectally or vaginally to the animal. In certain embodiments, systems containing canine exosomes may be mixed with food and delivered to dogs. In certain embodiments, systems containing feline exosomes may be mixed with food and delivered to cats. In certain embodiments, systems containing bovine exosomes may be mixed with food or nutritional feed and delivered to bovine species.

In certain embodiments, the present mammalian exosome delivery systems and compositions are for use in treating inflammatory or autoimmune disorders in humans. In certain embodiments, the inflammatory or autoimmune disorder is a gastrointestinal inflammatory or autoimmune disorder. In certain embodiments, the gastrointestinal inflammatory disorder or autoimmune disorder comprises inflammatory bowel disease or Crohn's disease. In certain embodiments, the gastrointestinal inflammatory disorder or autoimmune disorder comprises Crohn's disease.

In certain embodiments, the present mammalian exosome delivery systems and compositions are for use in treating inflammatory or autoimmune disorders in animals. In certain embodiments, the inflammatory or autoimmune disorder is a gastrointestinal inflammatory or autoimmune disorder. In certain embodiments, the animal is any one or more of a dog, cow, cat, pig, horse, or sheep.

Exosomes The exosomes described herein are derived from mammalian cells and are isolated and purified according to methods known in the art.

The non-parental, oral, rectal, or vaginal delivery systems and compositions for mammalian exosomes described herein are useful for delivering exosomes to the gastrointestinal tract of a subject. In certain embodiments, the exosomes include eukaryotic exosomes. In certain embodiments, the exosomes include eukaryotic exosomes that lack a cell wall. In certain embodiments, the exosomes include mammalian exosomes. In certain embodiments, the exosomes include human exosomes. In certain embodiments, the exosomes include domestic animal exosomes selected from cats, dogs, pigs, sheep, horses, cows, goats, yaks, and any combinations thereof. Exosomes contained in non-parental, oral, rectal, or vaginal delivery systems provide a therapeutic effect and are associated with a subject that has been diagnosed with or is suspected of having a disease. One that can stop, reduce, or ameliorate symptoms of at least one disease. In certain embodiments, the disease is a disease of the gastrointestinal tract selected from Crohn's disease or inflammatory bowel disease. In certain embodiments, the exosomes do not include prokaryotic exosomes.

Stem cells include certain types of cells useful for producing exosomes for inclusion in the non-parental, oral, rectal, or vaginal delivery systems described herein. In certain embodiments, the transrectal or vaginal delivery system comprises a plurality of stem cell exosomes derived from or isolated and purified from stem cells. In certain embodiments, the stem cells are embryonic stem cells, pluripotent stem cells, adult stem cells, induced pluripotent stem cells, mesenchymal stem cells, hematopoietic stem cells, myoblasts, fibroblasts, hepatic stem cells, renal stem cells, cardiac stem cells, or intestinal stem cells, or mixtures thereof. In certain embodiments, the cells include embryonic stem cells. In certain embodiments, the cells include mesenchymal stem cells. In certain embodiments, the cells include type I (pro-inflammatory) mesenchymal stem cells. In certain embodiments, the cells include type II (anti-inflammatory) mesenchymal stem cells. In certain embodiments, the cells include pluripotent stem cells or induced pluripotent stem cells. Type 1 or type 2 mesenchymal stem cells can be generated using TLR4 or TLR3 agonists, respectively. Methods for producing type 1 or type 2 mesenchymal stem cells are described in U.S. Patent Application Publication No. 2014/0017787 or WO 2016053758 (A1), which are incorporated herein by reference in their entirety. It will be done. In certain embodiments, type 2 MSCs express higher levels of CXCL9 than MSC2s that are not induced with TLR3.

Other types of cells useful for producing exosomes for inclusion in the non-parental, oral, rectal, or vaginal delivery systems described herein are immune cells. In certain embodiments, the non-parental, oral, rectal, or vaginal delivery system comprises a plurality of immune cells. In certain embodiments, the immune cells include B cells (eg, CD19+ cells), T cells (eg, CD3+ cells), CD8+ T cells, CD4+ T cells, NK cells, dendritic cells, or macrophage cells. In certain embodiments, the immune cells include T cells. In certain embodiments, the T cells include T cells with a regulatory T cell phenotype that express and secrete IL-10, express and secrete TGF-β, or express the transcription factor FoxP3. In certain embodiments, the immune cells include NK cells. In certain embodiments, the immune cells include macrophages. In certain embodiments, the macrophages include type M1 (pro-inflammatory) macrophages. In certain embodiments, the macrophages include type M2 (suppressor) macrophages.

In certain embodiments, the non-parental, oral, rectal, or vaginal delivery systems and compositions for mammalian exosomes described herein provide for delivery of different types of exosomes (e.g., exosomes from different types of cells). Contains mixtures. In certain embodiments, the mixture comprises 2, 3, 4, 5, 6, 7, 8, 9, or more different types of exosomes. In certain embodiments, the mixture comprises stem cell exosomes and immune cell exosomes. In certain embodiments, the mixture comprises different stem cell exosomes. In certain embodiments, the mixture includes different types of immune exosomes. In certain embodiments, the mixture comprises stem cell exosomes and immune cell exosomes. The mixture can be in any proportion suitable for therapeutic treatment. In certain embodiments, two different cell types are included, and the ratio of the first type to the second type is 1:1, 1:2, 1:3, 1:4, 1:5, 1 :6, 1:7, 1:8, or 1:9.

The non-parental, oral, rectal, or vaginal delivery systems and compositions of mammalian exosomes described herein include therapeutic amounts of exosomes. In certain embodiments, the therapeutic amount of exosomes comprises

Matrices Currently described compositions of exosomes and mammalian exosomes for non-parental, oral, rectal, or vaginal delivery systems include or are embedded in matrices, gels, or excipients. The matrix serves to protect exosomes from dehydration, osmotic stress, pH stress, protease degradation, and other stresses present in the digestive system. In certain embodiments, exosomes are embedded or suspended in a matrix. In certain embodiments, the matrix includes agar. In certain embodiments, the matrix includes methylcellulose. In certain embodiments, the matrix comprises alginate. In certain embodiments, the matrix includes methylcellulose and alginic acid. In certain embodiments, the matrix includes approximately equal amounts of methylcellulose and alginic acid. In certain embodiments, the matrix includes a hydrogel. Suitable hydrogels include those derived from collagen, hyaluronic acid, hyaluronan, fibrin, alginate, agarose, chitosan, and combinations thereof. In other embodiments, suitable hydrogels are synthetic polymers. In further embodiments, suitable matrices for embedding exosomes include those derived from poly(acrylic acid) and its derivatives, poly(ethylene oxide) and its copolymers, poly(vinyl alcohol), polyphosphazenes, and combinations thereof. Contains things. In various specific embodiments, the confinement material is a hydrogel, NovoGel™, agarose, alginate, gelatin, Matrigel™, hyaluronan, poloxamer, peptide hydrogel, poly(isopropyl n-polyacrylamide), selected from polyethylene glycol diacrylate (PEG-DA), hydroxyethyl methacrylate, polydimethylsiloxane, polyacrylamide, poly(lactic acid), or combinations thereof.

In certain embodiments, the matrix includes approximately equal amounts of alginate gel and methylcellulose.

In certain embodiments, the matrix comprises exosomes mixed with a predetermined ratio of alginate gel and methylcellulose. In certain embodiments, the ratio of alginate gel to methylcellulose in the matrix is about 1:0.1, about 1:0.2, about 1:0.3, about 1:0.4, about 1:0. .5, about 1:0.6, about 1:0.7, about 1:0.8, about 1:0.9, about 1:1, about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9, or The ratio is approximately 1:2. In certain embodiments, the ratio of methylcellulose to alginate gel in the matrix is about 1:0.1, about 1:0.2, about 1:0.3, about 1:0.4, about 1:0. .5, about 1:0.6, about 1:0.7, about 1:0.8, about 1:0.9, about 1:1, about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9, or The ratio is approximately 1:2.

Alternatively, in certain embodiments, the mixture of alginate gel and methylcellulose may contain different concentrations of methylcellulose. In certain embodiments, the concentration of the alginate gel is about 0.5% to about 5.0%. In certain embodiments, the concentration of the alginate gel is about 0.5% to about 4.0%. In certain embodiments, the concentration of the alginate gel is about 0.5% to about 3.0%. In certain embodiments, the concentration of alginate gel is at least about 0.5%. In certain embodiments, the concentration of alginate gel is up to about 3.0%. In certain embodiments, the concentration of the alginate gel is about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1 .1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9 %, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, About 2.8%, about 2.9%, or about 3.0%. In certain embodiments, the concentration of the alginate gel is at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1 0%, at least about 1.1%, at least about 1.2%, at least about 1.3%, or at least about 1.4%. In certain embodiments, the concentration of the alginate gel is up to about 3.0%, up to about 2.9%, up to about 2.8%, up to about 2.7%, up to about 2.6%, up to about 2. .5%, maximum approximately 2.4%, maximum approximately 2.3%, maximum approximately 2.2%, maximum approximately 2.1%, maximum approximately 2.0%, maximum approximately 1.9%, maximum approximately 1. 8%, up to about 1.7%, up to about 1.6%, or up to about 1.5%. In certain embodiments, the concentration of the alginate gel is about 0.5% to about 0.7%, about 0.5% to about 0.9%, about 0.5% to about 1.1%, about 0. .5% to about 1.3%, about 0.5% to about 1.5%, about 0.5% to about 1.7%, about 0.5% to about 1.9%, about 0.5 % to about 2.1%, about 0.5% to about 2.5%, about 0.5% to about 2.7%, about 0.5% to about 2.9%, about 0.7% to about 0.9%, about 0.7% to about 1.1%, about 0.7% to about 1.3%, about 0.7% to about 1.5%, about 0.7% to about 1 .7%, about 0.7% to about 1.9%, about 0.7% to about 2.1%, about 0.7% to about 2.5%, about 0.7% to about 2.7 %, about 0.7% to about 2.9%, about 0.9% to about 1.1%, about 0.9% to about 1.3%, about 0.9% to about 1.5%, About 0.9% to about 1.7%, about 0.9% to about 1.9%, about 0.9% to about 2.1%, about 0.9% to about 2.5%, about 0 .9% to about 2.7%, about 0.9% to about 2.9%, about 1.1% to about 1.3%, about 1.1% to about 1.5%, about 1.1 % to about 1.7%, about 1.1% to about 1.9%, about 1.1% to about 2.1%, about 1.1% to about 2.5%, about 1.1% to about about 2.7%, about 1.1% to about 2.9%, about 1.3% to about 1.5%, about 1.3% to about 1.7%, about 1.3% to about 1 .9%, about 1.3% to about 2.1%, about 1.3% to about 2.5%, about 1.3% to about 2.7%, about 1.3% to about 2.9 %, about 1.5% to about 1.7%, about 1.5% to about 1.9%, about 1.5% to about 2.1%, about 1.5% to about 2.5%, about 1.5% to about 2.7%, about 1.5% to about 2.9%, about 1.7% to about 1.9%, about 1.7% to about 2.1%, about 1 .7% to about 2.5%, about 1.7% to about 2.7%, about 1.7% to about 2.9%, about 1.9% to about 2.1%, about 1.9 % to about 2.5%, about 1.9% to about 2.7%, about 1.9% to about 2.9%, about 2.1% to about 2.5%, about 2.1% to about about 2.7%, about 2.1% to about 2.9%, about 2.5% to about 2.7%, about 2.5% to about 2.9%, or about 2.7% to about It is 2.9%.

In certain embodiments, the concentration of alginate gel is about 0.1% to about 1%. In certain embodiments, the concentration of the alginate gel is about 0.1% to about 0.25%, about 0.1% to about 0.33%, about 0.1% to about 0.5%, about 0. .1% to about 0.67%, about 0.1% to about 0.75%, about 0.1% to about 0.8%, about 0.1% to about 0.9%, about 0.1 % to about 1%, about 0.25% to about 0.33%, about 0.25% to about 0.5%, about 0.25% to about 0.67%, about 0.25% to about 0 .75%, about 0.25% to about 0.8%, about 0.25% to about 0.9%, about 0.25% to about 1%, about 0.33% to about 0.5%, About 0.33% to about 0.67%, about 0.33% to about 0.75%, about 0.33% to about 0.8%, about 0.33% to about 0.9%, about 0 .33% to about 1%, about 0.5% to about 0.67%, about 0.5% to about 0.75%, about 0.5% to about 0.8%, about 0.5% to About 0.9%, about 0.5% to about 1%, about 0.67% to about 0.75%, about 0.67% to about 0.8%, about 0.67% to about 0.9 %, about 0.67% to about 1%, about 0.75% to about 0.8%, about 0.75% to about 0.9%, about 0.75% to about 1%, about 0.8 % to about 0.9%, about 0.8% to about 1%, or about 0.9% to about 1%. In certain embodiments, the concentration of the alginate gel is about 0.1%, about 0.25%, about 0.33%, about 0.5%, about 0.67%, about 0.75%, about 0 .8%, about 0.9%, or about 1%. In certain embodiments, the concentration of the alginate gel is at least about 0.1%, about 0.25%, about 0.33%, about 0.5%, about 0.67%, about 0.75%, about 0.8%, or about 0.9%. In certain embodiments, the concentration of the alginate gel is at most about 0.25%, about 0.33%, about 0.5%, about 0.67%, about 0.75%, about 0.8%, It is about 0.9%, or about 1%.

In certain embodiments, the concentration of methylcellulose is about 0.5% to about 3.0%. In certain embodiments, the concentration of methylcellulose is at least about 0.5%. In certain embodiments, the concentration of methylcellulose is up to about 3.0%. In certain embodiments, the concentration of methylcellulose is about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1. 1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9% , about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, or about 3.0%. In certain embodiments, the concentration of methylcellulose is at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1. 0%, at least about 1.1%, at least about 1.2%, at least about 1.3%, or at least about 1.4%. In certain embodiments, the concentration of methylcellulose is up to about 3.0%, up to about 2.9%, up to about 2.8%, up to about 2.7%, up to about 2.6%, up to about 2.0%. 5%, maximum approximately 2.4%, maximum approximately 2.3%, maximum approximately 2.2%, maximum approximately 2.1%, maximum approximately 2.0%, maximum approximately 1.9%, maximum approximately 1.8 %, up to about 1.7%, up to about 1.6%, or up to about 1.5%. In certain embodiments, the concentration of methylcellulose is about 0.5% to about 0.7%, about 0.5% to about 0.9%, about 0.5% to about 1.1%, about 0. 5% to about 1.3%, about 0.5% to about 1.5%, about 0.5% to about 1.7%, about 0.5% to about 1.9%, about 0.5% ～about 2.1%, about 0.5% to about 2.5%, about 0.5% to about 2.7%, about 0.5% to about 2.9%, about 0.7% to about 0.9%, about 0.7% to about 1.1%, about 0.7% to about 1.3%, about 0.7% to about 1.5%, about 0.7% to about 1. 7%, about 0.7% to about 1.9%, about 0.7% to about 2.1%, about 0.7% to about 2.5%, about 0.7% to about 2.7% , about 0.7% to about 2.9%, about 0.9% to about 1.1%, about 0.9% to about 1.3%, about 0.9% to about 1.5%, about 0.9% to about 1.7%, about 0.9% to about 1.9%, about 0.9% to about 2.1%, about 0.9% to about 2.5%, about 0. 9% to about 2.7%, about 0.9% to about 2.9%, about 1.1% to about 1.3%, about 1.1% to about 1.5%, about 1.1% ～about 1.7%, about 1.1% to about 1.9%, about 1.1% to about 2.1%, about 1.1% to about 2.5%, about 1.1% to about 2.7%, about 1.1% to about 2.9%, about 1.3% to about 1.5%, about 1.3% to about 1.7%, about 1.3% to about 1. 9%, about 1.3% to about 2.1%, about 1.3% to about 2.5%, about 1.3% to about 2.7%, about 1.3% to about 2.9% , about 1.5% to about 1.7%, about 1.5% to about 1.9%, about 1.5% to about 2.1%, about 1.5% to about 2.5%, about 1.5% to about 2.7%, about 1.5% to about 2.9%, about 1.7% to about 1.9%, about 1.7% to about 2.1%, about 1. 7% to about 2.5%, about 1.7% to about 2.7%, about 1.7% to about 2.9%, about 1.9% to about 2.1%, about 1.9% ～about 2.5%, about 1.9% to about 2.7%, about 1.9% to about 2.9%, about 2.1% to about 2.5%, about 2.1% to about 2.7%, about 2.1% to about 2.9%, about 2.5% to about 2.7%, about 2.5% to about 2.9%, or about 2.7% to about 2 .9%.

In certain embodiments, the concentration of methylcellulose is about 0.1% to about 1%. In certain embodiments, the concentration of methylcellulose is about 0.1% to about 0.25%, about 0.1% to about 0.33%, about 0.1% to about 0.5%, about 0. .1% to about 0.67%, about 0.1% to about 0.75%, about 0.1% to about 0.8%, about 0.1% to about 0.9%, about 0.1 % to about 1%, about 0.25% to about 0.33%, about 0.25% to about 0.5%, about 0.25% to about 0.67%, about 0.25% to about 0 .75%, about 0.25% to about 0.8%, about 0.25% to about 0.9%, about 0.25% to about 1%, about 0.33% to about 0.5%, About 0.33% to about 0.67%, about 0.33% to about 0.75%, about 0.33% to about 0.8%, about 0.33% to about 0.9%, about 0 .33% to about 1%, about 0.5% to about 0.67%, about 0.5% to about 0.75%, about 0.5% to about 0.8%, about 0.5% to About 0.9%, about 0.5% to about 1%, about 0.67% to about 0.75%, about 0.67% to about 0.8%, about 0.67% to about 0.9 %, about 0.67% to about 1%, about 0.75% to about 0.8%, about 0.75% to about 0.9%, about 0.75% to about 1%, about 0.8 % to about 0.9%, about 0.8% to about 1%, or about 0.9% to about 1%. In certain embodiments, the concentration of methylcellulose is about 0.1%, about 0.25%, about 0.33%, about 0.5%, about 0.67%, about 0.75%, about 0 .8%, about 0.9%, or about 1%. In certain embodiments, the concentration of methylcellulose is at least about 0.1%, about 0.25%, about 0.33%, about 0.5%, about 0.67%, about 0.75%, about 0.8%, or about 0.9%. In certain embodiments, the concentration of methylcellulose is up to about 0.25%, about 0.33%, about 0.5%, about 0.67%, about 0.75%, about 0.8%, It is about 0.9%, or about 1%.

In some embodiments, the matrix comprises exosomes mixed with about 0.5% to about 3.0% alginate gel and about 0.5% to about 3.0% methylcellulose matrix. The cell gel mixture is then transferred to capsules with a lipophilic or hydrophilic coating. In certain embodiments, the capsules are coated with methyl methacrylate-methacrylic acid copolymer (1:1). In certain embodiments, the mammalian exosomes include mesenchymal stem cell exosomes.

In some embodiments, the matrix comprises mammalian exosomes mixed with a matrix of about 1.5% alginate gel and about 1.5% methylcellulose. The exosome gel mixture is then transferred to capsules with a lipophilic or hydrophilic coating. In certain embodiments, the capsules are coated with methyl methacrylate-methacrylic acid copolymer (1:1). In certain embodiments, the mammalian exosomes include mesenchymal stem cell exosomes.

In some embodiments, the matrix is about 0.5% to about 3.0% alginate gel and about 0.5% in a capsule coated with methyl methacrylate-methacrylic acid copolymer (1:1). Contains mammalian exosomes suspended in a mixture of ~3.0% methylcellulose. In certain embodiments, the mammalian exosomes include mesenchymal stem cell exosomes.

In some embodiments, the matrix is about 1.0% to about 2.5% alginate gel and about 1.0% in a capsule coated with methyl methacrylate-methacrylic acid copolymer (1:1). Contains mammalian exosomes suspended in a mixture of ~2.5% methylcellulose. In certain embodiments, the mammalian exosomes include mesenchymal stem cell exosomes.

In some embodiments, the matrix is about 1.0% to about 2.0% alginate gel and about 1.0% in a capsule coated with methyl methacrylate-methacrylic acid copolymer (1:1). Contains mammalian exosomes suspended in a mixture of ~2.0% methylcellulose. In certain embodiments, the mammalian exosomes include mesenchymal stem cell exosomes.

In some embodiments, the matrix is about 1.2% to about 1.8% alginate gel and about 1.2% in a capsule coated with methyl methacrylate-methacrylic acid copolymer (1:1). Contains mammalian exosomes suspended in a mixture of ~1.8% methylcellulose.

In some embodiments, the matrix comprises about 1.4% to about 1.6% alginate gel and about 1.4% alginate gel within a capsule coated with methyl methacrylate-methacrylic acid copolymer (1:1). Contains mammalian exosomes suspended in a mixture of ~1.6% methylcellulose. In certain embodiments, the mammalian exosomes include mesenchymal stem cell exosomes.

In some embodiments, the matrix is about 0.25% to about 0.75% alginate gel and about 0.25% in a capsule coated with methyl methacrylate-methacrylic acid copolymer (1:1). Contains mammalian exosomes suspended in a mixture of ~0.75% methylcellulose. In certain embodiments, the mammalian exosomes include mesenchymal stem cell exosomes.

In some embodiments, the matrix is about 0.1% to about 0.9% alginate gel and about 0.1% in a capsule coated with methyl methacrylate-methacrylic acid copolymer (1:1). Contains mammalian exosomes suspended in a mixture of ~0.9% methylcellulose. In certain embodiments, the mammalian exosomes include mesenchymal stem cell exosomes.

In some embodiments, the matrix is in a mixture of about 1.5% alginate gel and about 1.5% methylcellulose within a capsule coated with methyl methacrylate-methacrylic acid copolymer (1:1). Contains suspended mammalian exosomes. In certain embodiments, the mammalian exosomes include mesenchymal stem cell exosomes.

In certain embodiments, the matrix includes a predetermined concentration of exosomes and agar. In certain embodiments, the concentration of agar in the matrix is about 0.1% w/v to about 10% w/v, about 0.1% w/v to about 8% w/v, about 0.1 % w/v ~ approx. 5% w/v, approx. 0.1% w/v ~ approx. 4% w/v, approx. 0.1% w/v ~ approx. 3% w/v, approx. 0.1% w /v to about 2% w/v, about 0.1% w/v to about 1% w/v, about 0.2% w/v to about 10% w/v, about 0.2% w/v ~8% w/v, approx. 0.2% w/v ~ approx. 5% w/v, approx. 0.2% w/v ~ approx. 4% w/v, approx. 0.2% w/v ~ approx. 3% w/v, about 0.2% w/v to about 2% w/v, about 0.2% w/v to about 1% w/v, about 0.4% w/v to about 10% w/v, about 0.4% w/v to about 8% w/v, about 0.4% w/v to about 5% w/v, about 0.4% w/v to about 4% w/ v, about 0.4% w/v to about 3% w/v, about 0.4% w/v to about 2% w/v, about 0.4% w/v to about 1% w/v, About 0.5% w/v to about 10% w/v, about 0.5% w/v to about 8% w/v, about 0.5% w/v to about 5% w/v, about 0 .5% w/v to about 4% w/v, about 0.5% w/v to about 3% w/v, about 0.5% w/v to about 2% w/v, about 0.5 % w/v to about 1% w/v. In certain embodiments, the concentration of agar in the matrix is about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%. In certain embodiments, the concentration of agar in the matrix is about 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, or 10%.

The matrices described herein have a predetermined viscosity. In further embodiments, the matrix comprising exosomes has a particle size of about 500 to 1,000,000 centipoise, about 1000 to 1,000,000 centipoise, about 1,000 to 500,000 centipoise, about 10,000 to 500,000 centipoise. centipoise, having a viscosity of about 10,000 to 400,000 centipoise, about 10,000 to 3,000,000 centipoise, about 100,000 to 1,000,000 centipoise, or about 500,000 to 1,000,000 centipoise It is characterized by

In certain embodiments, the matrix comprises extracellular matrix proteins such as collagen or fibrin; carbohydrates such as glucose, dextrose, sucrose; fibers such as cellulose; vitamins or minerals; and additional components such as buffers, isotonic solutions, or amino acids. Contains molecules.

Exosomes of the present disclosure can be characterized in a variety of ways. In certain embodiments, the exosome comprises a protein or polypeptide selected from the list consisting of CD81, CD63, CD9, HSP60, HSP70, HSP90, VEGF, PGE2, and combinations thereof. Exosome particles can be characterized by standard methods known in the art, such as flow cytometry using specific antibodies compared to isotype control antibodies. In certain embodiments, the exome has a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more increase in CD81 protein compared to an isotype control antibody. It can be a feature. In certain embodiments, the exome has a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more increase in CD63 protein compared to an isotype control antibody. It can be a feature. In certain embodiments, the exome has a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more increase in CD9 protein compared to an isotype control antibody. It can be a feature. In certain embodiments, the exome has a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more increase in HSP60 protein compared to an isotype control antibody. It can be a feature. In certain embodiments, the exome has a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more increase in HSP70 protein compared to an isotype control antibody. It can be a feature. In certain embodiments, the exome has a 2-fold, 3-fold, 4-fold, 5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more increase in HHSP90 protein compared to an isotype control antibody. It can be a feature. In certain embodiments, the exome has a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more increase in VEGF protein compared to an isotype control antibody. It can be a feature. In certain embodiments, the exome has a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more increase in PGE2 protein compared to an isotype control antibody. It can be a feature.

The compositions described herein also allow for the recovery of higher numbers of exosomes after incubating the composition for 2 hours at pH 1.2. In certain embodiments, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more intact exosomes can be recovered after 2 hours of incubation at pH 1.2. In certain embodiments, 30% or more intact exosomes can be recovered after 2 hours of incubation at pH 1.2. In certain embodiments, 40% or more intact exosomes can be recovered after 2 hours of incubation at pH 1.2. In certain embodiments, 50% or more intact exosomes can be recovered after 2 hours of incubation at pH 1.2. In certain embodiments, 60% or more intact exosomes can be recovered after 2 hours of incubation at pH 1.2. Intact exosomes can be counted and confirmed using a microscope.

Coatings The oral delivery systems herein include coatings that are formulated to dissolve at a particular pH. For biphasic delivery, the oral delivery system includes two coatings prepared to dissolve at different pHs. In certain embodiments, the coating is soluble at a pH of about 5.5 to about 6.0 to target the duodenum; a pH of about 7.3 to about 8.0 to target the ileum. or, to target the colon, so as to dissolve at a pH of about 5.5 to about 6.5. In certain embodiments, the coating has a pH of about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4. , 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7 .7, 7.8, 7.9, or 8.0. In certain embodiments, for biphasic delivery, one coating is prepared at a pH of about 7.3 to about 8.0 to target the ileum; one coating is prepared at a pH of about 7.3 to about 8.0 to target the colon. Prepared at a pH of about 5.5 to about 6.5. In certain embodiments, for biphasic delivery, one coating is prepared at a pH of about 5.5 to about 6.0 to target the duodenum; one coating is prepared at a pH of about 5.5 to about 6.0 to target the colon. Prepared at a pH of about 5.5 to about 6.5. In certain embodiments, for two-phase delivery, one coating is prepared at a pH of about 5.5 to about 6.0 to target the duodenum; one coating is prepared at a pH of about 5.5 to about 6.0 to target the ileum. Prepared at a pH of about 7.3 to about 8.0.

Also contemplated are exosomes or preparations thereof, and enteric-coated exosome formulations comprising enteric-coated materials; and pharmaceutically acceptable carriers or excipients thereof. Enteric materials refer to polymers that are substantially insoluble in the acidic environment of the stomach, but are predominantly soluble in intestinal fluids at certain pHs. The small intestine is the part of the gastrointestinal tract (intestine) between the stomach and large intestine, and includes the duodenum, jejunum, and ileum. The pH of the duodenum is about 5.5, the pH of the jejunum is about 6.5, and the pH of the distal ileum is about 7.5. Thus, the enteric material may have a pH of, for example, about 5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, about 8.2, about 8.4, Not soluble up to about 8.6, about 8.8, about 9.0, about 9.2, about 9.4, about 9.6, about 9.8, or about 10.0. Exemplary enteric materials that can be included in the mammalian exosome oral delivery systems and compositions described herein include cellulose acetate phthalate (CAP), hydroxypropyl methylcellulose phthalate (HPMCP), polyvinyl acetate phthalate (PVAP). , hydroxypropyl methylcellulose acetate succinate (HPMCAS), cellulose acetate trimellitate, hydroxypropyl methylcellulose succinate, cellulose acetate succinate, cellulose acetate hexahydrophthalate, cellulose propionate phthalate, cellulose acetate maleate, cellulose acetate butyrate, Cellulose acetate propionate, copolymer of methyl methacrylate and methyl methacrylate, copolymer of methyl acrylate and methyl methacrylate and methacrylic acid, copolymer of methyl vinyl ether and maleic anhydride (Gantrez ES series), ethyl methyl acrylate-methyl methacrylate-chlorotrimethyl Natural resins such as ammonium ethyl acrylate copolymers, zein, shellac, copal colophonium, and several commercially available enteric dispersion systems (e.g., Eudragit L30D55, Eudragit FS30D, Eudragit L100, Eudragit S100, Kollicoat EMM30D, Estacry l 30D, Coateric, and Aquateric ). The solubility of each of the above substances is known or can be easily determined in vitro.

A biphasic capsule containing mammalian exosomes includes first and second enteric coatings. In certain embodiments, the first enteric coating comprises a copolymer of methacrylic acid, methyl methacrylate, cellulose acetate phthalate, cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, polyvinyl acetate phthalate, shellac, cellulose Contains acetate trimellitate, sodium alginate, or zein. In certain embodiments, the second enteric coating comprises a copolymer of methacrylic acid, methyl methacrylate, cellulose acetate phthalate, cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, polyvinyl acetate phthalate, shellac, cellulose Contains acetate trimellitate, sodium alginate, or zein. In certain embodiments, the first enteric coating comprises a copolymer of methacrylic acid. In certain embodiments, the second enteric coating comprises a copolymer of methacrylic acid.

In certain embodiments, the enteric coating of the mammalian exosome compositions described herein comprises methyl methacrylate-methacrylic acid copolymer (1:1).

In certain embodiments, the gel, matrix, or excipient together with the enteric coating increases the viability of the first plurality of mammalian exosomes and/or the second plurality of mammalian exosomes from about 80 to Maintain a survival rate of 85%. In certain embodiments, the gel, matrix, or excipient increases the viability of the first plurality of mammalian exosomes and/or the second plurality of exosomes by at least 2, 3, 4, 5 at 24°C. , 6, 7, 8, 9, 10 days at approximately 50%, 60%, 70%, or greater than 80% viability.

In certain embodiments, the gel, matrix, with an enteric coating or excipient, provides a T _max of about 0.4-0.6 hours. In certain embodiments, the gel, matrix or excipient is present for about 0.4 hours, about 0.41 hours, about 0.42 hours, about 0.43 hours, about 0.44 hours, about 0.45 hours. , about 0.46 hours, about 0.47 hours, about 0.48 hours, about 0.49 hours, about 0.50 hours, about 0.51 hours, about 0.52 hours, about 0.53 hours, about Provides a T _max of 0.54 hours, about 0.55 hours, about 0.56 hours, about 0.57 hours, about 0.58 hours, about 0.59 hours, or about 0.60 hours. In certain embodiments, the gel, matrix or excipient lasts for at least about 0.4 hours, at least about 0.4 hours, at least about 0.42 hours, at least about 0.43 hours, at least about 0.44 hours, or a T _max of at least about 0.45 hours. In certain embodiments, the gel, matrix or excipient lasts up to about 0.46 hours, up to about 0.47 hours, up to about 0.48 hours, up to about 0.49 hours, up to about 0.50 hours, Maximum of approximately 0.51 hours, Maximum of approximately 0.52 hours, Maximum of approximately 0.53 hours, Maximum of approximately 0.54 hours, Maximum of approximately 0.55 hours, Maximum of approximately 0.56 hours, Maximum of approximately 0.57 hours, Maximum Provides a T _max of about 0.58 hours, up to about 0.59 hours, or up to about 0.60 hours.

The non-parental, oral, rectal, or vaginal delivery systems herein include coatings that are formulated to dissolve at a particular pH. For biphasic delivery, the rectal or vaginal delivery system includes two coatings that are prepared to dissolve at different pHs. In certain embodiments, the coating is soluble at a pH of about 5.5 to about 6.0 to target the duodenum; a pH of about 7.3 to about 8.0 to target the ileum. or, to target the colon, so as to dissolve at a pH of about 5.5 to about 6.5. In certain embodiments, the coating has a pH of about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4. , 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7 .7, 7.8, 7.9, or 8.0. In certain embodiments, for biphasic delivery, one coating is prepared at a pH of about 7.3 to about 8.0 to target the ileum; one coating is prepared at a pH of about 7.3 to about 8.0 to target the colon. Prepared at a pH of about 5.5 to about 6.5. In certain embodiments, for biphasic delivery, one coating is prepared at a pH of about 5.5 to about 6.0 to target the duodenum; one coating is prepared at a pH of about 5.5 to about 6.0 to target the colon. Prepared at a pH of about 5.5 to about 6.5. In certain embodiments, for two-phase delivery, one coating is prepared at a pH of about 5.5 to about 6.0 to target the duodenum; one coating is prepared at a pH of about 5.5 to about 6.0 to target the ileum. Prepared at a pH of about 7.3 to about 8.0.

Also contemplated are enteric-coated formulations comprising the disclosed exosomes or preparations of exosomes and lipophilic or hydrophilic materials; and pharmaceutically acceptable carriers or excipients thereof. Enteric materials refer to polymers that are substantially insoluble in the acidic environment of the stomach, but are predominantly soluble in intestinal fluids at certain pHs. The small intestine is the part of the gastrointestinal tract (intestine) between the stomach and large intestine, and includes the duodenum, jejunum, and ileum. The pH of the duodenum is about 5.5, the pH of the jejunum is about 6.5, and the pH of the distal ileum is about 7.5. Thus, the enteric material may have a pH of, for example, about 5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, about 8.2, about 8.4, Not soluble up to about 8.6, about 8.8, about 9.0, about 9.2, about 9.4, about 9.6, about 9.8, or about 10.0. Exemplary enteric materials that can be included in the mammalian exosome transrectal or vaginal delivery systems and compositions described herein include cellulose acetate phthalate (CAP), hydroxypropyl methylcellulose phthalate (HPMCP), polyvinyl acetate. Phthalate (PVAP), hydroxypropyl methylcellulose acetate succinate (HPMCAS), cellulose acetate trimellitate, hydroxypropyl methylcellulose succinate, cellulose acetate succinate, cellulose acetate hexahydrophthalate, cellulose propionate phthalate, cellulose acetate maleate, cellulose Acetate butyrate, cellulose acetate propionate, copolymer of methyl methacrylate and methyl methacrylate, copolymer of methyl acrylate and methyl methacrylate and methacrylic acid, copolymer of methyl vinyl ether and maleic anhydride (Gantrez ES series), ethyl methyl acrylate-methyl Natural resins such as methacrylate-chlorotrimethylammonium ethyl acrylate copolymer, zein, shellac, copal colophonium, and several commercially available enteric dispersion systems (e.g., Eudragit L30D55, Eudragit FS30D, Eudragit L100, Eudragit S100, Kollicoat EMM30D) , Estacryl 30D, Coateric, and Aquateric). The solubility of each of the above substances is known or can be easily determined in vitro.

A biphasic capsule containing mammalian exosomes includes first and second enteric coatings. In certain embodiments, the first enteric coating comprises a copolymer of methacrylic acid, methyl methacrylate, cellulose acetate phthalate, cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, polyvinyl acetate phthalate, shellac, cellulose Contains acetate trimellitate, sodium alginate, or zein. In certain embodiments, the second enteric coating comprises a copolymer of methacrylic acid, methyl methacrylate, cellulose acetate phthalate, cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, polyvinyl acetate phthalate, shellac, cellulose Contains acetate trimellitate, sodium alginate, or zein. In certain embodiments, the first enteric coating comprises a copolymer of methacrylic acid. In certain embodiments, the second enteric coating comprises a copolymer of methacrylic acid.

In certain embodiments, the enteric coating of the mammalian exosome compositions described herein comprises methyl methacrylate-methacrylic acid copolymer (1:1).

In certain embodiments, the gel, matrix, or excipient, along with the enteric coating, maintains the stability and potency of the mammalian exosome and/or the second plurality of mammalian exosomes at about 80-85% potency. In certain embodiments, the gel, matrix or excipient increases the potency of the first plurality of mammalian exosomes and/or the second plurality of mammalian exosomes by at least 2, 3, 4, 5, 6 at 24°C. , 7, 8, 9, 10 days at greater than about 50%, 60%, 70%, or 80% efficacy. Potency or stability can be measured by any method suitable for determining the integrity of the membrane structure of exosomes, or the potency and stability (e.g., lack of degradation) of proteins or nucleic acids within exosomes.

The transrectal or vaginal delivery systems herein include one or more coatings that are prepared to melt at normothermia or human normal body temperature. For biphasic or single-phase delivery, the rectal or vaginal delivery system includes one or more coatings that are prepared to melt at different body temperatures. In certain embodiments, one or more coatings are prepared to melt at a temperature of about 36° C. or higher. In certain embodiments, the one or more coatings are prepared to melt at a temperature of about 36°C to about 47°C. In certain embodiments, the one or more coatings are prepared to melt at a temperature of about 32°C to about 41°C. In certain embodiments, the one or more coatings are prepared to melt at a temperature of about 34°C to about 45°C. In some embodiments, the one or more coatings are at least about 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, It can be prepared to melt at a temperature of 43°C, 44°C, 45°C, 46°C, or 47°C. In some embodiments, the one or more coatings have a temperature of about 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C. ℃, 44℃, 45℃, 46℃, or 47℃. In some embodiments, the one or more coatings have a temperature of up to about 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C. , 43°C, 44°C, 45°C, 46°C, or 47°C.

Also contemplated are the disclosed exosomes or preparations of exosomes and exosome formulations comprising lipophilic or hydrophilic materials; and pharmaceutically acceptable carriers or excipients thereof. A lipophilic or hydrophilic coating material prepared to melt at normal body temperature refers to a material that does not substantially melt at temperatures below normal body temperature, but primarily melts at temperatures above normal body temperature. Thus, a lipophilic or hydrophilic coating material prepared to melt at normal body temperature may, for example, be about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, It does not melt up to a temperature of about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, or about 47°C.

The transrectal or vaginal delivery systems herein include one or more coatings that are prepared to dissolve upon contact with an aqueous solution. For biphasic delivery, the rectal or vaginal delivery system includes one or more coatings prepared to dissolve in different amounts of aqueous solution. In certain embodiments, the one or more coatings are prepared to dissolve in an aqueous solution volume of about 5.6 mL or more. In certain embodiments, the one or more coatings are prepared to dissolve in an aqueous solution volume of about 5.5 mL to about 20.5 mL. In certain embodiments, the one or more coatings are prepared to dissolve in an aqueous solution volume of about 5.5 mL to about 13 mL. In certain embodiments, the one or more coatings are prepared to dissolve in an aqueous solution volume of about 10.5 mL to about 20.5 mL. In some embodiments, one or more coatings can be prepared to dissolve in an aqueous solution volume of at least about 5.5 mL, 8 mL, 10.5 mL, 13 mL, 15.5 mL, 18 mL, or 20.5 mL. In some embodiments, one or more coatings can be prepared to dissolve in an aqueous solution volume of about 5.5 mL, 8 mL, 10.5 mL, 13 mL, 15.5 mL, 18 mL, or 20.5 mL. In some embodiments, one or more coatings can be prepared to dissolve in an aqueous solution volume of up to about 5.5 mL, 8 mL, 10.5 mL, 13 mL, 15.5 mL, 18 mL, or 20.5 mL.

Also contemplated are the disclosed exosomes or preparations of exosomes and exosome formulations comprising lipophilic or hydrophilic materials; and pharmaceutically acceptable carriers or excipients thereof. A lipophilic or hydrophilic coating material prepared to be soluble in different volumes of aqueous solution refers to a material that is substantially insoluble in volumes less than normal body fluid volumes and predominantly soluble in volumes greater than or equal to normal body fluid volumes. Thus, lipophilic or hydrophilic coating materials prepared to dissolve in different volumes of aqueous solution may, for example, be about 5.5 mL, about 8 mL, about 10.5 mL, about 13 mL, about 15.5 mL, about 18 mL, or about 20 It does not dissolve up to a volume of .5 mL.

Non-parental administration of rectal or vaginal delivery systems herein includes suppositories and enemas. Suppositories are generally solid at room temperature and up to at least about 30°C, but are formulated to have a melting temperature below human normal body temperature of about 37°C. Suppository formulations in the present disclosure include about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, It may have a melting point of 44°C, 45°C, 46°C, 47°C or higher. Therefore, it is common to formulate suppositories using a fatty base, such as cocoa butter, that meets the melting point criteria described above. Cocoa butter is a mixture of saturated and unsaturated fatty acid triglycerides that can be treated as a solid at room temperature but completely melts at body temperature. Other common fatty bases for formulating suppositories with fatty bases include coconut oil, glycerinated gelatin, hydrogenated oils, polyethylene glycol (PEG), or fatty acid esters of PEG. Other materials used in the formulation of suppositories include hard fats, macrogol, and various gelatin mixtures consisting of, for example, gelatin, water, and glycerol. Commercially available fatty bases suitable for suppository formulations include those mentioned above, in particular lipophilic bases, which are semi-synthetic vegetable oil bases available in several grades such as Suppocire™ AS, AS2X, NA. Supocire™ (Gattefosse); Novata™ (Henkel Int.), e.g. Novata A, Novata B, and Novata BC; Witepsol™ (Dynamit Nobel Ab), e.g. Witepsol™ H5, H12 , H15, H32, H35, W25, W31, W32, W32, W35, and W45; Massa Estarinum™ (SASOL), such as Massa Estarinum™ grades B, BC, E, and 299; Japocire™ ), and Ovucire(TM). The suppositories of the present invention may suitably contain any of the above-mentioned materials as a base. Hydrophilic waxes such as polyethylene glycols (eg, PEG 1500, PEG 3000, PEG 4000 and mixtures thereof) can also be used in the present invention. Suppocire AP is an amphiphilic base containing saturated polyglycolyzed glycerides. In some embodiments, suppository forms may contain additional excipients such as, but not limited to, binders and adhesives, lubricants, disintegrants, colorants, and fillers. . In some embodiments, the suppository includes a combination of any of the above base materials.

Non-parenteral administration of the rectal or vaginal delivery systems herein may optionally include coating materials that can alter delivery of the therapeutic agent. Coating materials can facilitate administration without interfering with the therapeutic activity of the product.

In some embodiments, suppositories containing one or more fatty acids are prepared by: (i) coating the interior surface of a suppository mold with a coating material prior to molding the suppository; (ii) suppository molding; (iii) by molding or casting the coating material around the suppository, or (iv) by forming the coating by any other suitable method. . The coating may be based on fatty or oily bases such as cocoa butter, hard fats and hydrogenated vegetable oils, waxes, water-soluble or water-miscible bases such as polyethylene glycols, glycol-surfactant combinations and polyoxyethylene sorbitan fatty acids. Can include, but are not limited to, esters, as well as combinations thereof. Additionally, coated suppositories may be in the form of rectal capsules in which a core containing free fatty acids is coated with a water-soluble compound such as gelatin.

In some embodiments, the composition may further include cocoa butter, coconut oil, glycerinated gelatin, hydrogenated oils, polyethylene glycol (PEG), or fatty acid esters of PEG.

In some embodiments, commercially available materials suitable for coatings according to the present invention include semi-synthetic vegetable-based oils available in multiple grades, including, but not limited to, Suppocire™ AS, AS2X, NA. bases, lipophilic bases Supocire(TM) (Gattefosse); Novata(TM) (Henkel Int.), such as Novata A, Novata B, and Novata BC; Witepsol(TM) (Dynamit Nobel Ab), For example, Witepsol™ H5, H12, H15, H32, H35, W25, W31, W32, W32, W35, and W45; Massa Estarinum™ (SASOL), such as preferably Massa Estarinum™ grade B , BC, E, and 299; Japocire™, and Ovucire™. The coating of the present invention may suitably contain any of the above-mentioned materials as a base. Hydrophilic waxes such as polyethylene glycols (eg, PEG 1500, PEG 3000, PEG 4000 and mixtures thereof) can also be used in the present invention. Suppocire AP is an amphiphilic base containing saturated polyglycolated glycerides. Mixtures of the above are also useful in the present invention. In some embodiments, hard fat (eg, a suitable Suppocire grade material) constitutes a major component of the coating material. By varying the relative amounts of components in the coating, desired properties such as softening time and disintegration time can be tailored. Generally, increasing the amount of hydrophobic wax, such as beeswax, increases the softening time as well as the disintegration time.

It is also preferred that the coating material further includes a lubricant such as, but not limited to, glyceryl dibehenate. In some embodiments, one or more pharmaceutically active ingredients may be added to the coating, including, but not limited to, locally acting anesthetics and and/or analgesics, such as lidocaine, prilocaine, benzocaine, chloroprocaine, bupivacaine, levobupivacaine, or other ester or amide local anesthetics, or any combination thereof. but not limited to.

Other pharmaceutical compounds may be included in the coating, such as, but not limited to, hydrocortisone, particularly if rapid local delivery is desired. Small pediatric suppositories are also within the scope of this invention. The coating material may vary depending on the desired dose, overall suppository size, or rate of release of the therapeutic composition.

Typical sizes of molded or kneaded suppositories for adults may range from about 2 to 3 mL, such as about 2.0 mL, about 2.2 mL, or about 2.5 mL. Depending on the composition of the excipients, this generally corresponds to a weight of the uncoated core in the range of about 1.5 to about 3 g, and therefore suppositories according to the invention preferably have a weight within this weight range. eg, having a core of about 1.8 g, about 2.0 g, about 2.2 g, or about 2.5 g. Suitable sizes for pediatric suppositories are generally about half of the above sizes, such as in the range of 0.5 to 1.5 mL, such as about 0.5 mL, about 0.8 mL, about 1.0 mL, about 1.2 or about 1 It would be .5 mL. The typical thickness of the coating layer over the soft suppository core is about 0.1-3 mm.

The coating material has one or more carbon atoms having a chain length in the range from 4 to 36 carbon atoms, such as a chain length in the range from 4 to 24 carbon atoms, or a chain length in the range from 8 to 24 carbon atoms. may contain fatty acids. Preferably, the one or more fatty acids comprises a mixture of fatty acids, which may be derived from suitable natural lipid materials, such as oils of animal or vegetable origin, fractions thereof, or any combination thereof.

Coating materials include palmitoleic acid (16:1 n-7), cis-vaccenic acid (18:1 n-7), oleic acid (18:1 n-9), elaidic acid (18:1), and linoleic acid. (18:2 n-6), α-linolenic acid (18:3 n-3), γ-linolenic acid (18:3 n-6), moroctique acid (18:4 n-3), Arachidonic acid (20:4 n-6), gadoleic acid (20:1 n-11), gondic acid (20:1 n-9), cis-11 eicosenoic acid (20:11 n-7), eicosapentaenoic acid (20:5 n-3; EPA), erucic acid (22:1 n-9), cetoleic acid (22:1 n-11), culpanodonic acid (22:5 n-3), and docosahexaenoic acid (22:1 n-11). Unsaturated fatty acids such as 6n-3; DHA) may also be included. Vegetable oils used as raw materials for the fatty acids of the present invention include safflower oil, corn oil, almond oil, sesame oil, soybean oil, flaxseed oil, rapeseed oil, grapeseed oil, sunflower oil, wheat germ oil, hemp oil, or combinations thereof. can be mentioned. In some embodiments, the fatty acids are derived from oil materials (pharmaceutical grade oils) that are pharmaceutically acceptable and defined according to pharmacopoeial standards. Such oils may include marine omega oils such as Omega-3 Fish Oil (Lysi, Iceland).

In some embodiments, the suppository forms of the invention may include an excipient oil component, such as a triacylglyceride oil, to reduce discomfort during therapeutic administration associated with bodily functions such as defecation. good. The excipient oil may be configured to ensure the desired melting point of the overall composition of the dosage form.

Although the coating allows for a preservative-free suppository, the coating may also contain antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and ascorbic acid. Examples include, but are not limited to, acids or salts thereof, sulfatide salts, citric acid, propyl gallate, alpha-tocopherol, ascorbyl palmitate, or any combination thereof. In some embodiments, the coating comprises Ci_6-alkyl-p-hydroxy-benzoic acid, such as methyl-p-hydroxy-benzoic acid, ethyl-p-hydroxy-benzoic acid, propyl-p-hydroxy-benzoic acid, A preservative may be included, such as any of the group consisting of benzoic acid or its derivatives, such as butyl-p-hydroxy-benzoic acid, or any combination thereof.

In some embodiments, the coating comprises Ci_6-alkyl-p-hydroxy-benzoic acid, such as methyl-p-hydroxy-benzoic acid, ethyl-p-hydroxy-benzoic acid, propyl-p-hydroxy-benzoic acid, It may further include a preservative, such as any of the group consisting of benzoic acid or its derivatives, such as butyl-p-hydroxy-benzoic acid, or any combination thereof.

In certain embodiments, the preservative is a mixture of methyl-p-hydroxybenzoic acid and propyl-p-hydroxybenzoic acid. Preservatives may be present in the coating preparation at a concentration of about 0.05-0.2% by weight, or other concentrations that do not impair the activity of the lipids.

The exosome formulations described herein are useful for treating inflammatory and autoimmune disorders in individuals. In certain embodiments, the inflammatory or autoimmune disorder comprises an inflammatory or autoimmune disorder of the gastrointestinal tract. In certain embodiments, the inflammatory or autoimmune disorder of the gastrointestinal tract is inflammatory bowel disease. In certain embodiments, the inflammatory or autoimmune disorder of the gastrointestinal tract is inflammatory Crohn's disease. In certain embodiments, the inflammatory or autoimmune disorder of the gastrointestinal tract is colitis. In certain embodiments. In certain embodiments, the individual is a human individual. In certain embodiments, the individual is a cow, horse, sheep, goat, pig, cat, or dog.

The exosome formulations described herein help reduce inflammation in an individual. In certain embodiments, the inflammation is inflammation of the gastrointestinal tract. The exosome formulations described herein are useful in reducing neutrophil accumulation or activation in an individual's gastrointestinal tract. In certain embodiments, the individual is a human individual. In certain embodiments, the individual is a cow, horse, sheep, goat, pig, cat, or dog.

In certain embodiments, a therapeutic dose of an exosome composition comprises from about 1 billion exosome particles to about 2 trillion exosome particles. In certain embodiments, the therapeutic dose of the exosome composition comprises about 100 billion exosome particles to about 250 billion exosome particles, about 100 billion exosome particles to about 500 billion exosome particles, about 100 billion exosome particles. of exosome particles - about 750 billion exosome particles, about 100 billion exosome particles - about 1 billion exosome particles, about 100 billion exosome particles - about 1.25 trillion exosome particles, about 100 billion exosome particles of exosome particles ~ about 1.5 trillion exosome particles, about 100 billion exosome particles - about 2 trillion exosome particles, about 250 billion exosome particles - about 500 billion exosome particles, about 250 billion of exosome particles - about 750 billion exosome particles, about 250 billion exosome particles - about 1 billion exosome particles, about 250 billion exosome particles - about 1.25 trillion exosome particles, about 250 billion exosome particles of exosome particles - about 1.5 trillion exosome particles, about 250 billion exosome particles - about 2 trillion exosome particles, about 500 billion exosome particles - about 750 billion exosome particles, about 500 billion exosome particles of exosome particles - about 1 billion exosome particles, about 500 billion exosome particles - about 1.25 trillion exosome particles, about 500 billion exosome particles - about 1.5 trillion exosome particles, about 5000 1 billion exosome particles to about 2 trillion exosome particles, about 750 billion exosome particles to about 1 billion exosome particles, about 750 billion exosome particles to about 1.25 trillion exosome particles, about 750 billion exosome particles billion exosome particles to approximately 1.5 trillion exosome particles, approximately 750 billion exosome particles to approximately 2 trillion exosome particles, approximately 1 billion exosome particles to approximately 1.25 trillion exosome particles, Approximately 1 billion exosome particles ~ approx. 1.5 trillion exosome particles, approx. 1 billion exosome particles ~ approx. 2 trillion exosome particles, approx. 1.25 trillion exosome particles ~ approx. 1.5 trillion of exosome particles, about 1.25 trillion exosome particles to about 2 trillion exosome particles, or about 1.5 trillion exosome particles to about 2 trillion exosome particles. In certain embodiments, the therapeutic dose of the exosome composition comprises about 100 billion exosome particles, about 250 billion exosome particles, about 500 billion exosome particles, about 750 billion exosome particles, about 1 billion exosome particles. of exosome particles, about 1.25 trillion exosome particles, about 1.5 trillion exosome particles, or about 2 trillion exosome particles. In certain embodiments, the therapeutic dose of the exosome composition comprises at least about 100 billion exosome particles, about 250 billion exosome particles, about 500 billion exosome particles, about 750 billion exosome particles, about 1 billion exosome particles. exosome particles, about 1.25 trillion exosome particles, or about 1.5 trillion exosome particles. In certain embodiments, a therapeutic dose of an exosome composition comprises up to about 250 billion exosome particles, about 500 billion exosome particles, about 750 billion exosome particles, about 1 billion exosome particles, about 1 Contains 250 billion exosome particles, about 1.5 trillion exosome particles, or about 2 trillion exosome particles. Doses can be formulated for oral, rectal, or vaginal administration.

Methods of Making Transrectal or Vaginal Delivery Systems In certain embodiments, also described herein are methods of making oral delivery systems, which methods include mixing a population of living cells with a matrix described herein. and contacting the matrix:liable cell population composition with the enteric coating. In certain embodiments, the matrix:live cell composition is added to the capsule before the enteric coating is applied. In certain embodiments, also described herein is a method of making an oral delivery system, which method comprises mixing a population of living cells with a matrix comprising an agar described herein; contacting the composition with an enteric coating comprising a copolymer of methacrylic acid.

In another aspect, a method of making a composition of mammalian exosomes formulated for oral delivery comprises combining a plurality of mammalian exosomes with about 1.0% to about 2.0% methylcellulose and about 1.0% methylcellulose. mixing with a matrix comprising about 2.0% alginate gel to provide a cellular matrix; and applying an enteric coating to the cellular matrix. In certain embodiments, the matrix includes about 1.3% to about 1.8% methylcellulose and about 1.3% to about 1.8% alginate gel. In certain embodiments, the matrix includes about 1.5% methylcellulose and about 1.5% alginate gel. In certain embodiments, methylcellulose and alginate gel are present in a ratio of about 1:1. In certain embodiments, the enteric coating comprises methyl methacrylate-methacrylic acid copolymer (1:1). In certain embodiments, the plurality of mammalian exosomes comprises human exosomes. In certain embodiments, the plurality of mammalian exosomes comprises non-human exosomes.

In certain embodiments, methods of making transrectal or vaginal delivery systems are also described herein, which methods include mixing a matrix:exosome preparation with a lipophilic compound as described herein; or contacting with a hydrophilic coating. In certain embodiments, the matrix:exosome preparation is added to the capsule before the lipophilic or hydrophilic coating is applied. In certain embodiments, also described herein is a method of making a transrectal or vaginal delivery system, which method comprises: mixing an exosome preparation with an agar-comprising matrix described herein; , matrix: contacting the exosome preparation with a lipophilic or hydrophilic coating comprising an excipient or a copolymer of methacrylic acid.

In certain embodiments, also described herein is a method of making a transrectal or vaginal delivery system, which method comprises: mixing an exosome preparation with an agar-comprising matrix described herein; , matrix: contacting the exosome preparation with an excipient or a lipophilic or hydrophilic coating comprising cocoa butter, coconut oil, glycerinated gelatin, hydrogenated oil, polyethylene glycol (PEG), or a fatty acid ester of PEG; include.

In certain embodiments, also described herein is a method of making an oral delivery system, which method comprises mixing an exosome preparation with a matrix described herein; contacting the soluble coating. In certain embodiments, the matrix:exosome preparation is added to the capsule before the enteric coating is applied. In certain embodiments, also described herein is a method of making a transrectal delivery system, which method comprises mixing an exosome preparation with a matrix comprising agar described herein; contacting the exosome preparation with an enteric coating comprising an excipient or a copolymer of methacrylic acid.

In another aspect, a method of making a preparation of exosomes formulated for rectal or vaginal delivery comprises preparing a preparation of exosomes with about 0.5% to about 3.0% methylcellulose and about 0.5% to about 3.0% methylcellulose; .5% to about 3.0% alginate gel to provide a cellular matrix; and applying a lipophilic or hydrophilic coating to the cellular matrix. In certain embodiments, the matrix includes about 1.0% to about 2.0% methylcellulose and about 1.0% to about 2.0% alginate gel. In certain embodiments, the matrix includes about 1.3% to about 1.8% methylcellulose and about 1.3% to about 1.8% alginate gel. In certain embodiments, the matrix includes about 1.5% methylcellulose and about 1.5% alginate gel. In certain embodiments, methylcellulose and alginate gel are present in a ratio of about 1:1. In certain embodiments, the lipophilic or hydrophilic coating comprises a methyl methacrylate-methacrylic acid copolymer (1:1).
Specific Embodiment 1. A composition for non-parental and/or rectal or vaginal delivery of mammalian exosomes, the composition comprising a lipophilic or hydrophilic coating surrounding a plurality of mammalian exosomes mixed with a solid matrix. wherein the matrix comprises a matrix comprising about 0.5% to about 3.0% methylcellulose and about 0.5% to about 3.0% alginate gel.
2. The composition of embodiment 1, wherein the matrix comprises a matrix comprising about 1.0% to about 2.0% methylcellulose and about 1.0% to about 2.0% alginate gel.
3. The composition of embodiment 1, wherein the matrix comprises about 1.3% to about 1.8% methylcellulose and about 1.3% to about 1.8% alginate gel.
4. The composition of embodiment 1, wherein the matrix comprises about 1.5% methylcellulose and about 1.5% alginate gel.
5. 5. The composition of any one of embodiments 1-4, wherein the matrix comprises a ratio of methylcellulose to alginate gel of about 1:1.
6. The composition according to any one of embodiments 1 to 5, wherein the exosome is a mesenchymal stem cell exosome or a hematopoietic stem cell exosome.
7. 7. The composition according to embodiment 6, wherein the exosome is a hematopoietic stem cell exosome.
8. 7. The composition according to embodiment 6, wherein the exosome is a mesenchymal stem cell exosome.
9. 9. The composition according to embodiment 8, wherein the mesenchymal stem cells are type I mesenchymal stem cells.
10. 9. The composition according to embodiment 8, wherein the mesenchymal stem cells are type II mesenchymal stem cells.
11. The exosome according to any one of embodiments 8-10, wherein the exosome comprises a protein or nucleic acid selected from the list consisting of CD81, CD63, CD9, HSP60, HSP70, HSP90, VEGF, PGE2, and combinations thereof. Composition.
12. The composition of any one of embodiments 8-10, wherein the exosome comprises a nucleic acid selected from the list consisting of miRNA Let7, 155, 146, 122, and combinations thereof.
13. The composition of any one of embodiments 1-12, wherein the mammalian exosome has a diameter of about 30 nanometers to about 150 nanometers.
14. The composition according to any one of embodiments 1-13, wherein the mammalian exosome is a human exosome.
15. The composition according to any one of embodiments 1-13, wherein the mammalian exosome is a canine, feline, bovine, equine, ovine, or porcine exosome.
16. The composition according to any one of embodiments 1-15, wherein the composition is formulated for rectal delivery.
17. 16. The composition according to any one of embodiments 1-15, wherein the composition is formulated for vaginal delivery.
18. 17. The composition according to any one of embodiments 1-16, wherein the formulation does not swell upon contact with an aqueous medium.
19. 19. The composition according to any one of embodiments 1-18, wherein the lipophilic or hydrophilic coating melts at normal body temperature or above 36°C.
20. Embodiment 1, wherein the lipophilic or hydrophilic coating consists essentially of cocoa butter, coconut oil, glycerinated gelatin, hydrogenated oils, polyethylene glycol (PEG), fatty acid esters of PEG, or a Gattefosse lipophilic base. 20. The composition according to any one of items 1 to 19.
21. Embodiments 1-19, wherein the lipophilic or hydrophilic coating comprises cocoa butter, coconut oil, glycerinated gelatin, hydrogenated oil, polyethylene glycol (PEG), fatty acid ester of PEG, or a Gattefosse lipophilic base. A composition according to any one of the above.

The following illustrative examples are representative of embodiments of the compositions and methods described herein and are not intended to be limiting in any way.

Example 1 - Collection of Mammalian Exosomes from Mesenchymal Stem Cells Collection of MSC Exosomes MSCs are incubated at 37°C in a humidified atmosphere containing 5% _CO2 . Adherent cells are grown until they reach approximately 80% confluence. The morphology of isolated and expanded MSC cells is evaluated during cell culture using light microscopy and FACS analysis.

To generate and collect exosomes, cells are cultured in optimal growth media for up to 72 hours (<2% exosome-free). After culture and a subsequent priming period, cell supernatants are collected and exosomes are isolated using the Fab-TACS® Exosome Isolation Kit (IBA Lifesciences, ThermoFisher Scientific, CA).

Enrich CD9- and CD81-positive exosomes from MSC cell culture supernatant using the corresponding Fab-TACS® Exosome Isolation Kit according to the accompanying manual. Briefly, MSC cell culture supernatants are centrifuged at 3000×g for 10 minutes. Filter the supernatant through a 0.2 μm polyethersulfone filter. Filter the elution buffer and PBS through a cellulose acetate filter (0.2 μm). After draining the storage buffer, the column is loaded with Fab-Streps. Transfer 1 ml of the filtered culture medium to the column. After washing, apply filtered biotin elution buffer to the matrix to collect exosomes. Exosome size is analyzed on a NanoSight LM10 instrument (Malvern Instruments) and data is processed using Nanoparticle Tracking Analysis [NTA] software 2.3. Protein content is determined by standard protein assays or Western blot using monoclonal detection antibodies for CD9, Alix, and CD63.

Example 2 - Stability of exosomes at different pH and under different gastrointestinal conditions Exosomes [5 billion particles] from mesenchymal stem cells derived from human adult tissues were prepared in an oral formulation (0.67% w/v alginate and 0.67% w/v alginate). % w/v methylcellulose, methyl methacrylate-methacrylic acid copolymer (1:1 enteric coated). The enteric-coated capsules were then processed similar to the expected pH changes as the capsules travel from the oral cavity to the ileum and colon. That is, after incubating for 2 hours at pH=1.2, 30 minutes at pH 4.2, 30 minutes at pH 6.8, and finally over 2 hours at pH=7.4, exosomes were collected and expanded 10 times. It was concentrated and measured. Figures 6A and 6B show that the enteric-coated capsule did not release the payload until it became neutral above pH 6.8. Figure 6B shows the number of particles recovered and stained for exosome-specific markers CD63, CD81, and CD9 from each sample measured on a NanoSight NS300 and analyzed on an ExoViewAnalyzer 3.0 [R100, NanoView Biosciences, MA]. shows. FIG. 6C shows intact recovered exosomes by immunofluorescence microscopy.

Enteric-coated capsules containing exosomes [5 billion particles] were processed similar to the expected pH changes as the capsules travel from the oral cavity to the ileum and colon. Therefore, after incubation for 2 hours at pH=1 (USP [United States Pharmacopeia] standard, simulated gastric fluid, SIG), 2 hours in simulated intestinal fluid (USP [United States Pharmacopeia], SIF) at pH=7.5 Neutralization was performed, cells were harvested, and assayed as described above. Figures 7A and 7B show that the enteric capsule did not release the payload until a neutral SIF was reached. This figure shows the recovered, exosome-specific marker CD63 (red), The number of particles stained for CD81 (blue) and CD9 (green) is shown. FIG. 7C shows intact recovered exosomes by immunofluorescence microscopy.

The recovery of such exosomes after incubation at high pH (mimicking the stomach and upper gastrointestinal tract) and in simulated gastric fluid, at a pH that mimics the lower gastrointestinal tract, is surprising. This is because the above exosome composition comprising a matrix of alginate and methylcellulose has alginate gel pores with a size of 5-150 nm [25-200 kDa], while exosomes are approximately 70-150 nM. Nevertheless, it is believed that the matrix retains exosomes in a pH-dependent manner, allowing them to pass intact through the low pH/high proteolytic environment of the upper gastrointestinal tract and be delivered to the colon.

Example 3 - Treatment of IBD mouse model with oral exosome formulation The efficacy of the described oral exosome formulation was tested in a dextran sodium sulfate (DSS) induced colitis model. This model is an established and validated model of inflammatory bowel disease (IBD) and closely resembles human IBD. Li et al. “Mesenchymal stem cells and acellular products attenuate murine induced colitis.”, Stem Cell Research and Therapy (2020) 11:515 Please refer to . In these experiments, colitis is induced in mice by administering DSS at a concentration of 3% w/v in drinking water. Briefly, DSS (MW approximately 40,000, Sigma-Aldrich, St. Louis, MO) is administered daily in the drinking water throughout the study period. As shown in Figure 8A, for each study, mice (n=6 per group) were divided into (a) untreated wild-type control group, (b) DSS treatment only group, and (c) DSS oral (PO) exosomes. [10 billion particles] treatment group, (d) DSS PO MSC [million cells] treatment group, and (e) DSS and oral formulation vehicle control group. Mice were observed daily for body weight, clinical signs, stool consistency and color. On days 1, 3, and 5 of the study (DSS administration started on day 0), vehicle, exosomes, and MSCs were administered orally as indicated. Clinical scoring was performed using the modified scoring matrix described in Figure 8A. Fecal occult blood was measured using a test kit (Fisher Healthcare, Houston, TX). As shown in Figure 8B, this treatment with orally formulated exosomes resulted in a significant reduction in symptoms associated with DSS-induced colitis in mice.

Example 4 - Preservation of Broad Anti-Inflammatory Properties of Oral Exosome Preparations In preclinical diseased animal studies, the main therapeutic benefits of exosome therapy are attenuation of neutrophil infiltration and neutrophil-released inflammatory factors. It has been proven that this is due to A potency assay was devised to measure specific inflammatory factors released by activated neutrophils. Neutrophils were obtained from an established ATCC human neutrophil-like cell line [HL60] that can be banked and standardized. These cells were co-cultured alone (negative control), with anti-inflammatory MSCs, or with exosomes (positive control cells, starting exosome product), i.e., the recovered and 10-fold concentrated oral exosome product from the pH study. , the effect on the expression of specific inflammatory factors was evaluated by RT-qPCR assay.

Briefly, neutrophil-like induction of cells was achieved by culturing HL60 cells in RPMI followed by addition of 1.3% DMSO for 3 days. Next, 2.5 million cells/mL of neutrophil-like HL60 cells were added to 24-well plates containing adherent human BM MSCs [250,000 cells - 0.1x, positive control] or 5 billion EVs. cells and activated with 10 μg/mL LPS overnight or no activation [negative control]. Subsequently, HL60 cells were collected, RNA was extracted using the miRNAeasy kit [Qiagen, MD], and then real-time qPCR was performed using human gapdh as a normalized housekeeping gene using a BioRad CFX96 [BioRad, CA].

Exemplary data from such an assay is shown in FIG. 9 and shows that an important mechanism of action of the described exosomes is inhibition of neutrophil activity and thus inhibition of inflammation in general.

While preferred embodiments of the invention have been illustrated and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used in practicing the invention.

All publications, patent applications, issued patents, and other documents referenced herein are incorporated by reference in their entirety. are herein incorporated by reference as if specifically and individually indicated. Definitions contained in text incorporated by reference are excluded if they conflict with the definitions of this disclosure.

Claims (32)

A composition for oral delivery of mammalian exosomes, the composition comprising a plurality of mammalian exosomes mixed with a matrix and surrounded by an enteric coating, the matrix comprising about 0.1% to about A composition comprising 5.0% methylcellulose and about 0.1% to about 5.0% alginic acid. 2. The composition of claim 1, wherein the matrix comprises about 0.5% to about 1.0% methylcellulose and about 0.5% to about 1.0% alginic acid. 2. The composition of claim 1, wherein the matrix comprises about 0.25% to about 0.75% methylcellulose and about 0.25% to about 0.75% alginic acid. 2. The composition of claim 1, wherein the matrix comprises about 0.5% to about 3.0% methylcellulose and about 0.5% to about 3.0% alginic acid. 2. The composition of claim 1, wherein the matrix comprises about 1.0% to about 2.0% methylcellulose and about 1.0% to about 2.0% alginic acid. 2. The composition of claim 1, wherein the matrix comprises about 1.3% to about 1.8% methylcellulose and about 1.3% to about 1.8% alginic acid. 2. The composition of claim 1, wherein the matrix comprises about 1.5% methylcellulose and about 1.5% alginic acid. A composition according to any preceding claim, wherein the matrix comprises a ratio of methylcellulose to alginic acid of about 1:1. The composition according to any one of claims 1 to 8, wherein the exosome is a mesenchymal stem cell exosome or a hematopoietic stem cell exosome. The composition according to claim 9, wherein the exosome is a hematopoietic stem cell exosome. The composition according to claim 9, wherein the exosome is a mesenchymal stem cell exosome. 12. The composition according to claim 11, wherein the mesenchymal stem cells are type I mesenchymal stem cells. 12. The composition according to claim 11, wherein the mesenchymal stem cells are type II mesenchymal stem cells. 14. The exosome according to any one of claims 11 to 13, wherein the exosome comprises a protein or nucleic acid selected from the list consisting of CD81, CD63, CD9, HSP60, HSP70, HSP90, VEGF, PGE2, and combinations thereof. Composition. 14. The composition of any one of claims 1-13, wherein the exosome comprises a nucleic acid selected from the list consisting of miRNA Let7, 155, 146, 122, and combinations thereof. 16. The composition of any one of claims 1-15, wherein the mammalian exosome has a diameter of about 30 nanometers to about 150 nanometers. The composition according to any one of claims 1 to 16, wherein the mammalian exosome is a human exosome. The composition according to any one of claims 1 to 16, wherein the mammalian exosome is a canine, feline, bovine, equine, ovine, or porcine exosome. Composition according to any one of the preceding claims, wherein the enteric coating comprises a methyl methacrylate-methacrylic acid copolymer (1:1). Composition according to any one of the preceding claims, wherein the enteric coating consists essentially of methyl methacrylate-methacrylic acid copolymer (1:1). A composition according to any one of claims 1 to 20, wherein the composition comprises at least 100 billion exosomes. The composition according to any one of claims 1 to 20, wherein the composition comprises at least 100 billion to no more than 2 trillion exosomes. 23. A composition according to any one of claims 1 to 22, wherein after 2 hours of incubation at pH 1.2, more than 40% intact exosomes are recovered from the composition compared to the initial amount. 23. A composition according to any one of claims 1 to 22, wherein more than 50% of intact exosomes are recovered from the composition after 2 hours of incubation at pH 1.2 compared to the initial amount. Composition according to any one of claims 1 to 22, wherein more than 60% intact exosomes are recovered from the composition after 2 hours of incubation at pH 1.2 compared to the initial amount. 26. A method of reducing neutrophil accumulation or neutrophil activation in the gastrointestinal tract of an individual, comprising orally administering to said individual a composition according to any one of claims 1 to 25. ,Method. 26. A method of treating an inflammatory or autoimmune disorder in an individual, the method comprising orally administering to said individual a composition according to any one of claims 1 to 25. 28. The method according to claim 26 or 27, wherein the individual is a human individual. 28. The method according to claim 26 or 27, wherein the individual is a dog, cow, cat, pig, horse, or sheep. 28. The method of claim 27, wherein the inflammatory or autoimmune disorder is a gastrointestinal inflammatory or autoimmune disorder. 28. The method of claim 27, wherein the gastrointestinal inflammatory or autoimmune disorder comprises inflammatory bowel disease or Crohn's disease. 28. The method of claim 27, wherein the gastrointestinal inflammatory or autoimmune disorder comprises inflammatory bowel disease (IBD).