Methods for preventing or treating age-related conditions using extracellular vesicles derived from young mammals

Abstract

The present disclosure relates to methods of treating, preventing, or delaying age-related conditions or pathologies in mammals using extracellular vesicles (EVs) derived from young mammals.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from Australian Provisional Patent Application No. 2022903549, filed on 23 November 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure generally relates to oral compositions comprising extracellular vesicles (EVs) derived from young (e.g., juvenile) non-human animals and methods of orally administering them to subjects in need thereof to treat, prevent, or delay age-related conditions or pathologies. [Background technology]

[0003] Aging is associated with the progressive degeneration of tissues, resulting in deterioration in the structure and function of internal organs. Aging is characterized by a generalized decline in physical and cognitive function due to deterioration in cellular mechanisms that maintain health and stress resistance, resulting in a decline in homeostasis and the ability to respond to external stimuli. While aging itself is not considered a disease, aging is associated with several chronic diseases, such as cardiovascular disease, arthritis, and cancer. The balance between damage and repair rates determines how organ integrity is maintained and regulated by both genetic factors and extracellular and intracellular molecular factors. The deterioration of homeostatic mechanisms within the nervous, endocrine, and immune systems drives common features associated with aging, such as immunosenescence, inflammasesenescence, and cellular senescence. Currently, there are no specific pharmaceutical interventions to treat, prevent, or delay aging and age-related diseases.

[0004] Experiments using heterochronic parabiosis, heterochronic organ transplantation, and the injection of young plasma into aged mice and rats have demonstrated that aging phenotypes can be reversed by exposing tissues and organs of aged animals to a young environment. Although attempts to identify the specific factors involved in the juvenile effect of young blood have been unsuccessful, some studies suggest that EVs may contribute to the juvenile process. Conversely, other experiments using parabiosis and plasma injection have demonstrated the pro-aging effects of introducing an aging systemic environment into young rodents. Therefore, it has been suggested that circulating EVs in disease states, such as cancer and neurodegenerative diseases, can spread the corresponding disease state when transferred to other mice. Furthermore, senescent cell-derived EVs have also been shown to propagate inflammatory conditions by spreading the senescence-associated secretory phenotype (SASP) and increasing senescence in recipient tissues. These EVs increase over time in the aging EV pool. Senescent cells are known to accumulate in organs with age and contribute to tissue dysfunction. Thus, due to their ability to induce physiological changes in cells, EVs have been shown to play a significant role in aging and disease, as well as in juvenile transformation. The fact that this can occur through the addition of juvenile factors or other methods, such as exercise and calorie restriction, demonstrates that the age-related loss of repair processes is not irreversible and suggests that repair processes can be reactivated.

[0005] Most studies exploring the therapeutic effects of EVs have injected a single bolus of EVs into animals, potentially supraphysiologically, resulting in the bulk of EVs being directed to organs involved in clearance before reaching other target cells. A recent study by Horvath et al. (2023) in Geroscience demonstrated epigenetic age reversal in rats via intravenous administration of an exosome-containing fraction derived from young adult pig plasma. Similarly, WO 2017 / 189842 describes experiments in which intraperitoneal injection of EVs derived from mouse stem cells or serum was used to treat age-related symptoms caused by stem cell dysfunction or increased aging in a mouse model. Sanz-Ros et al. (2022) Sci Adv 8(42):1-18 demonstrated improvements in the health of aging mice using intravenous administration of small EVs derived from adipose mesenchymal stem cells (ADSCs) from young animals.

[0006] Despite growing evidence that EVs can have blastogenic effects when administered parenterally in laboratory settings, repeated parenteral administration by intravenous and intraperitoneal routes may not be a practical long-term strategy for delivery to humans and has the additional disadvantage of potentially initiating an immune response, particularly with xenogeneic EVs. Thus, there remains a need for therapeutic compositions and methods for treating, preventing, or delaying aging and age-related conditions or pathologies, where the therapeutic agents can be easily administered (e.g., self-administered) and are well tolerated long-term.

[0007] Any discussion of documents, acts, materials, devices, articles or the like which has been included in this specification should not be construed as an admission that any or all of such matters form part of the basis of the prior art or were common general knowledge in the art relevant to this disclosure as they existed before the priority date of each of the appended claims. Summary of the Invention

[0008] The present disclosure is based, inter alia, on the recognition by the inventors that EVs derived from young animals can exert a mitogenic effect when administered parenterally and, therefore, may be useful for treating, preventing, or delaying age-related conditions or pathologies. However, in order for EVs to become a viable therapeutic strategy for the treatment of age-related conditions and pathologies (or indeed, any condition for that matter), the inventors recognized that safer, more convenient, and less invasive methods of administering therapeutically effective amounts of EVs to subjects needed to be developed, particularly for long-term treatment strategies. Accordingly, the inventors set out to develop oral formulations comprising EVs derived from young animals, and methods for treating, preventing, or delaying aging and age-related conditions or pathologies by administering the formulations by the oral route. Although the literature has recognized that EVs derived from young animals can have blastogenic properties when administered parenterally, to the inventors' knowledge, there is no evidence that delivery of EVs derived from young animals via the oral route can confer the same or similar physiological effects as parenteral administration to aged animals, much less that oral administration of EVs is well tolerated. In particular, it is unclear whether there is sufficient uptake and bioavailability of EVs when delivered by the oral route, and it is unclear whether any orally delivered EVs retain their efficacy and efficiency after passing through the gastrointestinal tract. In this regard, there has been little progress in achieving systemic delivery of other intact proteins or nucleic acids via the oral route, and the pharmacodynamics and pharmacokinetics of orally delivered EVs have not been thoroughly studied or validated. Therefore, the inventors explored the blastogenic potential of EVs derived from young animals (e.g., young mice and young cows) when orally administered to aged mice. In doing so, the inventors show for the first time that treatment of aged mice with EVs derived from young animals, delivered via the oral route, can improve several important parameters associated with age-related diseases and pathologies, including cognitive function, physical performance, metabolic function, inflammation, and oxidative stress.Furthermore, the present inventors have demonstrated that EVs derived from young cattle (i.e., calves) are well tolerated when orally administered to aged mice, despite their xenogeneic origin, and that these EVs are capable of conferring the physiological improvements described above. Thus, the present inventors have demonstrated, for the first time and unexpectedly, that EVs derived from young animals (e.g., juveniles) have mitogenic properties when orally delivered to aged animals, even when these EVs are of xenogeneic origin.

[0009] An advantage of the therapeutic approach developed herein is that it leverages oral tolerance mechanisms, similar to those for food proteins, substantially reducing the likelihood of a subject developing an allergic response. In particular, all EV purification methods have been shown to result in some degree of protein and lipoprotein contamination, and the repeated systemic injections previously demonstrated in studies present a significant risk of developing antibody responses and potential allergic reactions to the foreign proteins. This is an important consideration when designing an administration strategy, because EV-based treatments aimed at blastogenesis or anti-aging may require frequent administration over extended periods of time. Furthermore, zoonotic viruses, such as latent endogenous viruses, in animal tissues from pigs and cattle represent a potential hazard for systemic administration. This is expected to present significant regulatory challenges for EVs sourced from animals and intended for systemic injection in humans. This challenge is mitigated by formulating EVs for oral administration, because plasma (or other tissue sources) can be manufactured to food-grade standards. Thus, the oral formulations of the present disclosure offer several advantages.

[0010] The inventors also hypothesize that the ability to source EVs from a variety of species, even from animal species different from the species of the subject being treated, presents certain advantages for EV production and procurement. To date, EV production has been limited to small-scale laboratory settings, which has been recognized as an obstacle to scaling up production for therapeutic applications. As a result, the inventors recognized the need for a readily available, substantial source of EVs that allows for simple extraction from raw materials, is cost-effective, and yields sufficient quantities of EVs. For example, by-products (e.g., blood, tissues, and organs) from livestock slaughtered for human consumption may provide a readily available source of EVs that can be processed and formulated for oral delivery to human and non-human subjects. A selected raw material supply chain, combined with simplified extraction and minimal purification acceptable for oral administration, avoids stringent and costly regulatory standards for injectable regenerative biotherapeutics. Thus, in addition to the therapeutic benefits identified by the inventors, the present disclosure provides additional novel uses for slaughterhouse waste.

[0011] Thus, in one example, the present disclosure provides an oral composition comprising a therapeutically effective amount of EVs derived from the blood, blood fractions, and / or tissues of a young non-human animal.

[0012] In one example, a young non-human animal is an animal that is at or includes the age of maximum sexual maturity. For example, a young non-human animal can be an animal that has not yet reached sexual maturity. For example, a young non-human animal can be a juvenile animal, e.g., an animal that has not yet reached its adult form, sexual maturity, and / or size.

[0013] In one example, the EVs may be derived from livestock species. For example, the EVs may be derived from mammalian livestock species. Exemplary non-human mammals may be selected from cattle, sheep, horses, goats, pigs, camelids, or deer species. In one example, the EVs are derived from bovine animals. In one example, the EVs are derived from ovine animals. In one example, the EVs are derived from equine animals. In one example, the EVs are derived from caprine animals. In one example, the EVs are derived from porcine animals. In one example, the EVs are derived from camelids. In one example, the EVs are derived from deer animals. Alternatively or in addition, the EVs may be derived from non-mammalian livestock species, such as poultry.

[0014] The EVs described herein may be selected from the group consisting of exosomes, microvesicles, membrane particles, membrane vesicles, exosome-like vesicles, extracellular vesicles, exovesicles, and combinations thereof.

[0015] As described herein, EVs can be derived from blood, blood fractions, and / or tissues of young non-human animals. In one example, EVs are derived from whole blood. In one example, EVs are derived from blood fractions, such as serum or plasma. In one example, EVs are derived from bone marrow. In another example, EVs are derived from tissues. For example, the tissues can be selected from adipose tissue, thymus, pancreas, lung, heart, liver, muscle, intestine, and any combination thereof. In a particular example, the tissues are selected from adipose tissue, thymus, and pancreas.

[0016] In some instances, EVs are present in the oral compositions described herein at a concentration that is greater than the concentration present in the blood, blood fraction, or tissue from which the EVs are derived.

[0017] In one example, the EVs are purified. According to this example, the EVs are purified from the blood, blood fraction, or tissue from which they are derived (i.e., separated from other component parts of the blood, blood fraction, or tissue, respectively) and formulated into an oral composition.

[0018] In one example, the EVs are freeze-dried. In another example, the EVs are spray-dried. In a further example, the EVs are gelled. In another example, the EVs are encapsulated. In some examples, the EVs are freeze-dried or spray-dried and then encapsulated.

[0019] In one example, the EVs are provided in liquid form.

[0020] In some instances, the EVs are provided within a capsule, i.e., in some instances, the oral composition is provided as a capsule containing the EVs described herein.

[0021] The present disclosure also provides a method of preparing the oral compositions described herein, comprising: a) obtaining blood, blood fractions, and / or tissues from a non-human mammal; b) isolating EVs from blood, blood fractions, and / or one or more other components of the tissue.

[0022] The EVs isolated from blood, blood fractions, and / or tissues can then be formulated in a form suitable for oral administration.

[0023] In some examples, the method of preparing an oral composition described herein further comprises performing one or more purification processes and / or one of more processes for concentrating EVs, followed by isolating the EVs from one or more other components of the blood, blood fraction, and / or tissue. The one or more purification processes may be selected from size exclusion chromatography, ultracentrifugation, polymer and calcium ion precipitation, or salt-induced precipitation, and ultrafiltration. In one example, the method comprises concentrating the EVs by ultrafiltration and centrifugation. In some examples, the method of the present disclosure comprises one or more purification processes and one of more processes for concentrating EVs (e.g., relative to the concentration of EVs in an isolate obtained from the blood, blood fraction, and / or tissue).

[0024] Alternatively or additionally, the methods described herein may further comprise lyophilizing and / or encapsulating the EVs. For example, the methods may comprise lyophilizing and encapsulating the EVs. In accordance with examples in which the EVs are purified and / or concentrated, the lyophilization and / or encapsulation steps may be performed on the purified and / or concentrated EVs.

[0025] The methods of preparing the oral compositions described herein may further include formulating the EVs in capsules that are suitable for oral administration.

[0026] The present disclosure also provides oral compositions when produced by the methods described herein.

[0027] The present disclosure also provides a method of treating, preventing, or delaying an age-related condition or pathology in a subject, comprising orally administering to the subject an oral composition described herein.

[0028] The present disclosure also provides the use of a composition described herein in the manufacture of a medicament for treating, preventing, or delaying an age-related condition or pathology in a subject, wherein the medicament is an oral medicament.

[0029] As described herein, an age-related condition or pathology can be associated with a decline in one or more of cognitive function, physical performance, and / or metabolic function.

[0030] In one example, the age-related condition or pathology is associated with a decline in cognitive function. For example, the decline in cognitive function can include a decline in one or more of arousal, executive function, memory, learning, visual-spatial processing using the frontal cortex, thalamus, dorsolateral prefrontal cortex, and / or hippocampus, and / or olfactory sensitivity.

[0031] Alternatively or additionally, the age-related condition or pathology is associated with a decline in physical performance. For example, the decline in physical performance may include a decline in one or more of musculoskeletal strength, motor skills, and / or balance, and / or an increase in frailty.

[0032] Alternatively or additionally, the age-related condition or pathology is associated with a decline in metabolic function. For example, the decline in metabolic function can include glucose intolerance and / or insulin insensitivity.

[0033] In some examples, age-related conditions or pathologies may be associated with increased inflammation and oxidative stress in the tissues of a subject. For example, inflammation and / or oxidative stress may be associated with kidney dysfunction, acute kidney injury, chronic kidney disease, or acute and chronic liver injury in a subject. Thus, the method or use of the present disclosure may be for treating, preventing, or delaying an age-related condition or pathology selected from kidney dysfunction, acute kidney injury, chronic kidney disease, or acute and chronic liver injury in a subject in need thereof. In one example, kidney dysfunction is caused by acute kidney injury. In another example, kidney dysfunction is caused by chronic kidney injury. In one example, liver dysfunction is caused by acute liver injury. In another example, liver dysfunction is caused by chronic liver injury.

[0034] The present disclosure also provides a method of improving kidney function in a subject suffering from impaired kidney function, acute kidney injury, or chronic kidney disease, comprising orally administering to the subject an oral composition described herein.

[0035] The present disclosure also provides use of a composition described herein in the manufacture of a medicament for improving renal function in a subject suffering from abnormal renal function, wherein improving renal function in the subject comprises orally administering the medicament to the subject.

[0036] In one example, the renal function abnormality is caused by acute kidney injury. Thus, the subject may be suffering from acute kidney injury. In another example, the renal function abnormality is caused by chronic kidney injury. Thus, the subject may be suffering from chronic kidney injury.

[0037] The present disclosure also provides a method of improving liver function in a subject suffering from abnormal liver function, the method comprising orally administering to the subject an oral composition described herein.

[0038] The present disclosure also provides the use of a composition described herein in the manufacture of a medicament for improving liver function in a subject suffering from a liver dysfunction disorder, wherein improving liver function in the subject comprises orally administering the medicament to the subject.

[0039] In one example, the liver function abnormality is caused by acute liver injury. Thus, the subject may suffer from acute liver injury. In another example, the liver function abnormality is caused by chronic liver injury. Thus, the subject may suffer from chronic liver injury.

[0040] The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0041] [Figure 1] Discrimination index scores recorded for mice in different treatment groups performing the Novel Object Recognition Test (NORT). [Figure 2] A. Mean escape latency (seconds) recorded for mice in different treatment groups during the trial period of the Barnes maze test. B. Number of correct trials (nose pokes) recorded for mice finding the escape hole in the maze. [Figure 3] Assessment of fear conditioned responding in mice from each treatment group as measured by the percentage of time spent freezing. [Figure 4] A. Olfactory sensitivity measured by total exploration time (seconds) of mice in different treatment groups at each odorant dilution. B. Latency (seconds) to find buried food for mice in different treatment groups. [Figure 5] Scores recorded for mice in different treatment groups performing the tight rope test. [Figure 6] A. Body mass (g) recorded for mice in different treatment groups before performing the four-limb suspension test. B. Suspension time (sec) measured for each mouse on the inverted screen. [Figure 7] Frailty index scores based on several parameters (detailed in the assessment form) for mice in the different treatment groups. [Figure 8] A. Blood glucose levels (mmol / L) in mice injected with a 20% glucose solution as measured every 15 minutes for 120 minutes. B. Insulin sensitivity and glucose homeostasis in mice from different treatment groups as measured by area under the curve (mmol / L x min). [Figure 9] Measurement of serum liver enzymes in mice from different treatment groups to assess liver function after exposure to CCL4 (A. Baseline levels of serum liver enzymes in untreated young and old mice; B. Alanine transaminase, ALT; C. Aspartate transaminase, AST; D. Alkaline phosphatase, ALP). E. Measurement of malondialdehyde (MDA) levels as a marker of oxidative stress in the livers of these mice. F. Measurement of hydroxyproline levels as a marker of fibrosis in the livers of these mice. [Figure 10] Measurement of serum renal parameters (A. urea, B. creatinine) in mice from different treatment groups to assess renal function after exposure to hemoglobin. C. Measurement of malondialdehyde (MDA) levels as a marker of oxidative stress in the kidneys of these mice. DETAILED DESCRIPTION OF THE INVENTION

[0042] General Techniques and Definitions Unless specifically defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., genomics, immunology, molecular biology, immunohistochemistry, biochemistry, oncology, and pharmacology).

[0043] The present disclosure is carried out without undue experimentation and, unless otherwise indicated, employs conventional techniques of molecular biology, microbiology, recombinant DNA technology, and immunology. Such procedures are described, for example, in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Fourth Edition (2012), volumes I, II and III in their entirety; DNA Cloning: A Practical Approach, volumes I and II (D.N. Glover, Second Edition, 1995), IRL Press, Oxford, the entire text; Oligonucleotide Synthesis: A Practical Approach (M.J. Gait, ed., 1984), IRL Press, Oxford, the entire text, in particular the articles by Gait, p. 11-22; Atkinson et al., pp. 35-81; Sproat et al., pp. 83-115; and Wu et al., pp. 135-151 therein; 4. Nucleic Acid Hybridization: A Practical Approach (B.D. Hames & S.J. Higgins, eds., 1985), IRL Press, Oxford, the entire text. Press, Oxford, entire text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, entire text; Perbal, B., A Practical Guide to Molecular Cloning (1984); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), entire series; and Remington's Pharmaceutical Sciences, 16th Ed., Mack Publishing Company, 1980.

[0044] Those skilled in the art will understand that the present disclosure is susceptible to variations and modifications other than those specifically described. The present disclosure is understood to include all such variations and modifications. The present disclosure also includes all steps, features, compositions, and compounds individually or collectively referred to or indicated herein, and any and all combinations of any two or more of any of such steps or features.

[0045] The present disclosure is not limited in scope by the specific embodiments described herein, which are intended for purposes of illustration only. Functionally equivalent products, compositions, and methods as described herein are clearly within the scope of the present disclosure.

[0046] Each feature of any particular aspect or embodiment or embodiment of the present disclosure may be applied mutatis mutandis to any other aspect or embodiment or embodiment of the present disclosure.

[0047] Throughout this specification, unless specifically stated otherwise or unless the context otherwise requires, references to a single step, composition of matter, group of steps, or group of compositions of matter will be interpreted as encompassing one and more (i.e., one or more) of that step, composition of matter, group of steps, or group of compositions of matter.

[0048] As used herein, the singular forms "a," "and," and "the" include the plurals of these words unless the context clearly dictates otherwise. For example, a reference to "a bacterium" includes a plurality of such bacterium, and a reference to "an allergen" is a reference to one or more allergens.

[0049] The term "and / or," e.g., "X and / or Y," is understood to mean either "X and Y" or "X or Y," and is interpreted as providing explicit support for both meanings or either meaning.

[0050] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, entity, or step, or group of elements, entities, or steps, but not the exclusion of any other element, entity, or step, or group of elements, entities, or steps.

[0051] The term "about" is used herein to mean approximately. When used in conjunction with a numerical range, the term "about" modifies the range by extending the upper and lower limits of the recited numerical values. In general, the term "about" is used herein to modify numerical values, such as lengths of time, concentrations, temperatures, etc., to ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% above and below the stated value from the specified value, as appropriate for carrying out the disclosed methods.

[0052] Oral Composition This disclosure highlights the inventors' innovative approach in developing oral therapeutic compositions designed to treat, prevent, and / or delay age-related conditions, focusing on ease of administration, sustained tolerability, and therapeutic efficacy. Specifically, this approach addresses uncertainties regarding both the bioavailability and therapeutic efficacy of EVs when administered orally. Indeed, the application of several orally delivered EV compositions (e.g., dairy EVs and plant EVs) has been investigated, but results regarding systemic absorption, biodistribution, and bioavailability are uncertain.

[0053] As described herein, the inventors have shown for the first time that oral delivery of EVs derived from the blood, blood fractions, and / or tissues of young non-human animals can improve one or more parameters associated with age-related diseases and pathologies in aging animals, including, but not limited to, cognitive function, physical performance, metabolic function, inflammation, and oxidative stress. Accordingly, the present disclosure provides oral compositions comprising a therapeutically effective amount of extracellular vesicles (EVs) derived from the blood, blood fractions, and / or tissues of young non-human animals, and uses of such compositions for treating, preventing, or delaying an age-related condition or pathology in a mammalian subject via the oral route.

[0054] The terms "extracellular vesicles," "EVs," or "microvesicles" are understood to refer to nanosized membrane-bound structures with a lipid bilayer that are released by cells. EVs range in size from 1 micron or larger in diameter, approaching the size of the smallest physically possible unilamellar liposomes (approximately 20-30 nm). EVs are capable of transporting cargo, including proteins, nucleic acids, lipids, metabolites, and even organelles, from the parent cells into which they are released. Note that the terms "extracellular vesicles" and "microvesicles" are used interchangeably herein to describe all cell-derived membrane-bound structures except apoptotic bodies.

[0055] A wide variety of EV subtypes have been proposed, and EV subtypes are variously defined by size, neoplastic pathway, cargo, cellular source, and function. There is also some diversity in the nomenclature used to define various EV subtypes. It is contemplated that the oral compositions of the present disclosure may contain EVs present in the blood, blood fractions, and / or tissues of young non-human animals. Exemplary EV subtypes include, but are not limited to, exosomes, microvesicles, oncosomes, ectosomes, prostasomes, matrix / calcified vesicles, trellosomes, cardiosomes, and vexosomes, as well as any combination thereof. Extracellular vesicles and their respective properties are discussed in Lotvall et al. (2014) Journal of Extracellular Vesicles 3:26913 and Zempleni et al. (2013) Nature Reviews Drug Discovery 12:347-357, the contents of which are incorporated herein by reference.

[0056] As described herein, EVs vary in size, with a given sample of EVs having individual vesicles with an average diameter that varies within a range. EVs in compositions of the present disclosure can range in size from a lower size limit of about 20-50 nanometers (nm) in diameter to an upper size limit of about 1 micron in diameter.

[0057] The EVs used to formulate the oral compositions of the present disclosure may be derived from blood, blood fractions, and / or tissues of young non-human animals. As used herein, the term "blood" is understood to include whole blood, which is typically composed of unclotted plasma and cellular components. Plasma typically represents about 50% to about 60% of the volume, while the cellular components, i.e., erythrocytes (red blood cells or RBCs), leucocytes (white blood cells or WBCs), and thrombocytes (platelets), represent about 40% to about 50% of the volume. In some examples, the EVs in the oral compositions of the present disclosure are derived from whole blood. In other examples, the EVs in the oral compositions are derived from "blood fractions" or components of whole blood. For example, the EVs of the present disclosure may be derived from plasma, serum, red blood cells, platelets, fluids containing any of these substances, and / or fluids derived from any of these substances. As used herein, the terms "blood plasma" or "plasma" refer to the liquid fraction of blood and lymph, which constitutes approximately half of the blood's volume (e.g., about 50 to about 60% by volume). Plasma lacks cells and, unlike serum, is not coagulated. Plasma is a clear, yellowish liquid containing about 90 to about 95% by volume of water. As used herein, the terms "blood serum" or "serum" refer to the clear liquid that separates from blood upon complete clotting, and thus is plasma from which, among other things, fibrinogen has been removed during clotting. Like plasma, serum is pale yellow in color.

[0058] Alternatively or additionally, the EVs contained in the compositions of the present disclosure may be derived from non-human animal tissues. In one example, the tissue is a blood-related tissue, such as bone marrow. As used herein, the term "bone marrow" refers to the gelatinous tissue found in mammalian and avian bones, particularly the hollow interiors of mammals. In other examples, the tissues from which the EVs are derived are the thymus, liver, spleen, lung, brain, kidney, pancreas, gastrointestinal tract, small intestine, colon, stomach, heart, and any combination thereof.

[0059] As described herein, the EVs contained in the oral compositions of the present disclosure are derived from non-human animals. In one example, the EVs may be derived from livestock species. As used herein, the term "livestock" refers to domesticated animals raised in agricultural settings for commercial purposes (e.g., meat, wool, milk, etc.). Livestock species may be mammalian livestock species or avian livestock species. However, in one particular example, the composition contains EVs derived from a mammalian livestock species. Exemplary mammalian livestock species include bovine, ovine, equine, caprine, porcine, camelid, or cervid species. Alternatively or additionally, the EVs in the compositions of the present disclosure may be derived from a non-mammalian livestock species, such as poultry.

[0060] The non-human animal from which the EVs are derived is a young non-human animal. In this regard, the present disclosure is based on the unexpected finding that EVs derived from a young animal have juvenile potential when orally administered to an aged animal. As used herein, the term "young" refers to an immature animal. For example, a young animal may be a juvenile animal, e.g., an animal that has not yet reached its adult form, sexual maturity, and / or size. In some instances, a "young" animal is understood to refer to an animal that has not yet reached sexual maturity. Thus, in some instances, a young animal may be an animal that is at or includes the age of maximum sexual maturity.

[0061] As used herein, the term "sexual maturity" is understood to refer to the age or developmental stage at which an organism (e.g., an animal) is able to reproduce sexually. It will be understood by those skilled in the art that the age of sexual maturity may vary between species, breeds, and / or sexes, as well as between different individuals within any one or more of these categories.

[0062] In examples where the animal is a bovine animal, the bovine animal can be less than about 24 months old (e.g., less than about 18 months old, or less than about 12 months old, or less than about 9 months old, or less than about 6 months old). In one example, the bovine animal is less than about 24 months old. For example, the bovine animal is between about 1 month and about 24 months old (e.g., between about 1 month and about 6 months old, or between about 1 month and about 12 months old, or between about 1 month and about 18 months old, or between about 6 months and about 12 months old, or between about 6 months and about 18 months old, or between about 12 months and about 18 months old, or between about 12 months and about 24 months old, or between about 18 months and about 24 months old).

[0063] In examples where the animal is an ovine animal, the ovine animal is less than about 15 months old (e.g., less than about 9 months old, or less than about 6 months old, or less than about 4 months old). In one example, the ovine animal is less than about 15 months old. For example, the ovine animal is between about 1 month and about 15 months old (e.g., between about 1 month and about 6 months old, or between about 1 month and about 9 months old, or between about 6 months and about 9 months old, or between about 6 months and about 15 months old, or between about 9 months and about 15 months old).

[0064] In examples where the animal is an equine animal, the equine animal is less than about 48 months or 60 months old. However, in some examples, the equine animal is less than about 36 months old (e.g., less than about 24 months old, or less than about 18 months old, or less than about 12 months old). In one example, the equine animal is less than about 36 months old. For example, the equine animal is between about 1 month and about 36 months old (e.g., between about 1 month and about 6 months old, or between about 6 months and about 12 months old, or between about 6 months and about 18 months old, or between about 6 months and about 24 months old, or between about 6 months and about 36 months old, or between about 12 months and about 24 months old, or between about 12 months and about 24 months old, or between about 12 months and about 36 months old, or between about 18 months and about 24 months old, or between about 18 months and about 36 months old, or between about 24 months and about 36 months old).

[0065] In examples where the animal is a porcine animal, the porcine animal is less than 12 months old (e.g., less than about 11 months old, or less than about 10 months old, or less than about 9 months old, or less than about 8 months old, or less than about 7 months old, or less than about 6 months old, or less than about 5 months old, or less than about 4 months old). In one example, the porcine animal is less than about 12 months old. In one example, the porcine animal is less than about 6 months old. For example, the porcine animal can be about 1 month to about 12 months old (e.g., about 1 month to about 3 months old, or about 1 month to about 6 months old, or about 1 month to about 9 months old, or about 3 months to about 9 months old, or about 6 months to about 12 months old).

[0066] In examples where the animal is a camelid, the camelid is less than about 36 months old (e.g., less than about 24 months old, or less than about 18 months old, or less than about 12 months old, or less than 6 months old). In one example, the camelid is less than about 36 months old. For example, the camelid is between about 1 month and about 36 months old (e.g., between about 1 month and about 6 months old, or between about 1 month and about 12 months old, or between about 1 month and about 18 months old, or between about 1 month and about 24 months old, or between about 6 months and about 18 months old, or between about 6 months and about 24 months old, or between about 6 months and about 36 months old, or between about 12 months and about 24 months old, or between about 12 months and about 36 months old, or between about 18 months and about 36 months old, or between about 24 months and about 36 months old).

[0067] In examples where the animal is a deer animal, the deer animal may be less than about 18 months old (e.g., less than about 12 months old, or less than about 9 months old, or less than about 6 months old). In a particular example, the deer animal is less than about 18 months old. For example, the deer animal is between about 1 month and about 18 months old (e.g., between about 1 month and about 6 months old, or between about 1 month and about 12 months old, or between about 6 months and about 12 months old, or between about 6 months and about 18 months old, or between about 12 months and about 18 months old).

[0068] The oral composition may include a therapeutically effective amount of EVs from a young non-human animal to treat, prevent, or delay an age-related condition or pathology in a subject. As used herein, the term "therapeutically effective amount" describes an amount of a specified agent, e.g., EVs, that is sufficient to achieve a desired therapeutic, biological, or physiological effect in a subject being treated with the agent. In the context of the compositions of the present disclosure, a "therapeutically effective" amount of EVs from a young non-human animal may be an amount sufficient to reduce, alleviate, and / or prevent an age-related condition or pathology over the course of a treatment period. For example, a "therapeutically effective" amount of EVs from a young non-human animal may be an amount effective to reduce, prevent, or delay the progression of an age-related condition or pathology associated with a decline in one or more of cognitive function, physical performance, and / or metabolic function, or associated with oxidative stress and / or inflammation in a subject's tissues.

[0069] Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce a desired result without causing substantial cytotoxic effects in the subject. The effective amount of an agent useful for reducing, preventing, and / or delaying the age-related conditions and pathologies described herein will depend on the subject being treated, the type and severity (e.g., condition or pathological progression) of any associated disease, disorder, and / or condition, and the mode of administration of the therapeutic composition.

[0070] The EVs in the oral compositions of the present disclosure may be present at a concentration greater than that present in the blood, blood fraction, or tissue from which the EVs are derived. In this regard, the EVs may be subjected to one or more enrichment or purification steps (e.g., to separate the EVs from other component parts of the blood, blood fraction, or tissue, respectively) before the oral composition is formulated.

[0071] The oral compositions of the present disclosure contain at least 10 5 , 5×10 5 , 10 6 , 5×10 6 , 10 7 , 5×107 , 10 8 , 5×10 8 , 10 9 , 5×10 9 , 10 10 , 5×10 10 , 10 11 , 5×10 11 , or 10 12 , or 5 x 10 12 In some examples, the oral composition comprises 10 EVs derived from the blood, blood fractions, or tissues of a young non-human animal described herein. 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 ~10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 In one particular example, the oral composition may comprise EVs derived from the blood, blood fractions, or tissues of a young non-human animal described herein, such as at least 10 6 Or at least 10 8 The EVs include those derived from the blood, blood fractions, or tissues of the young non-human animals described herein.

[0072] The EVs present in the oral composition may be lyophilized, for example, to improve the stability of the EVs within the composition. As used herein, the term "lyophilized" is understood to mean that the EVs have been subjected to a lyophilization (i.e., freeze-drying) step. Thus, a lyophilized EV preparation may refer to a dried EV preparation or a preparation of EVs that has been subjected to lyophilization and then resuspended in an aqueous solution.

[0073] In some instances, the EVs present in the oral composition may be spray-dried. As used herein, the term "spray-dried" is understood to mean that the EVs have been converted from a fluid state to a dry powder form by spraying a liquid feed into a hot drying medium.

[0074] In other instances, the EVs present in the oral composition may be gelled. As used herein, the term "gelled" is understood to mean that the EVs have been processed to form a three-dimensional network, thus transitioning from a liquid composition to a viscous composition.

[0075] The oral compositions of the present disclosure may be formulated and provided in solid or liquid form.

[0076] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the lyophilized dried EV is mixed with at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate, dicalcium phosphate, or casein / caseinate, and / or a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants, such as glycerol, d) disintegrants, such as cryopreservatives, and / or glycerol-containing ethanol. The formulation may be mixed with starch, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution inhibitors such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain a buffering agent.

[0077] Solid compositions of a similar type can also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings, release-controlling coatings, and other coatings well known in the pharmaceutical formulation art. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0078] Liquid forms of oral compositions include, but are not limited to, sterile solutions and suspensions. For example, oral compositions may contain the EVs described herein formulated with a pharmaceutically and / or nutritionally acceptable carrier for administration to a mammal. Examples of such carriers include, but are not limited to, sterile aqueous or non-aqueous solutions, solvents, suspensions, and emulsions. Examples of non-aqueous solvents include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils, and organic esters. Aqueous carriers include, but are not limited to, water, alcohol, saline, and buffer solutions. Acceptable carriers may also include physiologically acceptable aqueous vehicles (e.g., saline) or other carriers used for oral administration.

[0079] The term "pharmaceutically and / or nutritionally acceptable carrier" refers to a non-toxic carrier or vehicle that does not destroy the pharmacological activity of the compound (or extracellular vesicle, as the case may be) with which it is formulated.

[0080] Preparation of oral compositions containing EVs The present disclosure provides a method for preparing the compositions described herein, comprising: a) Obtaining blood, blood fractions, and / or tissues from a young non-human mammal; b). isolating EVs from blood, blood fractions, and / or one or more other components of a tissue.

[0081] Blood, blood fractions, and / or tissues of young non-human animals can be obtained from any suitable source, and EVs are then isolated therefrom. In one example, EVs can be isolated from the blood, blood fractions, and / or tissues of young livestock species. In this regard, by-products from livestock slaughtered for human consumption, such as blood, tissues, and organs, can provide a readily available source of materials from which EVs can be isolated and formulated for oral delivery to human and non-human subjects. Thus, in addition to the therapeutic benefits identified by the inventors, the present disclosure contemplates that the compositions and processes for producing them described herein can provide novel uses for slaughterhouse waste. In one example, the livestock species can be a mammalian livestock species. Exemplary mammalian livestock species include, but are not limited to, cattle, sheep, horses, goats, pigs, or camelid species. In one example, EVs are isolated from the blood, blood fractions, and / or tissues of young bovine animals. In one example, EVs are isolated from the blood, blood fractions, and / or tissues of a young sheep animal. In one example, EVs are isolated from the blood, blood fractions, and / or tissues of a young horse animal. In one example, EVs are isolated from the blood, blood fractions, and / or tissues of a young caprine animal. In one example, EVs are isolated from the blood, blood fractions, and / or tissues of a young porcine animal. In one example, EVs are isolated from the blood, blood fractions, and / or tissues of a young camelid animal. Alternatively or additionally, EVs are isolated from the blood, blood fractions, and / or tissues of a young non-mammalian livestock species, such as poultry.

[0082] As described herein, the non-human animal from which blood, blood fractions, and / or tissues are obtained is a young non-human animal. In this regard, the present disclosure is based on the principle that EVs derived from blood, blood fractions, and / or tissues obtained from a young animal may have mitogenic potential or properties, and is based on the unexpected discovery by the inventors that these mitogenic properties can confer anti-aging effects (e.g., treatment, prevention, or delay of age-related conditions or pathologies) upon oral administration to an aging animal. The young non-human animal from which blood, blood fractions, and / or tissues are obtained may be an immature animal. For example, the young animal may be a juvenile animal, e.g., an animal that has not yet reached its adult morphology, sexual maturity, and / or size. In some examples, the young non-human animal may be an animal that has not yet reached sexual maturity. Thus, in some examples, the young animal from which blood, blood fractions, and / or tissues are obtained may be an animal that is at or includes the age of maximum sexual maturity.

[0083] Exemplary young non-human animals from which blood, blood fractions, and / or tissues may be obtained for the isolation of EVs are described herein and are intended to apply mutatis mutandis to each and every example describing methods for producing oral compositions of the present disclosure.

[0084] In one example, the method includes obtaining whole blood from the non-human animal and isolating EVs from the whole blood. In one example, the method includes obtaining a blood fraction, such as serum or plasma, from the non-human animal and isolating EVs from the blood fraction. For example, the EVs of the present disclosure can be isolated from plasma, serum, red blood cells, platelets, fluids containing any of these substances, and / or fluids derived from any of these substances. In another example, the method includes obtaining a tissue from the non-human animal and isolating EVs from the tissue. Suitable tissues include, but are not limited to, adipose tissue, bone marrow, thymus, liver, spleen, lung, brain, kidney, pancreas, gastrointestinal tract, small intestine, colon, stomach, heart, and any combination thereof.

[0085] As used herein, the term "isolating" refers to a process in which EVs are removed from their natural state, e.g., blood, blood fractions, and / or tissues. Thus, "isolated" EVs can be substantially or essentially free from components (e.g., cells and / or cell debris) that normally accompany EVs in their natural state.

[0086] The process of isolating (or harvesting) EVs from blood, blood fractions, and / or one or more other components of a tissue includes: (i) EVs in isolated form and with a greater concentration compared to the starting material (e.g., enriched), (ii) removal of any amount or type of impurity from the starting material (e.g., purification); (iii) an increase in the ratio of the amount of EVs to the amount of any unwanted components in the starting material (e.g., enrichment); (iv) any artificial process for removing EVs from their natural source or location; (v) any artificial process (e.g., purification) for separating EVs from at least one other component with which they are normally associated; or (vi) Any combination of (i), (ii), (iii), (iv), or (v) may result.

[0087] In some instances, isolating EVs from blood, blood fractions, and / or tissues purifies the EVs. However, in other instances, one or more additional purification steps may be performed. As used herein, the terms "purifying," "purify," "purification," or similar terms refer to a process for removing at least one impurity or contaminant from a starting material. For example, purifying a molecule of interest (e.g., EVs) from a starting material refers to a process for removing at least one impurity from the starting material to produce a relatively purer form of the molecule of interest.

[0088] EVs can be isolated and / or purified by any means known in the art, including centrifugation (e.g., differential ultracentrifugation), size-exclusion filtration, size-exclusion chromatography, affinity chromatography, density gradient centrifugation, immunoaffinity capture, polymer-based precipitation, calcium ion precipitation, salt-induced precipitation, and by using microfluidic techniques. In some examples, EV isolation and / or purification involves centrifugation of cells and / or cell-conditioned medium. In some examples, ultracentrifugation is used. In some examples, EV isolation and / or purification is carried out via size-exclusion filtration. In some examples, EV isolation and / or purification from a population of cells involves the use of discontinuous density gradients, immunoaffinity, ultrafiltration, and / or high-performance liquid chromatography (HPLC).

[0089] In some instances, differential ultracentrifugation may be used to isolate and / or purify EVs from cells or tissues, including centrifugal forces of at least 1000×g, 2000×g, 3000×g, 4000×g, 5000×g, 6000×g, 7000×g, 8000×g, or 9000×g to 2000×g, 3000×g, 4000×g, 5000×g, 6000×g, 7000×g, 8000×g, 9000×g, 10,000×g, or more to separate larger sized particles from cell- or tissue-derived EVs.

[0090] In some instances, isolation and / or purification of EVs from a population of cells involves the use of filtration or ultrafiltration. In certain instances, size-exclusion membranes with different pore sizes are used. For example, size-exclusion membranes can include the use of filters with pore sizes of at least 0.1, 0.5 μm, 1.0 μm, 2.5 μm, 5 μm to 0.5 μm, 1.0 μm, 2.5 μm, 5 μm, or larger. In some instances, the pore size is about 0.2 μm. In some examples, filtration or ultrafiltration involves size exclusion in the range of 0.1 kDa, 0.5 kDa, 1 kDa, 2 kDa, 5 kDa, 10 kDa, 25 kDa, 50 kDa, 100 kDa, or 250 kDa to 0.5 kDa, 1 kDa, 2 kDa, 5 kDa, 10 kDa, 25 kDa, 50 kDa, 100 kDa, 250 kDa, 500 kDa, or more.

[0091] In some instances, the isolated and / or purified EVs are filter sterilized, for example, with a 0.22 μm microbial exclusion filter. In some instances, the isolated and / or purified EVs are filtered using a 0.45 μm filter to remove cellular debris.

[0092] In some instances, isolation of EVs from a population of cells involves the use of a tangential flow filtration (TFF) system to purify and / or concentrate the EV fraction. In other instances, preparation of EVs from a population of cells involves the use of HPLC to purify EVs into uniformly sized particles. In other instances, centrifugation in a density gradient, e.g., a sucrose density gradient, or application of a separate sugar cushion in the preparation may be used.

[0093] In some instances, the preparation of EVs from a population of cells may involve the use of a precipitation reagent. For example, a precipitation reagent, such as EXOQUICK®, protamine, sodium acetate, magnesium sulfate, ammonium sulfate, sodium chloride, calcium chloride, potassium carbonate, and calcium sulfate, or a combination thereof, may be added to a solution containing a population of cells to rapidly precipitate a population of exosomes. In some instances, the preparation of EVs from a population of cells involves the use of a volume-excluding polymer (e.g., polyethylene glycol (PEG)). In yet other instances, the preparation of EVs from a population of cells involves the use of flow field-flow fractionation (FIFFF), an elution-based technique.

[0094] In some instances, agents that cause aggregation and / or precipitation of EVs may be added to the preparation before or after purification.

[0095] Additionally, any one or more of the processes for isolating or purifying EVs can be used to enrich the EVs, thereby increasing the concentration of EVs in the oral composition. Thus, in some instances, the methods of the present disclosure include an enrichment step, whereby the concentration of EVs in the oral composition is greater than the concentration present in the blood, blood fraction, or tissue from which the EVs are derived.

[0096] The identity, purity, and integrity of isolated and / or purified EVs can be confirmed using any number of methods known in the art, such as Western blot, nanoparticle tracking analysis, enzyme-linked immunosorbent assay (ELISA), flow cytometry, and transmission electron microscopy. The absence of aggregation and EV purity can be assessed as recommended by the International Society for Extracellular Vesicles (Lotvall J, et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracellular Vesicles 2014;3:26913).

[0097] As described herein, the oral formulations of the present disclosure comprise a therapeutically effective amount of EVs derived from the blood, blood fractions, or tissues of a young non-human animal. This is a quantity or amount of EVs sufficient to achieve a juvenile or anti-aging effect in a subject to which the oral composition is administered. Thus, the methods of the present disclosure can include formulating a specific dosage form of an oral composition, where the dosage form comprises a therapeutically effective amount of EVs isolated from the blood, blood fractions, or tissues of a young non-human animal. For example, the method can include formulating an oral composition, whereby the dosage form comprises at least 10 5 , 5×10 5 , 10 6 , 5×10 6 , 10 7 , 5×10 7 , 10 8 , 5×10 8 , 10 9 , 5×10 9 , 10 10 , 5×10 10 , 10 11 , 5×10 11 , or 10 12, or 5 x 10 12 In some examples, the method includes producing a dosage form of an oral composition comprising EVs isolated from the blood, blood fractions, or tissues of a young non-human animal described herein. 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 ~10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 In one particular example, the oral composition produced by the methods of the present disclosure comprises at least 10 EVs isolated from the blood, blood fractions, or tissues of a young non-human animal described herein. 6 Or at least 10 8 The EVs may include EVs isolated from the blood, blood fractions, or tissues of the young non-human animals described herein.

[0098] As described herein, the oral compositions of the present disclosure may contain EVs in freeze-dried, spray-dried, or gelled form. Therefore, the method for preparing an oral composition may further include freeze-drying the EVs. "Freeze-drying" or "lyophilization" refers to a drying method in which a water-containing sample is frozen and placed under reduced pressure to sublimate and remove the water in the sample, and the sample is then placed in long-term storage. Protectants for freeze-dried EVs include methionine, mannitol, and trehalose, and may be used in the form of an aqueous solution containing ascorbic acid and retinol. The aqueous solution may be water for injection, saline, phosphate buffer, purified water, or deionized water.

[0099] According to an example in which the oral composition of the present disclosure comprises spray-dried EV, the method for preparing the oral composition can include a step of spray-drying EV. Methods for spray-drying are known in the art. Spray-drying generally involves a process of converting a material from a fluid state to a dry form by spraying a liquid feed into a hot drying medium. The resulting product is generally and most preferably a powder. In some instances, the powder may exhibit some degree of granulation and / or agglomeration.

[0100] In accordance with the example where the oral composition of the present disclosure comprises gelled EVs, the method for preparing the oral composition may include a gelling step. Methods for gelling are known in the art and generally involve the formation of a three-dimensional network through the cross-linking of branched polymers, referred to as gelling agents. Using these gelling agents, a viscous EV composition, or gel, can be formed from the liquid composition.

[0101] If the EV composition is lyophilized or spray-dried, the composition may be maintained as a dry EV preparation prior to formulation into an oral composition, or may be resuspended in an aqueous solution prior to formulation into an oral composition. Thus, the oral compositions produced by the methods of the present disclosure may be in solid or liquid form.

[0102] Solid dosage forms of oral compositions that can be produced include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the lyophilized dried EV is mixed with at least one inert pharmaceutically acceptable excipient or carrier, such as sodium citrate, dicalcium phosphate, or casein / caseinate, and / or a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants, such as glycerol, d) disintegrants, such as cryopreservatives, and / or glycerol. The formulation may be mixed with starch, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution inhibitors such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain a buffering agent.

[0103] Solid compositions of a similar type can also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings, release-controlling coatings, and other coatings well known in the pharmaceutical formulation art. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0104] Liquid forms of oral compositions that can be produced include, but are not limited to, sterile solutions and suspensions. For example, oral compositions can include the EVs described herein formulated with a pharmaceutically and / or nutritionally acceptable carrier for administration to a mammal. Examples of such carriers include, but are not limited to, sterile aqueous or non-aqueous solutions, solvents, suspensions, and emulsions. Examples of non-aqueous solvents include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils, and organic esters. Aqueous carriers include, but are not limited to, water, alcohol, saline, and buffer solutions. Acceptable carriers can also include physiologically acceptable aqueous vehicles (e.g., saline) or other carriers used for oral administration.

[0105] The term "pharmaceutically and / or nutritionally acceptable carrier" refers to a non-toxic carrier or vehicle that does not destroy the pharmacological activity of the compound (or extracellular vesicle, as the case may be) with which it is formulated.

[0106] How to use Aging is a physiological process mediated by many biological and genetic pathways that is directly related to lifespan and is the driving force behind all age-related diseases. Aging is the irreversible, progressive decline of physiological, physical, and cognitive functions that ultimately leads to a myriad of age-related diseases, such as cardiovascular disease, musculoskeletal disorders, neurodegenerative diseases, organ dysfunction, diabetes, and dementia. The present disclosure is based, inter alia, on the unexpected discovery by the inventors that treatment of mature aging animals via the oral route using EV preparations isolated from blood obtained from young animals can improve several important parameters associated with age-related diseases and pathologies, including cognitive function, physical performance, metabolic function, inflammation, and oxidative stress. Accordingly, the present disclosure contemplates the use of the oral compositions described herein for the prophylactic, preventative, and / or therapeutic treatment of age-related conditions and / or pathologies in mammals.

[0107] As used herein, the terms "treating," "treat," or "treatment" include (i) preventing a pathological condition (e.g., fever or inflammation) from occurring (e.g., prophylaxis), (ii) inhibiting or arresting the onset of a pathological condition (e.g., fever or inflammation), and (iii) alleviating a pathological condition (e.g., alleviating symptoms associated with fever or inflammation).

[0108] In one example, the present disclosure provides a method of treating, preventing, or delaying an age-related condition or pathology in a subject, comprising orally administering to the subject an oral composition described herein.

[0109] In another example, the disclosure provides the use of a composition described herein in the manufacture of a medicament for treating, preventing, or delaying an age-related condition or pathology in a subject, wherein the medicament is formulated for oral administration.

[0110] An age-related condition or pathology may be associated with a decline in one or more of cognitive function, physical performance, and / or metabolic function.

[0111] In one example, the subject to whom the oral composition or medicament is administered suffers from or has a predisposition to an age-related condition or pathology associated with a decline in the subject's cognitive function. For example, the decline in cognitive function may include a decline in one or more of arousal, executive function, memory, learning, visual-spatial processing using the frontal cortex, thalamus, dorsolateral prefrontal cortex, and / or hippocampus, and / or olfactory sensitivity. In some examples, administering the oral composition or medicament to a subject may enhance the subject's neurological process and / or cognitive function compared to a corresponding subject not administered the oral composition or medicament. For example, administering the oral composition or medicament of the present disclosure to a subject may result in an improvement in cognitive function in the subject compared to a corresponding subject not administered the oral composition or medicament, and the improvement in cognitive function may include one or more of an improvement in recognition memory, enhanced learning and memory based on spatial / visual cues, an improved ability to learn and remember associations between environmental cues and aversive experiences, and increased olfactory sensitivity compared to a corresponding subject not administered the oral composition or medicament.

[0112] In another example, the subject to whom the oral composition or medicament is administered suffers from or has a predisposition to an age-related condition or pathology associated with a decline in physical ability. For example, the decline in physical ability may include a decline in one or more of musculoskeletal strength, motor skills, and / or balance, and / or an increase in frailty. In some examples, administering the oral composition or medicament to a subject may enhance the subject's physical ability or muscle strength compared to a corresponding subject not administered the oral composition or medicament. For example, administering the oral composition or medicament of the present disclosure to a subject may result in an improvement in the subject's physical ability compared to a corresponding subject not administered the oral composition or medicament, where the improvement in physical ability includes one or more of an increase in strength, an increase in agility, and / or a decrease in frailty compared to a corresponding subject not administered the oral composition or medicament.

[0113] In yet another example, the subject to whom the oral composition or medicament is administered suffers from or has a predisposition to an age-related condition or pathology associated with a decline in metabolic function. For example, the decline in metabolic function may include impaired glucose tolerance and / or insulin insensitivity. Thus, in some examples, administering an oral composition or medicament to a subject may enhance one or more metabolic functions in the subject's cells compared to a corresponding subject not administered the oral composition or medicament. For example, administering an oral composition or medicament of the present disclosure to a subject may result in an improvement in the metabolic function of the subject's cells compared to a corresponding subject not administered the oral composition or medicament, and the improvement in metabolic function may include one or more of an increase in glucose sensitivity and / or an improvement in glucose homeostasis compared to a corresponding subject not administered the oral composition or medicament.

[0114] In yet another example, the subject to whom the oral composition or medicament is administered suffers from or is predisposed to an age-related condition or pathology associated with a decline in the subject's immune system. Thus, in some examples, administering the oral composition or medicament to the subject can enhance the subject's immune system or response compared to a corresponding subject not administered the oral composition or medicament.

[0115] The present inventors have also provided evidence that oral delivery of the compositions or medicaments described herein may be useful in treating age-related conditions or pathologies associated with inflammation and / or oxidative stress. In this regard, the present inventors have shown that treatment of aged mice with the oral compositions of the present disclosure can reduce markers of inflammation and / or oxidative stress in the liver and kidney of aged mice. Thus, in some instances, the age-related condition or pathology being treated may be associated with increased inflammation and oxidative stress in a subject's tissue (e.g., kidney or liver). For example, inflammation and / or oxidative stress may be associated with abnormal kidney function, acute kidney injury, chronic kidney disease, or acute and chronic liver injury in a subject.

[0116] Thus, in one example, the present disclosure provides a method of improving kidney function in a subject suffering from impaired kidney function, acute kidney injury, or chronic kidney disease, comprising orally administering to the subject an oral composition described herein.

[0117] The present disclosure also provides use of a composition described herein in the manufacture of a medicament for improving renal function in a subject suffering from abnormal renal function, wherein improving renal function in the subject comprises orally administering the medicament to the subject.

[0118] Improved renal function may include faster resolution of acute renal stress in a subject suffering from acute kidney injury, or improvement in renal health in a subject suffering from chronic kidney injury. Resolution of acute renal stress or overall renal health can be determined by any means known in the art, such as measuring serum urea and / or creatinine levels in a subject after acute kidney injury (e.g., 24 hours after injury, 48 hours after injury, or 72 hours after injury). In one example, administration of an oral composition or medicament of the present disclosure to a subject suffering from acute kidney injury may result in faster resolution of renal dysfunction compared to a corresponding subject suffering from acute kidney injury but not administered the oral composition or medicament. For example, faster resolution of renal dysfunction may be determined by a decrease in serum urea and / or creatinine at least 24 hours (e.g., at least 48 hours) after renal stress relative to the serum urea and / or creatinine levels in a corresponding subject not administered the oral composition or medicament. In one example, improved renal function may include a faster resolution of acute renal stress in a subject who has suffered acute kidney injury. The level of resolution of acute renal stress can be determined by any means known in the art, for example, by measuring the level of serum urea and / or creatinine and / or reactive oxygen species (ROS) and / or reactive nitrogen species (RNS) in a subject after acute kidney injury (e.g., 24 hours after injury, 48 hours after injury, or 72 hours after injury). In this regard, increased and / or sustained levels of serum urea, serum creatinine, ROS, and / or RNS indicate renal stress and related renal dysfunction. Conversely, a decrease in one or more of serum urea, serum creatinine, ROS, and / or RNS in a subject after acute kidney injury (e.g., 24 hours after injury, 48 hours after injury, or 72 hours after injury) indicates improved renal function and resolution of renal stress.

[0119] In one example, the renal dysfunction is caused by acute kidney injury. Thus, the subject may be suffering from acute kidney injury. Acute kidney injury typically develops within hours or days and can be caused by physical trauma, acute poisoning, multiple organ failure, or various other diseases, such as infection. Acute kidney injury can be prerenal renal failure caused by hemorrhage, dehydration, osmotic diuresis, excessive diuretic use, sepsis, anaphylaxis, pancreatitis, afferent arteriolar vasoconstriction caused by NSAIDS, ACE inhibitors, and other vasoconstrictors. Acute kidney injury can also be intrinsic renal failure caused by primary or secondary glomerulonephritis (infectious, autoimmune, inflammatory), ischemia, nephrotoxicity, or sepsis. Acute kidney injury can also be postrenal renal failure caused by kidney stones or malignant tumors, bladder dysfunction, prostate hyperplasia, uterine disease, or catheter obstruction.

[0120] In another example, renal dysfunction is caused by chronic kidney injury.Chronic kidney disease typically develops over several years and causes irreversible damage.Causes can include chronic bacterial infections that cause kidney inflammation, polycystic kidney disease, various autoimmune diseases, hypertension, diabetes, persistent renal obstruction, chronic exposure to toxic chemicals, toxins, or drugs, nephrosclerosis, interstitial nephritis, or pyelonephritis.Therefore, the subject to be treated can suffer from chronic kidney disease.

[0121] The present disclosure also provides a method of improving liver function in a subject suffering from abnormal liver function, the method comprising orally administering to the subject an oral composition described herein.

[0122] The present disclosure also provides for the use of a composition described herein in the manufacture of a medicament for improving liver function in a subject suffering from liver dysfunction, wherein improving liver function in the subject comprises orally administering the medicament to the subject.

[0123] The degree of liver dysfunction and any improvement in liver function after treatment can be determined by means known in the art, for example, by measuring the levels of biomarkers related to liver inflammation (e.g., serum aminotransferases (ALT, AST) and / or alkaline phosphatase (ALP)), oxidative stress (e.g., malondialdehyde (MDA)), and / or fibrosis (e.g., hydroxyproline). In this regard, elevated levels of these biomarkers indicate liver injury and / or liver dysfunction. As described herein, treating a subject suffering from liver injury (acute or chronic) by administering an oral composition or medicament of the present disclosure can result in a reduction in the levels of one or more of ALT, AST, ALP, MDA, and / or hydroxyproline in the subject, for example, compared to a corresponding subject suffering from liver injury (acute or chronic) but not administered the oral composition or medicament.

[0124] In some cases, liver dysfunction may be the result of acute liver injury.Acute liver injury may be caused by drugs such as acetaminophen, NSAIDs, isoniazid and halothane, or toxins such as compounds derived from amanita phalloides or herbal medicines, viral infections such as hepatitis A, B, E or herpes simplex virus, shock, heatstroke or vascular problems caused by invasive malignant tumors, metabolic dysfunction such as acute fatty liver or alpha 1 antitrypsin deficiency.

[0125] In other examples, liver dysfunction may be the result of chronic liver disease, which may be caused by hemochromatosis, ischemia, sepsis, drugs, alcohol, viral infection with hepatitis A, B, or C, cirrhosis, fibrosis, cancer, nonalcoholic steatohepatitis, or autoimmune hepatitis.

[0126] As used herein, "treating," "treat," or "treatment" refers to a therapeutic intervention, course of action, or protocol that at least ameliorates the symptoms of such disease, disorder, or condition after the disease, disorder, or condition, and / or its symptoms, have at least begun to develop. As used herein, "preventing," "prevent," or "prevention" refers to a therapeutic intervention, course of action, or protocol that is initiated prior to the onset of an age-related condition or condition, and that prevents, inhibits, or delays the onset or progression of such condition, condition, or symptom thereof.

[0127] According to the above-mentioned use, the oral composition of the present disclosure can be administered to any animal. For example, the animal (or subject) to which the oral composition is administered can be a mammal, a bird, a chordate, an amphibian, or a reptile. In one example, the animal is a mammal. Exemplary mammalian subjects include, but are not limited to, humans, primates, livestock (e.g., sheep, cows, chickens, horses, camelids, goats, donkeys, pigs), companion animals (e.g., dogs, cats, horses), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), and captive wild animals (e.g., foxes, deer). In a specific example, the subject to which the oral composition is orally administered is a human.

[0128] The subject to whom the oral composition is orally administered is preferably an adult subject, for example, a sexually mature subject.In some cases, the subject suffers from one or more of the age-related conditions or pathologies described herein.In other cases, the subject does not suffer from one or more of the age-related conditions or pathologies described herein, but wishes to slow down the aging process and prevent or delay the onset of the age-related conditions or pathologies described herein.

[0129] The composition of the present disclosure can be orally administered to a subject in need thereof in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions or solutions.For oral use of tablets, commonly used carriers include lactose and corn starch.Also, lubricants such as magnesium stearate are typically added.For oral administration in capsule form, useful diluents include lactose and dried corn starch.

[0130] It is understood that the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, as well as the judgment of the treating physician and the severity of the particular age-related condition or condition being treated. Moreover, specific dosages may be varied over time, as needed, to maintain efficacy and / or minimize any adverse events.

[0131] The oral composition of the present disclosure can be administered in multiple doses at different times with defined intervals.For example, oral composition can be administered to a subject, for example, every day, or every 2 days, or every 3 days, or every 4 days, or every 5 days, or every 6 days, or every week, or every other week, or every month.The frequency of treatment can be adjusted as needed to ensure efficacy and / or minimize adverse events.For subjects who do not respond adequately to treatment, multiple doses can be administered within a week.Alternatively or additionally, escalating doses can be administered. [Example]

[0132] Example 1. Evaluation of cognitive function in aged mice treated with EVs derived from young and aged bovine animals In this example, the inventors evaluated whether treatment with EVs derived from young bovine animals improved cognitive function in aged mice compared to those treated with EVs derived from old bovine animals.

[0133] Cognitive performance encompasses attention / alertness, executive function, memory, and visuospatial processing, which utilize the frontal cortex, thalamus, dorsolateral prefrontal cortex, and hippocampus. Function in these regions is known to decline with age. When assessing age sensitivity for memory and learning, it is important to select tests that avoid food restriction or significant stressors that may affect the ability of older animals to perform the task and confound the results. The Novel Objection Recognition Test (NORT), Barnes maze test, fear recognition response test, and olfactory test were used here to assess cognitive function in aged mice treated with EVs derived from either young or aged bovine animals.

[0134] Materials and Methods EV extraction from bovine animals Blood was collected from young (weaned calves under 12 months of age) and old (8-9 years old) bovine animals by intravenous venipuncture into serum collection bags. The bags were centrifuged at 3,000 × g for 10 minutes at 4°C to remove cellular debris and sedimented red blood cells. The supernatant was then centrifuged at 12,000 × g for 45 minutes at 4°C to remove apoptotic bodies and clarified using a 0.45 μm filter.

[0135] The flow-through fraction was then subjected to filtration using a 100-300 kDa membrane to reduce the level of protein contamination and impurities smaller than 20 nm (i.e., smaller than exosomes). Depending on the volume required, this can be performed using a syringe filter, centrifugal filter, or tangential flow filtration. The filtrate was then concentrated in a swinging bucket rotor centrifuge with a Jumbosep centrifugal filter (Pall, NY, USA). To reduce the risk of EV aggregation during concentration, 0.45 μm filtered plasma was diluted 1:1 with 50 nM trehalose solution and then concentrated. After concentration, the presence of EVs was determined using an ExoELISA CD63 detection kit (Systems Biosciences, CA, USA). The EV concentrate was then resuspended in 50 nM trehalose solution containing 8% reconstituted micellar casein to create a 300 μl dose for oral gavage in mice.

[0136] treatment group Aged wild-type mice (22-24 months old) were divided into two groups and treated with EV concentrate every other day. A minimum of 300 μl of concentrate was administered to mice by oral gavage every other morning after overnight food deprivation. Food was then reintroduced one hour later.

[0137] Aged wild-type mice were divided into two groups: 1. Orally administered EVs procured from young cattle (EV+). 2. Orally administered EVs procured from aged bovine animals (EV-).

[0138] Young wild-type mice (3-4 months old) were used as controls for the test parameters. Mice were treated for a period of 30 days before any tests were performed.

[0139] NORT The NORT is used to assess primary visual learning and cognition, particularly recognition memory. The test is designed to assess short-term or long-term recognition memory in mice and rats and relies on rodents' innate preference for novelty. Aging mice have been shown to exhibit deficits in short-term memory, which affects their ability to recognize different objects. Performance is recorded by calculating a discrimination index based on the difference in time spent exploring existing and novel objects.

[0140] Barnes maze test The Barnes maze assesses hippocampal function and tests for spatial memory abnormalities. The Barnes maze assesses learning and memory primarily based on spatial / visual cues. The test utilizes mice's natural tendency to escape from a brightly lit, exposed area. Performance is measured by the latency and number of errors made by the mice in the maze. Mice are motivated to move from the exposed open platform to the escape hole. Because mice have a preference for solving mazes using visual cues, mice are trained to find the location of the escape hole using visual cues. The arena is a circular platform with holes around the perimeter. One contains an escape box in which the mouse can hide. There are spatial cues that the mouse can use to find the location of the escape hole. All mice undergo a 4-day training period before the test period. Performance is measured by the latency to escape and the number of attempts to find the escape hole, measured as a nose poke in the correct hole when the box is removed.

[0141] Fear Conditioned Response This test assesses hippocampal function and is capable of eliciting a contextual fear response when mice learn to associate a chamber with a mild foot shock, manifested as freezing behavior. A. spp.-dependent cued fear conditioning pairs a foot shock with light and sound cues in different chambers.

[0142] Mice are conditioned to the test chamber and then exposed to a tone in the test chamber, followed by a foot shock. Mice are placed in the chamber and the amount of time spent freezing in the chamber is recorded (contextual freezing). On the final day, mice are placed in a different chamber and exposed to the conditioning stimulus (tone), and the amount of time spent freezing is recorded (cued response). Freezing is defined as a complete lack of movement for a minimum of 0.75 seconds, and the percentage of freezing within each period is reported.

[0143] Olfactory test Neurogenesis, along with brain function and cognition, is known to decline with age. Age-related declines in neurogenesis are reflected in a decrease in olfactory sensitivity. To determine whether there was functional improvement after treatment with EV, both groups of aged mice were subjected to an olfactory sensitivity test (Witt RM et al. J Vis Exp. 2009;(23):949). This test measures the ability of mice to detect decreasing concentrations of odors and locate food through odor detection. Briefly, mice are assessed against serial dilutions of a specific scent (e.g., cinnamon), and the total exploration time at each dilution is recorded. The longer the exploration time, the higher the olfactory sensitivity. Mice were also subjected to a "buried food test" (Dan X. et al. Ageing Res Rev. 2021;70:101416). This test, widely used to assess overall olfactory ability and integration, measures how well mice detect and locate food buried under bedding. Mice are deprived of food before the experiment, and then given a very small amount of food to be used in the test. A small amount (less than 2 gm) of food is then buried under the bedding. The latency to initial excavation and the latency to find food are recorded. Olfactory function is evaluated by these latencies, and in the evaluation, better function is associated with a shorter latency.

[0144] result NORT EV+ mice demonstrated a high discrimination index when exploring between objects compared with EV- mice, similar to that observed in young control mice (Figure 1, p<0.01). These data indicated an improvement in recognition memory in EV+ mice.

[0145] Barnes maze test Mice in all treatment groups showed improved performance in escape latency during the trial period, with minimal age or treatment effects, and no significant differences in latency by day 4 (Figure 2A). However, EV+ mice exhibited similar numbers of trials (nose pokes) to those observed in young control mice, with an approximately two-fold increase in nose pokes compared to EV- mice (Figure 2B, p<0.05). These results demonstrated enhanced learning and memory based on spatial / visual cues in EV+ mice.

[0146] Fear Conditioned Response Although no significant difference in freezing time for the cue response was observed between EV+ and EV- mice, EV+ mice demonstrated a significant increase in freezing time for the contextual response compared with EV- mice (Fig. 3, p<0.05). These results indicated that EV+ mice had an improved ability to learn and remember associations between environmental cues and aversive experiences.

[0147] Olfactory test EV+ and young control mice were exposed to the two lowest dilutions of odorant (1 × 10 -5 and 1 x 10 -4 ) compared to EV- mice, whereas EV+ mice demonstrated significantly longer exploration times compared to EV- mice (Figure 4A, 1 × 10 -5 , p<0.01 and 1 × 10 -4 , p<0.05). Furthermore, a shorter latency period in finding the buried food reward was also observed in EV+ mice compared with EV- mice (Fig. 4B, p<0.01). These results suggested that EV+ mice had increased olfactory sensitivity compared with EV- mice.

[0148] conclusion EVs derived from young bovine animals are able to increase cognitive performance in aged mice compared to those treated with EVs derived from old bovine animals.

[0149] Example 2. Evaluation of physical performance in aged mice treated with EVs derived from young and aged bovine animals In this example, the inventors evaluated whether treatment with EVs derived from young bovine animals improved physical performance in aged mice compared to those treated with EVs derived from old bovine animals.

[0150] With aging, skeletal muscle shrinks, associated with a decrease in muscle fiber size and number, which contributes to muscle weakness. Loss of physical performance can be observed by decreased strength in the limbs and overall physical fitness, as well as increased frailty. Several methods were used here to assess physical performance in aged mice after treatment with EVs derived from either young or old bovine animals.

[0151] Materials and Methods Tight rope test It assesses both physical strength and motor skills. A 60 cm long rope is suspended between two platforms, and a mouse is suspended by its forelimbs from the center of the rope; the mouse must reach either platform within a specified time. Mice are scored based on the time spent and whether they succeeded in reaching the platform.

[0152] Four-limb suspension test This is a strength and endurance test in which mice support their body weight by hanging upside down on all four limbs. Mice are reluctant to fall from this position, which motivates them to persist until fatigued before falling. Mice have a high strength / weight ratio, and most young mice can persist for the maximum duration of the test. Here, the mouse is placed on a screen, which is then inverted and suspended on soft bedding. The latency to fall is recorded. A maximum score is recorded when the mouse does not fall within a given time frame.

[0153] frailty To assess frailty, several clinical signs of deterioration / deficiency were evaluated for each mouse. A score of 0 to 1 was assigned for each parameter, with 0 = absent, 0.5 = mild, and 1 = severe. Each mouse was weighed and its body surface temperature was measured with an infrared temperature probe. Parameters scored included: outer skin Age-related baldness and / or alopecia hair loss due to hair thinning Loss of coat color or a change in coat color from black to gray or brown Skin inflammation, excessive grooming, trimming, or scratches that cause skin sores Loss of whisker hair due to aging and / or whisker trimming Coat condition (ruffled and / or matte) Body / Musculoskeletal The development of a tumor or mass anywhere in the body Abdominal distension or enlargement Kyphosis (excessive outward curvature of the lower cervical / thoracic spine) ○Tail hardening Gait disturbance (poor coordination) ○ Rotation or relaxation Tremor (involuntary shaking at rest or during movement) Decrease in limb strength (fall from a suspended inverted screen) Body condition score (visual signs of muscle wasting or obesity based on the amount of flesh covering bony prominences) Vestibular Cochlea / Hearing Vestibular disorders (disruptions in the ability to perceive movement and gravity, resulting in problems with balance, orientation, and acceleration) Hearing loss (inability to respond to sudden sounds) eyes / nose ○ Cataract ○Cornea clouding ○Eye discharge / swelling ○Microphthalmia Loss of vision (indicated by an inability to reach the ground when lowered by the tail) The threat reflex (rapid blinking and closure of the palpebral fissure in response to a non-tactile visual threat to the eye) Runny nose Gastrointestinal / genitourinary ○Malocclusion (overgrowth or uneven incisors) ○Rectal prolapse ○Vaginal / uterine / penile prolapse Diarrhea respiratory system o Breathing rate / depth (dyspnea, rales, and / or tachypnea) discomfort ○ Mouse Grimace Scale (pain / discomfort based on facial expression. Assessment of five facial features: orbital tightening, nasal prominence, cheek prominence, ear position (retraction), or vibrissa changes (either posterior or anterior) Piloerection (involuntary hair growth due to activation of the sympathetic nervous system) others ○Increase or decrease in body temperature Weight gain or loss

[0154] result Tight rope test A significant increase in performance scores of EV+ mice, similar to those in young control mice, was observed compared to EV- mice (Figure 5, p<0.01), demonstrating that EV+ mice had increased strength and agility compared to EV- mice.

[0155] Four-limb suspension test The weight of each mouse was recorded (Figure 6A) and adjusted for the time elapsed before falling for each mouse. It was observed that EV+ mice and young control mice demonstrated similar lengths of suspension time, in contrast to EV- mice, which had significantly shorter suspension times than the other two groups (Figure 6B, p<0.05). This indicates increased strength and endurance capabilities in EV+ mice compared to EV- mice.

[0156] frailty Frailty scores were calculated for each mouse and plotted as median values ​​with SEM for the three groups. As expected, young control mice obtained a low index score of approximately 0.02. EV+ mice obtained a score of approximately 0.14 on the index, which was nearly 2.5-fold lower than EV- mice, which obtained a score of approximately 0.34 on the frailty index (Figure 7, p<0.001). These results indicate that EVs from young bovine animals can reduce frailty in aged mice.

[0157] conclusion EVs derived from young bovine animals were able to increase physical performance in aged mice compared to those treated with EVs derived from old bovine animals.

[0158] Example 3. Evaluation of metabolic function in aged mice treated with EVs derived from young and aged bovine animals In this example, we evaluated whether treatment with EVs derived from young bovine animals improved metabolic function in aged mice compared to those treated with EVs derived from old bovine animals.

[0159] A common hallmark of aging is metabolic dysfunction, and one of the most involved nutrient signaling pathways is the insulin / IGF1 pathway, which affects energy homeostasis. Glucose intolerance and insulin sensitivity have been observed in aging mice. Energy homeostasis is required to maintain energy balance in a steady state, resulting in optimal metabolic function in organisms. Ongoing energy imbalance leads to the development of metabolic diseases, such as obesity and diabetes.

[0160] The glucose tolerance test (GTT), which measures the clearance of glucose, was used here to assess metabolic function in aged mice treated with EVs derived from either young or old bovine animals.

[0161] Materials and Methods Mice were fasted for 16 hours prior to administration, and baseline fasting blood glucose levels were recorded via tail vein sampling. A 20% glucose solution (2 mg / kg body weight) was injected intraperitoneally, and serial blood glucose measurements using a clinical blood glucose meter were performed every 15-30 minutes over a 2-hour period following injection to determine peak levels, clearance rate, and area under the curve (AUC) to assess insulin sensitivity and glucose homeostasis.

[0162] result Similar trends in blood glucose levels after glucose administration were observed in the different treatment groups (Figure 8A), but EV- mice maintained higher blood glucose levels for a longer period compared to EV+ and young control mice, suggesting abnormal or reduced glucose clearance in these mice (Figure 8B, p<0.01).

[0163] conclusion EVs derived from young bovine animals were able to improve metabolic function by increasing glucose sensitivity in aged mice compared to those treated with EVs derived from old bovine animals.

[0164] Example 4. Evaluation of levels of oxidative stress and inflammation in aged mice treated with EVs derived from young and aged bovine animals In this example, we evaluated whether treatment with EVs derived from young bovine animals improved kidney and liver dysfunction in aged mice compared to those treated with EVs derived from old bovine animals.

[0165] Chronic inflammation is known to increase with age, leading to a condition considered "inflammage-aging" associated with age-related pathologies. Excess reactive oxygen or nitrite species (ROS / RNS) generated during inflammation can drive oxidative damage to cellular lipids and proteins. With aging, these processes lead to structural changes in organs, such as the kidney and liver, which can significantly affect their function and the overall health and lifespan of the organism. To demonstrate the effects of EVs on rejuvenating organ function, two commonly used experimental models of oxidative stress and inflammation in the kidney and liver were used.

[0166] Materials and Methods Models for Liver Injury Previous studies have demonstrated that aging livers are more susceptible to acute liver injury than young livers. Furthermore, the fibrotic response resulting from the inflammatory response to injury is significantly greater in aged mice. To mimic a model of liver injury, carbon tetrachloride (CCL4) was injected intraperitoneally (0.2 ml / kg body weight as a 20% solution in corn oil) into all three treatment groups. Mice were euthanized 96 hours after injection, and several markers were measured from serum and liver tissue.

[0167] Models for kidney injury Acute kidney injury is known to increase the risk of subsequent chronic kidney disease, especially with aging. Hemoglobin can induce acute kidney injury, and aging kidneys are particularly sensitive to hemoproteins, as evidenced by increases in serum creatinine, serum urea, and lipid oxidation markers after exposure. Three groups of mice were administered bovine hemoglobin solution (2 gm / kg body weight) via the tail vein, and the mice were euthanized 48 hours later. Blood and tissues were then collected, and parameters related to kidney function were measured. An additional control group containing young mice receiving only saline was included to determine baseline values.

[0168] result liver Serum liver enzymes commonly used for clinical assessment of liver function, such as serum aminotransferases (ALT, AST) and alkaline phosphatase (ALP), were measured, as well as the oxidation markers malondialdehyde (MDA) and the degree of fibrosis (hydroxyproline).

[0169] ALT, AST, and ALP levels in EV- mice were still significantly elevated after 96 hours, whereas EV+ and young control mice (data not shown) showed decreased levels of these markers, indicating resolution of oxidative stress and inflammation (Figure 9A-D, ALT p<0.05, AST p<0.01, ALP p<0.01). Residual lipid peroxidation, as determined by MDA detection, was still significantly higher in EV- mice compared with EV+ and young control mice, indicating abnormal clearance of oxidative damage and persistent oxidative damage (Figure 9E, p<0.05). Hydroxyproline levels in EV- mice were also significantly higher compared with EV+ and young control mice, indicating a prolonged fibrotic response in these mice (Figure 9F, p<0.01).

[0170] kidney Although no differences were observed between EV+ and young control mice, EV- mice demonstrated significant and sustained increases in serum urea and creatinine 48 hours after hemoglobin administration compared with EV+ and young control mice (Figures 10A and 10B, p<0.01 and p<0.05, respectively), indicating slower resolution of acute insult in these mice. Also, MDA levels persisted over time in EV- mice compared with the other two treatment groups, indicating sustained oxidative damage to the kidneys of these mice (Figure 10C, p<0.01).

[0171] conclusion EVs derived from young bovine animals were able to reduce oxidative stress and inflammation-induced damage in aged mice compared to those treated with EVs derived from old bovine animals.

Claims

1. An oral composition comprising a therapeutically effective amount of extracellular vesicles (EVs) derived from the blood, blood fractions, and / or tissues of a young non-human animal.

2. The composition of claim 1 , wherein the young non-human animal has not yet reached sexual maturity.

3. The composition of claim 1 or 2, wherein the EV is selected from the group consisting of exosomes, microvesicles, membrane particles, membrane vesicles, exosome-like vesicles, extracellular vesicles, exovesicles, and combinations thereof.

4. The composition of any one of claims 1 to 3, wherein the EV is derived from a livestock species.

5. The composition according to any one of claims 1 to 4, wherein the EV is derived from a non-human mammal.

6. The composition of claim 5 , wherein the non-human mammal is selected from cattle, sheep, horses, goats, pigs, or camelids.

7. The composition according to any one of claims 1 to 4, wherein the EV is derived from an avian livestock species.

8. The composition of any one of claims 1 to 7, wherein the EVs are derived from whole blood, serum, plasma, and / or bone marrow.

9. 8. The composition of claim 1, wherein the EVs are derived from at least one tissue selected from the group consisting of adipose tissue, thymus, pancreas, lung, heart, liver, muscle, intestine, and combinations thereof.

10. The composition according to any one of claims 1 to 7 and 9, wherein the EVs are derived from adipose tissue.

11. 11. The composition of any one of claims 1 to 10, wherein the EVs are present in the composition at a concentration that is greater than the concentration that is present in the blood, blood fraction, or tissue from which the EVs are derived.

12. The composition of any one of claims 1 to 11, wherein the EV is purified.

13. The composition of any one of claims 1 to 12, wherein the EV is freeze-dried, spray-dried, or gelled.

14. The composition according to any one of claims 1 to 13, wherein the EV is encapsulated.

15. The composition of any one of claims 1 to 13, wherein the EV is provided in a capsule.

16. The composition of any one of claims 1 to 12, wherein the EV is provided in liquid form.

17. A method for preparing a composition according to any one of claims 1 to 16, comprising the steps of: a. Obtaining blood, blood fractions, and / or tissue from a non-human mammal; b. isolating said EVs from one or more other components of said blood, blood fraction, and / or tissue.

18. 20. The method of claim 17, further comprising performing one of one or more purification processes and / or more processes to enrich the EVs.

19. 20. The method of claim 18, wherein the one or more purification processes are selected from size exclusion chromatography, ultracentrifugation, polymer or calcium ion or salt induced precipitation, and ultrafiltration.

20. 19. The method of claim 18, wherein the EVs are concentrated by ultrafiltration and centrifugation.

21. 21. The method of any one of claims 17 to 20, further comprising freeze-drying the EVs.

22. A composition when produced by the method of any one of claims 17 to 21.

23. 23. A method of treating, preventing, or delaying an age-related condition or pathology in a subject, comprising orally administering to the subject a composition of any one of claims 1-16 or 22.

24. 17. Use of a composition according to any one of claims 1 to 16 in the manufacture of a medicament for treating, preventing or delaying an age-related condition or pathology in a subject.

25. 25. The method of claim 23 or the use of claim 24, wherein the age-related condition or pathology is associated with a decline in one or more of cognitive function, physical performance, and / or metabolic function.

26. 26. The method or use of claim 25, wherein the decline in cognitive function comprises a decline in one or more of arousal, executive function, memory, learning, visuospatial processing using the frontal cortex, thalamus, dorsolateral prefrontal cortex, and / or hippocampus, and / or olfactory sensitivity.

27. 27. The method or use of claim 25 or 26, wherein the decline in physical performance comprises a decline in one or more of musculoskeletal strength, motor skills, and / or balance, and / or an increase in frailty.

28. 28. The method or use according to any one of claims 25 to 27, wherein the reduction in metabolic function comprises impaired glucose tolerance and / or insulin insensitivity.

29. 25. The method of claim 23 or the use of claim 24, wherein the age-related condition or pathology is associated with inflammation and / or oxidative stress.

30. 30. The method or use of claim 29, wherein the inflammation and / or oxidative stress is associated with impaired kidney function, acute kidney injury, chronic kidney disease, or acute and chronic liver injury in the subject.

31. 23. A method of improving kidney and / or liver function in a subject suffering from a kidney and / or liver condition, comprising orally administering to said subject a composition of any one of claims 1 to 16 or 22.

32. 20. Use of a composition according to any one of claims 1 to 16 in the manufacture of a medicament for improving kidney and / or liver function in a subject suffering from a kidney and / or liver condition.

33. 33. The method of claim 31 or the use of claim 32, wherein the renal condition is acute kidney injury and / or the hepatic condition is acute liver injury.

34. 33. The method of claim 31 or the use of claim 32, wherein the renal condition is chronic kidney disease and / or the liver condition is chronic liver disease.

35. 17. A method of ameliorating decline in one or more of cognitive function, physical performance, and / or metabolic function by orally administering to a subject in need thereof a composition according to any one of claims 1 to 16.

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