Amino acid composition and use thereof

Abstract

The present disclosure provides compositions and methods for promoting stem cell and / or progenitor cell proliferation and / or differentiation. The compositions provided can be used to treat diseases or conditions such as wound healing associated with mucosal barrier function in subjects in need, treat skin disorders (e.g., atopic dermatitis, psoriasis, bedsores or conditions associated with skin aging), treat pulmonary disorders (e.g., asthma), conditions associated with improving mucosal barrier function and / or treat GI mucosal damage. The present disclosure also provides methods for promoting the proliferation and / or differentiation of stem cells and / or progenitor cells in subjects in need of such treatment by administering compositions. The ability to stimulate the proliferation and / or differentiation of stem cells and / or progenitor cells in vivo, in vitro and / or in vitro provides great benefits. The present disclosure can be used to increase stem cell populations in vivo, in vitro and / or in vitro. Stem cell transplantation provides treatment and / or cure for many disease states, degeneration and / or damage.

Description

[0001] Case information

[0002] This application is a divisional application of the invention patent application with application number 201780074351.9 filed on October 4, 2017 and invention name “Amino Acid Compositions and Uses Thereof”.

[0003] Related Applications

[0004] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application USSN 62 / 403,965, filed on October 4, 2016, and U.S. Provisional Application USSN 62 / 421,443, filed on November 14, 2016, each of which is incorporated herein by reference. Background Art

[0005] Human intestinal epithelial cells are generated from a fixed stem cell population functionally located in the lower part of the intestinal crypts, which in mice includes rapidly cycling crypt base columnar cells (CBCs) and more quiescent "+4" cells above Paneth cells. 14-16 These stem cells give rise to absorptive enterocytes, mucus cells, Paneth cells, and enteroendocrine cells. 17 Differentiation of each cell type occurs as cells migrate upward to the villi (absorptive cells, mucus cells, and endocrine cells) or concentrate downward at the bottom of the crypts (Paneth cells). The multiple mechanisms involved in these complex events are not fully understood.

[0006] Radiation and / or chemotherapy can cause severe damage to the lining of the gastrointestinal (GI) tract. Moderate to high doses of radiation and / or chemotherapy result in the destruction of cells with clonogenic potential that are necessary for the continuous replacement of cells shed from the villus tips during normal proliferation, maturation, and differentiation.

[0007] Toxic effects of radiation exposure and / or chemotherapy on the gastrointestinal system cause symptoms such as nausea, vomiting, diarrhea, electrolyte imbalance, and dehydration, and adversely affect the patient's health during cancer treatment. Radiation exposure affects the intestinal epithelial cells that undergo rapid mitosis in the submucosal crypts. In therapeutic radiation exposure, gastrointestinal toxicity often becomes the dose-limiting factor of treatment and may affect the patient's quality of life. Therapeutic compounds and supportive care are often used to minimize toxicity, but these approaches are not completely effective.

[0008] There is increasing interest in developing agents to mitigate short-term and long-term GI toxicity in cancer patients and victims of radiation disasters. 5,15,34,37 There are only two FDA-approved agents; The two FDA-approved medical countermeasures currently available for the treatment of radiation syndrome are tadalafil and pegfilgrastim (granulocyte colony-stimulating factor). Both are used to improve the survival of patients exposed to myelosuppressive doses of radiation. However, there are no agents that specifically address gastrointestinal toxicity. Treatment of GI toxicity is primarily symptomatic, with antidiarrheals used to prevent fluid loss, montmorillonite used to absorb bile salts, opioids used to relieve stomach or rectal pain, steroids used to relieve inflammation, and in extreme cases parenteral feeding used to correct malabsorption of nutrients and electrolytes. Other agents that could potentially be used to mitigate GI toxicity are: 1) statins and / or angiotensin-converting enzymes, which have been found to be effective when used during radical pelvic radiation therapy through their anti-inflammatory, antifibrotic, and antithrombotic effects; 2) antioxidants such as vitamin E and / or selenium; 3) teduglutide, which is a glucagon-like peptide-2 analog that must be given before radiation; 4) sucralfate, which is a highly sulfated polyanionic disaccharide that aids epithelial healing but has not been shown to be useful for radiation-induced GI toxicity; 5) nitroxides (n itroxides such as hydroxylamine (tempol), which act through their antioxidant properties; 6) dithiolthione (Oltipraz), which acts by increasing sulfhydryl groups in the cell; 7) isoflavones (genistein), which are tyrosine kinase inhibitors and antioxidants; 8) Cox inhibitors (celecoxib, aspirin), which act by increasing Cox2 activity and prostaglandin synthesis; and 8) prebiotics, which are preparations containing large amounts of live and well defined microorganisms that are used to alter the host microflora and may have some effect on radiation-induced GI toxicity, 5 (Stacey, R. & Green, J TR Radiation-induced small bowel disease: latest developments and clinical guidance. Ther. Adv. Chronic Disease, 5, 15-29, doi: 10.1177 / 2040622313510730 (2014)).

[0009] Crypt to villus migration takes 5-7 days. Therefore, gastrointestinal toxicity is manifested in the first week after radiation exposure and / or chemotherapy and is the most important dose-limiting factor in cancer treatment. Even at low doses, continuous loss of villus and brush border of the small intestine is observed within a few days after irradiation and / or chemotherapy. Although crypt cells can quickly rebuild (repopulate) this area after mild to moderate doses of irradiation and / or chemotherapy, they are lost at a logarithmic rate after irradiation and / or chemotherapy at high doses.

[0010] Irradiation and / or chemotherapy are particularly destructive to the villus epithelium that absorbs nutrients and electrolytes. The villus epithelium undergoes a continuous cell loss and regeneration process, in which a constant supply of immature enterocytes originating from progenitor cells in the lower pole of the crypt of Lieberkuhn migrates out of the proliferation compartment at the crypt base to the top of the villus. In their short life span, these enterocytes gradually mature into villus cells along the crypt-villus axis. Radiation and / or chemotherapy not only destroy existing villus cells, but also destroy stem cells and / or progenitor cells that form new villus cells, thus depleting almost the entire villus epithelium even at moderate doses.

[0011] Mature and differentiated villous cells are involved in fluid absorption secondary to sodium, chloride, and nutrient absorption, whereas less differentiated, immature epithelial cells located in the crypts are primarily involved in chloride absorption. - ) secretion and fluid loss. The lack of absorptive villous epithelial cells results in a malabsorptive state in which unabsorbed nutrients, electrolytes, and water are dumped into the distal GI tract, resulting in nausea, vomiting, and diarrhea.

[0012] Stem cell-mediated reconstitution of villous cells, either by proliferation of cells in situ and / or potential migration into the tissue from distant sites via the circulation of progenitor cells, is relevant to recovery from the effects of acute irradiation and / or chemotherapy at the tissue level. 11-13 Therefore, the loss of crypt stem cells or villus endothelial cells is thought to be involved in radiation- and / or chemotherapy-induced intestinal damage.

[0013] Damage to the GI tract not only leads to malabsorption and the loss of nutrients, minerals and fluids, but also destroys the intestinal barrier function. Leaky gut allows pathogens and other antigenic substances to easily enter the systemic compartment from food, passing through the mucosal barrier, thereby causing inflammation, bacteremia and endotoxemia. For example, even if the dose is as low as 5-12Gy (the conventional fractionation process of radiation uses 1.8-2Gy per fraction), acute radiation enteritis, diarrhea and abdominal pain can be developed within a few days after irradiation, but GI toxicity usually occurs at higher doses. Chronic radiation enteritis can be developed between 18 months and 6 years after radiotherapy, but may even be developed after 15 years. 27-29

[0014] Treatment options for radiation- and / or chemotherapy-induced enteritis are limited. Conventional treatment options include administration of antidiarrheal drugs to prevent fluid loss, administration of montmorillonite as a bile salt adsorbent, administration of opioids to relieve stomach or rectal pain, and administration of steroids to relieve inflammation.

[0015] A common approach in the treatment of radiation- and / or chemotherapy-induced enteritis is to provide bowel rest using total parenteral nutrition (TPN); however, whether parenteral nutrition meets the nutritional needs of patients or is actually therapeutic for radiation- and / or chemotherapy-induced enteritis remains to be determined. Although TPN can correct nutritional imbalances in some patients, severe radiation- and / or chemotherapy-induced enteritis may still develop. 37 TPN also causes intestinal atrophy, which usually occurs within 48 hours of administration. TPN also weakens the mechanical and immune barriers. 38

[0016] The formulation used in U.S. Pat. No. 8,993,522 works by correcting rehydration via amino acid coupled sodium transport, reducing anion secretion from the crypts by selecting a set of amino acids with antisecretory properties, and tightening the mucosa by using amino acids shown to tighten the mucosal barrier.

[0017] Improved compositions are needed for treating irradiation and / or chemotherapy-induced GI damage secondary to the loss of proliferating stem cells and / or progenitor cells. Also needed are compositions for treating diseases or conditions associated with mucosal barrier function such as wound healing, treating skin conditions (e.g., atopic dermatitis, psoriasis, bedsores, or conditions associated with skin aging), treating lung disorders (e.g., asthma), improving mucosal barrier function, and / or treating GI mucosal damage in subjects in need thereof. Summary of the invention

[0018] Amino acid compositions for treating GI, lung and skin disorders are described herein. In one aspect, the disclosure provides compositions and methods for promoting cell survival, proliferation, migration, maturation and / or cell differentiation. In certain embodiments, the disclosure provides compositions and methods for promoting stem cell and / or progenitor cell survival, proliferation and / or development. Cell development may include, for example, migration, maturation and / or differentiation. The disclosure also provides compositions and methods for treating diseases or conditions related to mucosal barrier function such as wound healing in subjects in need, treating skin disorders (e.g., atopic dermatitis, psoriasis, bedsores or conditions related to skin aging), treating lung disorders (e.g., asthma), improving mucosal barrier function and / or treating GI mucosal damage.

[0019] The cell proliferation and / or development can be used to improve the proliferation and function of stem cells and progenitor cells located in various organ systems and positions in the body. Stem cells and / or progenitor cells can be, for example, in the small intestine or from other tissues, including but not limited to skin, bone marrow, lungs, neurons, pancreas, muscle, bone tissue, vascular endothelial cells and corneal epithelial cells. Stem cells and / or progenitor cells can be adult stem cells, embryonic stem cells or tumor stem cells. In one embodiment, the composition that can be used to treat the disease described herein comprises one or more free amino acids selected from the group consisting of threonine, valine, tyrosine, tryptophan, aspartic acid and serine; and optionally an acceptable carrier.

[0020] In certain embodiments, the composition of the present disclosure does not include one or more amino acids selected from the group consisting of lysine, glycine, isoleucine and asparagine. In certain embodiments, the composition does not include lysine, glycine, aspartic acid, isoleucine and asparagine. In another specific embodiment, the composition does not include or only includes negligible amounts of serine, lysine, glycine, aspartic acid, isoleucine and asparagine. In certain embodiments, the composition does not include glutamine and / or methionine; and any dipeptide, oligopeptide or polypeptide or protein that can be hydrolyzed into glutamine and / or methionine. In certain embodiments, the composition does not include methionine.

[0021] In some embodiments, the composition comprises at least one amino acid, and the amino acid residues thereof are present in the composition. Alternatively, in certain embodiments, even if these amino acids are present in the composition, they are also present in an amount that will not suppress stem cell and / or progenitor cell survival, proliferation and / or development. In some embodiments, the composition does not include serine. In some embodiments, the composition does not include cysteine. In certain embodiments, even if these amino acids are present in the composition, their amount will not affect the treatment of diseases or illnesses such as wound healing related to mucosal barrier function, treatment of skin disorders (for example, atopic dermatitis, psoriasis, bedsores or the illnesses related to skin aging), treatment of pulmonary disorders (for example, asthma), improvement of mucosal barrier function and / or treatment of GI mucosal damage.

[0022] If present in the composition, the amino acids can be present, for example, in the following concentrations: threonine is about 0.4 to about 1.5, about 0.7 to about 1.3, or about 0.9 to about 1.1 g / L; valine is about 0.7 to about 1.7, about 0.9 to about 1.5, or about 1.1 to about 1.3 g / L; serine is about 0.6 to about 1.6, about 0.8 to about 1.4, about 1.0 to about 1.2 g / L; tyrosine is about 0.05 to about 0.4, or about 0.1 to about 0.3 g / L; and tryptophan is about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 g / L. In certain embodiments, the concentration is grams of amino acid per liter of solution. In certain embodiments, the solution comprises water. In certain embodiments, the therapeutic composition comprises threonine (about 1.0 g / L), valine (about 1.2 g / L), serine (about 1.1 g / L), tyrosine (about 0.2 g / L), and tryptophan (about 1.6 g / L). In one embodiment, the composition does not comprise serine. In some embodiments, the composition does not comprise methionine. In some embodiments, the composition does not comprise cysteine.

[0023] In certain embodiments, the total osmolarity of the composition is from about 100 mosm to about 280 mosm or from about 150 to about 280 mosm.

[0024] The composition may have, for example, a pH of about 2.5 to about 8.5. In certain embodiments, the composition has a pH of about 2.5 to about 6.5, about 3.0 to about 6.0, about 3.5 to about 5.5, about 3.9 to about 5.0, or about 4.2 to about 4.6. In other embodiments, the pH is about 6.5 to about 8.5, about 7.0 to about 8.0, or about 7.2 to about 7.8.

[0025] In certain embodiments, the composition is in the form of a solution or beverage, powder, pill, gel, cream, ointment, applied as part of a matrix or applied on a bandage. In certain embodiments, the composition is applied as part of a matrix delivery system.

[0026] Said composition can be systemically or topically applied. In certain embodiments, said composition is used to promote ex vivo or in vitro cell survival, proliferation and / or development. In certain embodiments, said composition is used to treat diseases or conditions related to mucosal barrier function such as wound healing, treat skin disorders (e.g., atopic dermatitis, psoriasis, bedsores or conditions related to skin aging), treat lung disorders (e.g., asthma), improve mucosal barrier function and / or treat GI mucosal damage.

[0027] In certain embodiments, the composition further comprises additives (eg, nutrients, electrolytes, vitamins, minerals, etc.).

[0028] Without wishing to be bound by any particular theory, it is believed that the compositions and methods can prevent DNA damage and are therefore useful for preventing damage to DNA and / or repairing damaged DNA. In another embodiment, the compositions and methods promote the survival, proliferation and development of stem cells by preventing damage to DNA and / or repairing damaged DNA.

[0029] In another aspect, the present disclosure provides a method for treating a lung disorder in a subject in need. In another aspect, the present disclosure provides compositions and methods for improving lung healing, reducing pneumonia, reducing airway resistance and / or improving lung function in a subject in need. In certain embodiments, the lung disorder is lung injury, pneumonia or asthma. In certain embodiments, the lung disorder is associated with increased airway resistance. In certain embodiments, the lung disorder is associated with reduced barrier function. In certain embodiments, the composition is systemically administered or administered via inhalation. In certain embodiments, the composition and method can be used, for example, to improve lung healing, reduce pneumonia, reduce airway resistance and improve lung function after radiation exposure and / or chemotherapy.

[0030] In another embodiment, the compositions and methods of the present disclosure are used to treat bone marrow suppression caused, for example, by drugs, viruses, bacteria, toxins, chemicals, or vitamin deficiencies. For example, in certain embodiments, the compositions and methods are used to treat patients with low platelet counts (thrombocytopenia) caused, for example, by dengue virus infection.

[0031] In another aspect, the present disclosure provides a method for treating GI disorders or GI tract disorders (e.g., radiation damage, ischemic colitis, infection, trauma) or any other condition related to mucosal barrier function and / or integrity. In certain embodiments, a method for treating a disease or condition related to mucosal barrier function is provided. In certain embodiments, a disease or condition related to mucosal barrier function is a functional disorder of a mucosal barrier. In certain embodiments, the treatment of a disease or condition related to mucosal barrier function is wound healing in a subject in need, treating damage to GI mucosa. In certain embodiments, a method for treating GI mucosal damage in a subject in need is provided. In certain embodiments, GI mucosa is small intestinal mucosa. In certain embodiments, the composition is used for treatment after surgery (e.g., intestinal surgery). In certain embodiments, the composition is used for treating any disease or condition that will cause intestinal ischemia, including, for example, hypotension, shock, thrombosis, intestinal obstruction, etc.

[0032] One composition that can be used to treat radiation enteritis is an amino acid-based oral rehydration solution (AA-ORS) described in U.S. Pat. No. 8,993,522, which is incorporated herein by reference. AA-ORS is a composition for improving small intestinal health, wherein the composition is formulated for enteral administration and comprises threonine, valine, tryptophan, serine and tyrosine as free amino acids; and water; wherein the composition does not contain the free amino acid glutamine or a dipeptide containing glutamine, or if free amino acid glutamine and / or a dipeptide containing glutamine is present, the total concentration of the free amino acid glutamine and the dipeptide containing glutamine is less than 50 mg / L; wherein the composition does not contain glucose, or if glucose is present, the concentration of glucose is less than 1 g / L; and wherein the composition does not contain the free amino acid methionine or a peptide containing methionine, optionally lysine, glycine, aspartic acid and / or isoleucine, and optionally electrolytes, vitamins, minerals and / or flavoring agents.

[0033] In another aspect, the disclosure provides a method for treating a skin disorder (e.g., atopic dermatitis, psoriasis, or a disorder associated with skin aging) in a subject in need thereof, the method comprising administering a composition as described herein to the subject. In certain embodiments, the skin disorder is atopic dermatitis, psoriasis, skin aging, a disorder associated with skin aging, or a bedsore. In certain embodiments, the compositions and methods described herein can be used in cosmetic applications, where, for example, rejuvenation of the skin layers and / or underlying tissue is desired.

[0034] definition

[0035] The terms "improving a skin condition" or "treating a skin condition" include prophylactically preventing or therapeutically treating a skin condition, and may involve one or more of the following benefits: thickening of the skin, prevention of loss of skin elasticity, and reduction of lines or wrinkles.

[0036] As used herein, the term "epidermis" or "epidermal" refers to the outermost layer of the skin.

[0037] As used herein, the term "external application" means applying or spreading the composition of the present invention on the surface of epidermal tissue.

[0038] As used herein, the term "dermatologically acceptable" means that the compositions so described, or components thereof, are suitable for use in contact with mammalian epidermal tissue without undue toxicity, incompatibility, instability, allergic response, and the like.

[0039] As used herein, the term "therapeutically effective amount" refers to an amount of a compound or composition sufficient to induce a positive benefit, preferably a positive skin appearance and / or feel. According to the present disclosure, the therapeutically effective amount is the amount of amino acids alone or in combination with other agents that regulate and / or improve the skin.

[0040] As used herein, the term "improvement" or any grammatical variant thereof (e.g., "ameliorate," "ameliorating," and "amelioration," etc.) includes, but is not limited to, delaying the onset of a disease or condition or reducing the severity of a disease or condition. As used herein, improvement does not require complete absence of symptoms.

[0041] As used herein, the term "effective amount" or "significant amount" refers to an amount that can treat or improve a disease or condition or otherwise produce the desired therapeutic effect.

[0042] The term "health functional food" refers to food prepared or processed into tablets, capsules, powders, granules, liquids, pills or any other forms using raw materials or ingredients that have useful functions for the human body.

[0043] The term "functional" means a beneficial effect on human health, such as structural or functional modulation of nutrients, the immune system, inflammation, fluid balance, physiological effects, and the like.

[0044] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

[0045] As used herein, the term "treat" or any grammatical variations thereof (e.g., "treat," "treating," and "treatment," etc.) includes, but is not limited to, alleviating the symptoms of a disease or disorder; and / or reducing, suppressing, inhibiting, alleviating or affecting the progression, severity and / or scope of a disease or disorder.

[0046] As used herein, the term "consisting essentially of" limits the scope of ingredients and steps to the specified materials or steps and those that do not materially affect one or more of the basic and novel characteristics of the present invention (i.e., compositions and methods for promoting stem cell development). BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1A to Figure 1B Shown are the increased crypt counts and villus lengths by AA-ORS after irradiation. Physiological saline (saline) was used as a control; saline and AA-ORS were given by gastric gavage. Six mice were included in each treated or untreated irradiation group (0, 1, 3, 5, 6, 7, 9, 13, and 15 Gy). Figure 1A Semi-log survival curves showing the effect of AA-ORS on crypt counts are shown. AA-ORS shifted the graph to the left. Crypt survival curves were modeled using a single-hit multiple-target cell survival model to assess biological effects. The equation [S = 1-(1-e^-D / D 0 ) n The survival probability of mitotic cells in crypts after radiation was calculated. S represents the fraction of mitotic cells in crypts that survived each radiation dose, and D represents the radiation dose; D 0 A measure of the intrinsic radioresistance of the crypt germinal unit. The Dq values ​​of saline-treated mice and AA-ORS-treated mice are indicated by black and gray arrows, respectively. Dq is given by the formula Dq = D 0 In n calculation. Without limiting the constant cell sensitivity, the N values ​​were 10.4 ± 0.2 and 5.3 ± 0.1 (P < 0.001), indicating that the number of progenitor units per circumference was almost doubled compared with the control. When limiting the constant D 0 (4.8±0.1Gy), the difference was still significant at 8.8±0.4 to 6.1±0.3 (P<0.001). Figure 1BVillus height after treatment with saline and AA-ORS in irradiated mice is shown. Villus height was significantly increased in mice treated with AA-ORS compared to mice receiving saline as treatment. Crypts per circumference were counted and villus length was measured from 10 sections obtained from the ileum. Mean ± SEM data for 6 mice per group are shown. * indicates a statistically significant difference (P < 0.01). Normal saline (saline) was used as a control, and both saline and AA-ORS were given by gastric gavage.

[0048] Figure 2A Confocal microscopy of longitudinal ileal sections is shown with significantly stained epithelial cells along the length of the villi. 5-μm thick paraffin-embedded tissue was used. Nuclei were stained with DAPI (dark gray) and Edu-positive epithelial cells were stained light gray. The distance that Edu-positive cells migrated along the height of the villi was measured using Image Pro Plus software. Bar - 50 μm. At least five well-oriented villi were counted for each tissue section and the results were averaged. Edu-positive cells were seen all the way to the tip of the villi in the 5 Gy irradiated tissue, but not in the AA-ORS treated mice.

[0049] Figure 2B The migration distance of EdU positive cells measured at 72 hours is shown. Compared with 0Gy, 5Gy irradiated saline treated mice had significantly reduced cell migration distance (black bars); when compared with non-irradiated and irradiated saline treated mice, AA-ORS treated mice had increased migration distance. Values ​​are mean ± SEM of 6 mice per group.

[0050] FIG. 3A to FIG. 3B Shows the use of 22 Na and 36 Ussing chamber flux studies of C1 showing the effect of AA-ORS on sodium and chloride absorption. AA-ORS increased net sodium (J) in 0 Gy and 5 Gy irradiated tissues (n=8). net Na) and chlorine (J net C1) Absorption.

[0051] Figure 3C is an enlarged view of immunohistochemistry showing NHE3 expression (light gray) along the brush border membrane (BBM) (white arrows) of villous epithelial cells. 5-μm thick paraffin-embedded tissue was used. Nuclei were stained with DAPI (dark gray). At least five well-oriented villi were used.

[0052] Figure 3D Western blot analysis of NHE3 protein is shown.

[0053] Figure 3E A graphic representation of NHE3 protein density in intestinal tissue of mice treated with saline (black bars) or AA-ORS (hatched bars) after 0 or 5 Gy irradiation is shown. Immunoblots were repeated four times. Values ​​are mean ± SEM from n = 4; * indicates a statistically significant difference compared to saline-treated animals (P < 0.05).

[0054] Figure 3F NHE3 transcript levels in intestinal tissue of mice treated with saline (black bars) or AA-ORS (hatched bars) after 0 or 5 Gy irradiation are shown. Values ​​are mean ± SEM from n = 6; * indicates a statistically significant difference (P < 0.05) compared to saline-treated animals. Saline or AA-ORS were administered for 6 days.

[0055] FIG. 4A to FIG. 4D Glucose-stimulated sodium absorption and SGLT1 protein levels are shown: (4A) Chamber flux studies using 22Na showing the effect of AA-ORS on glucose-coupled sodium absorption. AA-ORS treatment increased JnetNa absorption in 5Gy irradiated tissues (n=8). (4B) Western blot analysis for SGLT1 protein and β-galactosidase showed that AA-ORS treatment increased protein levels in villous cells of 0Gy and 5Gy mice. The immunoblot was repeated four times. (4C) Normalized SGLT1 protein levels for Western blot analysis. Significant differences in SGLT1 protein levels were observed in 5Gy irradiated mice treated with AA-ORS compared to 5Gy mice. (4D) SGLT1 transcript levels in intestinal tissues of mice treated with saline (black bars) or AA-ORS (hatched bars) after 0 or 5Gy irradiation. Values ​​are mean ± SEM from n=6; * indicates a statistically significant difference (P<0.05) compared to saline-treated animals. Saline or AA-ORS were administered for 6 days.

[0056] FIG. 5A to FIG. 5G Shown are the protein levels and mRNA expression of Lgr5, BMI1, p-AKT, AKT, pERK, and ERK in villous epithelial cells of saline- and AA-ORS-treated mice after 0 and 5 Gy irradiation. Figure 5HWestern blot analysis of Lgr5, BMI1, p-AKT, AKT, p-ERK, and ERK is shown. Immunoblotting was repeated at least four times, and q-PCR was repeated at least 6 times. (5A) Western blot analysis of stem cell and proliferation markers (Lgr5, BMI1, p-AKT, AKT, p-ERK, ERK, and PCNA). The protein bands of interest were normalized to the total amount of protein in each lane using Coomassie blue stain. (5B) Western blot analysis of apoptotic proteins (Bcl2, Bax, cleaved caspase-3, caspase-3, and p53). (5C) Lgr5 mRNA levels in mice treated with saline or AA-ORS and irradiated with 0 Gy or 5 Gy. (5D) Changes in BMI1 mRNA levels in mice treated with saline or AA-ORS and irradiated with 0 Gy or 5 Gy. (5E) Changes in ERK mRNA levels in mice treated with saline or AA-ORS and irradiated with 0 Gy or 5 Gy. (5F) Changes in AKT mRNA levels in mice treated with saline or AA-ORS and irradiated with 0 Gy or 5 Gy. (5G) mRNA expression of caspase-3. Values ​​are mean ± SEM from n = 6 different mice in triplicate. #P < 0.05 and *P < 0.001 compared to saline control. (5H) Western blot analysis of Lgr5, BMI1, p-AKT, AKT, p-ERK, and ERK. The protein bands of interest were normalized to the total amount of protein in each lane using Ponsceau S stain.

[0057] FIG. 6A to FIG. 6G Shown is the expression of Lgr5 in the ileal mucosa at 0 Gy (left) and 5 Gy (right) after treatment with saline (top) or AA-ORS (bottom). + , Ki-67 + and PCNA + Representative micrographs of cell distribution. (6A) Immunostaining for Lgr5: Lgr5 was observed in the lower 1 / 3 of the crypt. + Mice irradiated with 5 Gy resulted in the expression of Lgr5 in ileal crypts. + Stem cells were significantly reduced, and AA-ORS increased Lgr5 + Stem cells. Scale bar indicates 25 μm. (6B) Average Lgr5 expressed in crypts + Mean number. Error bars represent SEM. (6C) Immunostaining for Ki-67: The number of cells expressing Ki-67 (a proliferation marker) in 0 Gy irradiated mice treated with AA-ORS did not show significant differences when compared to the saline treated group. In the case of AA-ORS treatment, 5 Gy irradiated mice showed Ki-67 +(6D) Average number of cells expressing Ki-67 in crypt and / or villus cells. Error bars represent SEM. (6E) Immunostaining for PCNA: PCNA + The number and distribution of PCNA in mice after 5Gy irradiation + Cells decreased in crypts and / or villus cells, but increased in the case of AA-ORS treatment. Scale bar represents 100 μm. (6F) Average number of cells expressing PCNA in crypts and / or villus cells. Error bars represent SEM. (6G) Protein levels and mRNA expression of cleaved caspase 3, total caspase 3, and p53 in villous epithelial cells of mice treated with saline and AA-ORS after 0 and 5 Gy irradiation of mice. Immunoblotting was repeated at least four times and q-PCR was repeated at least 6 times. Western blot analysis was performed for cleaved caspase 3, total caspase 3, and p53. The protein bands of interest were normalized to the total amount of protein in each lane using Ponceau S stain and β-actin.

[0058] Figure 7 Schematic diagram of the small intestinal villi and enterocytes is shown: AA-ORS treatment rapidly increased Lgr5-positive dividing stem cells, as well as proliferation markers p-ERK, p-AKT, and PCNA. The treatment also increased cleaved caspase 3, p53, and Bcl-2. AA-ORS treatment increased villus height and increased the expression of NHE3, SGLT1, and β-galactosidase, thereby increasing electrolyte absorption, sodium-coupled glucose absorption, and disaccharide decomposition at the brush border membrane, respectively. The cartoon image of the enterocyte in the upper right shows NHE3-mediated Na + Functional improvement of absorption and glucose-coupled sodium transport.

[0059] Figure 8 shows protein density. Protein density was normalized to b-actin. Mice treated with saline after 0 or 5 Gy irradiation are shown as black bars and mice treated with AA-ORS are shown as shaded bars. Values ​​are mean ± SEM from n = 4 different mice in triplicate. #P < 0.05 compared to saline control. ( Fig. 8A ) Lgr5 protein; (8B) Bmi1 protein; (8C) p-ERK protein; (8D) p-AKT protein; (8E) AKT protein; (8F) caspase-3 protein (normalized to b-actin).

[0060] Fig.9A Shown are the results of Western blot analysis showing that gastric gavage with valine increased Lgr5 protein levels in 0 Gy-irradiated mice and 5 Gy-irradiated mice. Fig. 9BA graphical representation of the protein levels of Lgr5 protein in mice treated with valine for a period of 6 days is shown. Fig.13 , data from using individual amino acids ( Fig. 9 (valine); Fig. 10 (tryptophan); Fig. 11 (serine); Fig. 12 (tyrosine); Fig.13 Male NIH Swiss mice (8 weeks) were treated with threonine (Threonine) (e.g., by gavage (300 μl OD) for 6 days). Animals were euthanized by CO2 on day 6 and tissues were collected for protein analysis by Western blot analysis. Lgr5 (100 KD) was used as a marker for crypt stem cell proliferation. These experiments were repeated at least 4 times from 4 different mice (data shown).

[0061] Fig. 10A Shown are changes in Lgr5 protein levels upon tryptophan treatment over a 6-day period. Fig. 10B Shown is a graphical representation of Lgr5 protein levels in mice treated with tryptophan for a period of 6 days.

[0062] Fig.11A Shown are changes in Lgr5 protein levels with serine treatment over a 6-day period. Fig. 11B Shown is a graphical representation of Lgr5 protein levels in mice treated with serine for a period of 6 days.

[0063] Fig. 12A Shown are changes in Lgr5 protein levels with tyrosine treatment over a 6-day period. Fig. 12B Shown is a graphical representation of Lgr5 protein levels in mice treated with tyrosine for a period of 6 days.

[0064] Fig.13 Shown are changes in Lgr5 protein levels upon threonine treatment over a 6-day period.

[0065] Figures 14 to 20The results of allergic asthma and anti-inflammatory airway function of the treatment with amino acid preparations in the sheep model are shown. In this study, animals will show early and late airway responses to inhalation stimulation with Ascaris suum antigens. Venous blood samples (about 3 ml) can be obtained from the external jugular vein for pharmacokinetic data. Measurement of airway mechanics: Unsedated sheep are confined in a prone position in a cart with their heads fixed. After local anesthesia of the nasal passages with a 2% lidocaine solution, a balloon catheter will be passed through one nostril into the lower esophagus. A cuffed endotracheal tube is passed through the other nostril of the animal. (The cuff of the endotracheal tube will only be inflated for airway mechanics measurements and inflated during aerosol stimulation to prevent excessive discomfort. This process has no effect on airway mechanics). Pleural pressure is estimated with an esophageal balloon catheter (filled with 1 ml of air), which will be located 5-10 cm from the gastroesophageal junction. At this position, the end-expiratory pleural pressure is between -2 and -5 cm H 2 O. Once the balloon is placed, it will be fixed so that it remains in place during the experiment. The lateral pressure in the trachea will be measured using a side hole catheter (inner dimensions, 2.5mm), which passes through the endotracheal tube tip and is located at its distal end. The transpulmonary pressure, i.e. the difference between the tracheal and pleural pressures, will be measured using a differential pressure sensor catheter system. For the measurement of pulmonary resistance (RL), the proximal end of the endotracheal tube will be connected to a pneumotachograph. The signal of flow and transpulmonary pressure will be recorded on an oscilloscope recorder, which is connected to a computer for online calculation of RL from transpulmonary pressure, respiratory volume (obtained by digital integration) and flow. The analysis of 5-10 breaths will be used to calculate the RL in L x cm H. 2 O / L / S determination RL.

[0066] Effects on antigen-induced airway responses: Baseline dose-response curves for aerosol carbachol were obtained 1-3 days prior to antigen challenge. On the day of challenge, baseline values ​​for lung resistance (RL) were obtained, and then sheep were challenged with Ascaris suum antigen. Measurements of RL were obtained immediately after challenge, hourly from 1-6h after challenge, and every half hour from 6.5-8h after challenge. Measurements of RL were obtained 24h after challenge, followed by a 24h dose-response curve. Figures 14 to 19 For graphs showing airway responsiveness (PC400), BSL is baseline and PASC is after antigen stimulation.

[0067] For preliminary studies, sheep were treated with aerosolized compounds using the above-described aerosolization system 30 minutes before antigen stimulation, 1 hour before antigen stimulation, 30 minutes after antigen stimulation, or 2 hours after antigen stimulation. The aerosolized compound was an amino acid preparation consisting of tyrosine (1.2 mM), threonine (8 mM), valine (10 mM), serine (10 mM), and tryptophan (8 mM), wherein 4 mL of the amino acid preparation solution was administered. The measurement of RL was repeated after treatment. Thereafter, the next study will evaluate the effect after oral administration of the compound. In the oral study, sheep received an amino acid preparation consisting of tyrosine (1.2 mM), aspartic acid (8 mM), threonine (8 mM), valine (10 mM), and serine (10 mM), wherein 8 oz of the amino acid preparation solution was administered orally, and RL and airway responsiveness (PC400) were measured as described above.

[0068] Fig.14 Results are shown for lung resistance (RL) and PC 400 (respiratory units) in sheep that received nebulized compound one hour prior to antigen challenge compared to controls.

[0069] Fig.15 Results are shown for lung resistance (RL) and PC 400 (respiratory units) in sheep treated with nebulized compound two hours after antigen challenge compared to controls.

[0070] Fig.16 Results are shown for lung resistance (RL) and PC 400 (respiratory units) in sheep treated with aerosolized compound 30 minutes prior to antigen challenge compared to controls.

[0071] Fig.17 Results are shown for lung resistance (RL) and PC 400 (respiratory units) in sheep receiving nebulized compound 30 minutes after antigen challenge compared to controls.

[0072] Fig.18 Results are shown for lung resistance (RL) and PC 400 (respiratory units) in sheep given compound formulations orally 30 minutes after antigen challenge compared to controls.

[0073] Fig.19 Results are shown for lung resistance (RL) and PC 400 (respiratory units) in sheep given formulations orally two hours after antigen challenge compared to controls.

[0074] Fig. 20A summary of the results of an asthma study in sheep for anti-inflammatory improved airway function is shown, including the mean late airway response ("LAR") and airway hyperresponsiveness ("AHR") of controls and sheep treated with aerosolized compounds (5-8 hours) or given amino acid formulations orally before and after aerosol antigen challenge. "*" indicates the mean late airway response (5-8 hours). "+" indicates PC 400 after / before challenge. A ratio close to 1 indicates the absence of airway hyperresponsiveness (AHR).

[0075] Fig.21 The results of wound healing studies are shown: the dorsal surface of mice selected for surgery was shaved to remove hair. All surgical procedures were performed under anesthesia using 5% isoflurane in oxygen, and the surgical plane of anesthesia was maintained using 1%-3% isoflurane. A 3mm punch biopsy was performed on the back of 8-week-old NIH Swiss mice. The mouse wound model may be affected by wound contraction due to the presence of the subcutaneous muscle layer (i.e., a layer not present in the human body). To prevent wound contraction, a silicon O-ring was used as a splint and fixed in place by interrupted sutures. AA-ORS or saline was used in a transparent occlusive dressing for the wound. The dressing was changed every day after measuring the wound area using a caliper. Therefore, the procedure measures re-epithelialization and simulates wound healing in the human body. The results are shown in the figure. Data are from n=6 mice per group. DETAILED DESCRIPTION

[0076] Amino acid compositions for treating GI, lung and skin disorders are described herein. In one aspect, compositions and methods for promoting cell proliferation and / or development are described herein. In a certain embodiment, the cell is a stem cell and / or a progenitor cell. As used herein, reference to "development" may include, for example, migration, maturation and / or differentiation of cells. The disclosure also provides compositions and methods for treating wounds, skin disorders (e.g., atopic dermatitis, psoriasis, bedsores, disorders related to skin aging, cosmetic disorders), lung disorders (e.g., lung injury, pneumonia or asthma), GI disorders (e.g., radiation damage, ischemic colitis, infection, trauma) or any other disorders related to mucosal barrier function and / or integrity.

[0077] The compositions and methods can be used to enhance stem and / or progenitor cell populations in vivo, ex vivo and / or in vitro. These cells can be used to treat a variety of disease states, degeneration and injury.

[0078] In one embodiment, provided herein are methods of promoting the proliferation and / or development of stem and / or progenitor cells in a subject in need of such treatment by administering to the subject a composition of the present disclosure.

[0079] The subject can be a patient who needs to promote the proliferation and / or development of stem cells and / or progenitor cells. Due to gastrointestinal toxicity induced by, for example, malabsorption, radiation or chemotherapy, or secondary to infection, cancer or cancer treatment, the patient may have this need. In one embodiment, the patient is asymptomatic. The subject can be any animal, including, for example, humans. Except humans, animals can be, for example, mammals, such as cattle, horses, sheep, pigs, goats, dogs and cats. Animals can also be, for example, chickens, turkeys or fish.

[0080] In certain embodiments, the composition comprises one or more free amino acids selected from the group consisting of threonine, valine, tyrosine, tryptophan, aspartic acid and serine. In certain embodiments, the composition comprises one or more free amino acids selected from the group consisting of threonine, valine, tyrosine, tryptophan, aspartic acid, serine and derivatives thereof. In certain embodiments, the composition comprises one or more free amino acids selected from the group consisting of threonine, valine, tyrosine, tryptophan and aspartic acid. The composition preferably comprises one or more free amino acids selected from the group consisting of threonine, valine, serine, tyrosine and tryptophan. In certain embodiments, the composition comprises two or more free amino acids selected from the group consisting of threonine, valine, tyrosine, tryptophan, aspartic acid and serine. In certain embodiments, the composition comprises three or more free amino acids selected from the group consisting of threonine, valine, tyrosine, tryptophan, aspartic acid and serine. In certain embodiments, the composition comprises four or more free amino acids selected from the group consisting of threonine, valine, tyrosine, tryptophan, aspartic acid and serine. In certain embodiments, the composition comprises threonine, valine, tyrosine, tryptophan and aspartic acid. In certain embodiments, the composition comprises the free amino acids of threonine, valine, tyrosine, tryptophan and serine.

[0081] In one embodiment, the therapeutic composition comprises, consists essentially of, or consists of: one or more free amino acids selected from the group consisting of: threonine, valine, tyrosine, tryptophan, serine, aspartic acid, and derivatives thereof; and optionally, for example, pharmaceutically acceptable carriers, adjuvants, and other active agents. In certain embodiments, the composition comprises one or more free amino acids selected from the group consisting of: threonine, valine, tyrosine, tryptophan, serine, and aspartic acid; and optionally, for example, pharmaceutically acceptable carriers, adjuvants, other active agents, and additives (e.g., sugars, electrolytes, vitamins, minerals, etc.).

[0082] In one aspect, the compositions described herein comprise one or more free amino acids selected from the group consisting of threonine, valine, tyrosine, tryptophan, aspartic acid, or serine.

[0083] In some specific embodiments, the composition does not include one or more amino acids selected from the group consisting of lysine, glycine, isoleucine and asparagine. In certain embodiments, the composition does not include lysine. In certain embodiments, the composition does not include glycine. In certain embodiments, the composition does not include isoleucine. In certain embodiments, the composition does not include asparagine. In certain embodiments, the composition does not include lysine and glycine. In certain embodiments, the composition does not include lysine and isoleucine. In certain embodiments, the composition does not include lysine and asparagine. In certain embodiments, the composition does not include lysine, glycine and isoleucine. In certain embodiments, the composition does not include lysine, glycine and asparagine. In certain embodiments, the composition does not include lysine, glycine, isoleucine and asparagine. In certain embodiments, the composition does not include glycine and isoleucine. In certain embodiments, the composition does not include glycine and asparagine. In certain embodiments, the composition does not include glycine, isoleucine and asparagine. In certain embodiments, the composition does not include isoleucine and asparagine. In certain embodiments, the composition does not include lysine, glycine, aspartic acid, isoleucine and asparagine. In another specific embodiment, the composition does not include serine, lysine, glycine, aspartic acid, isoleucine and asparagine, or only includes negligible amounts of serine, lysine, glycine, aspartic acid, isoleucine and asparagine. In certain embodiments, the composition does not include glutamine and / or methionine; and any dipeptide, oligopeptide or polypeptide or protein that can be hydrolyzed into glutamine and / or methionine.

[0084] Alternatively, in certain embodiments, even if these amino acids are present in the composition, they are present in an amount that does not inhibit stem cell and / or progenitor cell survival, proliferation and / or development. In some embodiments, the composition does not have serine, or the amount of serine it has is negligible. "Negligible" means that the serine present has no effect on stem cell survival, proliferation and / or development. "Negligible" means that the serine present has no effect on diseases or conditions related to mucosal barrier function in a subject in need, such as wound healing, skin disorders (e.g., atopic dermatitis, psoriasis, bedsores or conditions related to skin aging), pulmonary disorders (e.g., asthma), mucosal barrier function and / or GI mucosal damage.

[0085] If present in the composition, the amino acids can be present, for example, at the following concentrations: threonine at about 0.4 to about 1.5, about 0.7 to about 1.3, or about 0.9 to about 1.1 g / L; valine at about 0.7 to about 1.7, about 0.9 to about 1.5, or about 1.1 to about 1.3 g / L; serine at about 0.6 to about 1.6, about 0.8 to about 1.4, about 1.0 to about 1.2 g / L; tyrosine at about 0.05 to about 0.4, or about 0.1 to about 0.3 g / L; and tryptophan at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 g / L. In certain embodiments, the composition comprises threonine (about 1.0 g / L), valine (about 1.2 g / L), serine (about 1.1 g / L), tyrosine (about 0.2 g / L), tryptophan (about 1.6 g / L), and aspartic acid (about 0.4 to 3.6 g / L). In certain embodiments, the composition has no serine or has negligible serine. In certain embodiments, the concentration is grams of amino acid / liter of solution. In certain embodiments, the solution comprises water.

[0086] In one embodiment, the total osmolarity of the composition is from about 100 mosm to about 280 mosm, or preferably from about 150 to about 260 mosm.

[0087] The pH of the composition can be from about 2.5 to about 8.5. In certain embodiments, the pH range of the composition is from about 2.5 to about 6.5, from about 3.0 to about 6.0, from about 3.5 to about 5.5, from about 3.9 to about 5.0, or from about 4.2 to about 4.6. In other embodiments, the pH range of the composition is from about 6.5 to about 8.5, from about 7.0 to about 8.0, or from about 7.2 to about 7.8.

[0088] In certain embodiments, the pH of the composition is, for example, from about 2.5 to about 8.5. In certain embodiments, the pH of the composition is from about 2.5 to about 6.5, from about 2.5 to about 6.0, from about 3.0 to about 6.0, from about 3.5 to about 6.0, from about 3.9 to about 6.0, from about 4.2 to about 6.0, from about 3.5 to about 5.5, from about 3.9 to about 5.0, or from about 4.2 to about 4.6. In other embodiments, the pH is from about 6.5 to about 8.5, from about 7.0 to about 8.5, from about 7.0 to about 8.0, from about 7.2 to about 8.0, or from about 7.2 to about 7.8.

[0089] In some embodiments, the composition is systemic or topical. In certain embodiments, the composition is used for promoting cell survival, proliferation and / or development in vitro or in vitro. In certain embodiments, the composition is used for treating diseases or conditions related to mucosal barrier function such as wound healing, treating skin disorders (e.g., atopic dermatitis, psoriasis, bedsores or conditions related to skin aging), treating lung disorders (e.g., asthma), improving mucosal barrier function and / or treating GI mucosal damage. Therapeutic compositions can be administered by enteral route or parenteral or external administration or by inhalation. In certain embodiments, the composition is a therapeutic composition, a cosmetic composition or a nutritional composition.

[0090] In some embodiments, the compositions described herein (e.g., amino acid-based oral rehydration solutions (AA-ORS)) act by correcting functional changes that occur on the GI mucosa after radiation. In certain embodiments, the composition is a solution. In certain embodiments, the solution is an amino acid-based oral rehydration solution (AA-ORS). The amino acids are selected to counteract increased paracellular permeability, increased Cl after radiation. - secretion and reduced electrolyte absorption. Since the compositions described herein (e.g., AA-ORS) correct functional changes in the GI mucosa, their effects are believed to be upstream of current agents in the pipeline. The compositions can be administered with other therapeutic agents. It has recently been found that electrolytes, glucose, and some amino acids are poorly absorbed in the gastrointestinal tract after irradiation. In addition, glucose and some amino acids have been observed to stimulate the removal of Na + Electrogenic Cl - secreted and can increase paracellular permeability, which further complicates radiation-induced diarrhea and increased intestinal permeability. 51,52 Increased paracellular permeability is known to increase the translocation of antigenic material from the intestinal lumen into systemic compartments, leading to an increase in proinflammatory cytokines. 51

[0091] In one embodiment, the compositions of the present disclosure do not include a large amount of glucose, glutamine, methionine and / or lactose. In certain embodiments, the compositions do not include a large amount of glucose. In certain embodiments, the compositions do not include a large amount of glutamine. In certain embodiments, the compositions do not include a large amount of methionine. In certain embodiments, the compositions do not include methionine. In certain embodiments, the compositions do not include a large amount of lactose.

[0092] In one embodiment, the compositions described herein are used as compositions for culturing cells to promote the survival, development and / or proliferation of stem cells and / or progenitor cells. Compositions for culturing cells can be used to obtain stem cells and / or progenitor cells in an increased amount to treat various diseases. Compositions can also be applied to stem cells and / or progenitor cells immediately before and / or after transplantation. Compositions can also be used to increase the proliferation of natural stem cells present in various parts of the body.

[0093] In one embodiment, the present disclosure provides a method for improving the treatment outcome of implanted stem cells and / or progenitor cells, which comprises administering the composition together with the implantation of stem cells and / or progenitor cells, or administering the composition as a maintenance or supportive therapy after, for example, bone marrow or liver transplantation. In certain embodiments, maintenance or supportive therapy after, for example, bone marrow or liver transplantation is provided herein. The administration of the composition can be carried out at or near the target stem cell and / or progenitor cell implantation site in humans or non-human animals. In optional embodiments, the environment can be further changed by providing an influencing factor or by cleaning an environment that may affect the administered stem cells and / or progenitor cells in an undesirable manner. Unwanted or toxic reagents.

[0094] The cells administered may be unmodified, or may be engineered to bias a target differentiation endpoint. U.S. Patent Publication Nos. 2006 / 0134789 and 2006 / 0110440 are incorporated herein by reference in their entirety to provide examples of stem cells engineered for negative and positive differentiation preferences that are contemplated for use with the methods taught herein.

[0095] When the composition is applied to stem cells and / or progenitor cells in culture or in situ, secretory proteins (e.g., cell survival-related factors and cell proliferation-related factors) and transcription factors change. As a result, cell activity is changed, and specifically, cell proliferation, survival and / or development are enhanced. Therefore, stem cells and / or progenitor cells produced on an enhanced scale by means of the composition of the present invention can be transplanted into diseases or other parts as cell therapeutic agents to promote cell regeneration and effectively treat various diseases.

[0096] In one embodiment, the compositions described herein stimulate the survival, proliferation and / or development of stem and / or progenitor cells as evidenced by one or more of the following: 1) an increase in proliferation markers (e.g., p-ERK and p-AKT) at the mRNA and / or protein level, 2) an increase in stem cell markers (e.g., BMI1 and Lgr5) at the mRNA and / or protein level, 3) activation of protein kinases (e.g., MEK and ERK); 4) a decrease in apoptosis markers (e.g., cleaved caspase 3).

[0097] ERK is known to transmit cell surface signals to the nucleus for mediating the protein of transcription and translation changes required for proliferation. ERK1 and ERK2 are 44kDa and 42kDa proteins, which are important subfamily of protein kinases that control numerous cell activities and physiological processes (including cell proliferation and differentiation) by down-regulating pro-apoptotic molecules and up-regulating anti-apoptotic molecules. The activation of MEK1 / 2 leads to the phosphorylation of ERK1 and ERK2. After stimulation, ERK1 / 2 is phosphorylated on threonine and tyrosine residues, and the phosphorylation on the tyrosine residues causes ERK1 / 2 to dissociate from MEK1 / 2. Then ERK1 / 2 translocates to the nucleus. In one embodiment, the compositions described herein help maintain mitogenic stimulation until late G1, so that successful entry into the S phase.

[0098] AKT is a serine / threonine-specific protein kinase that plays a role in cell proliferation and survival and inhibits apoptosis and metabolism. Phosphorylation of AKT activates AKT. Like pERK, AKT is also known to play a role in the cell cycle. AKT can also promote growth factor-mediated cell survival. Various studies have documented the key role of the Akt pathway in preventing apoptotic cell death. 9 PCNA is a unique protein associated with DNA replication, which is therefore used as a proliferation marker, and PCNA was measured with AA-ORS or saline treatment. AA-ORS increased PCNA in 0Gy and 5Gy irradiated mice, but not in saline treated mice. An increase in PCNA is an early sign of small intestinal epithelial proliferation. Together, these studies suggest that treatment with AA-ORS promotes proliferation.

[0099] Caspase-3 is the executioner or effector of apoptosis, as cleavage of intracellular protein substrates leads to morphological changes associated with apoptosis, including DNA degradation and chromatin condensation, as well as membrane blebbing to trigger the apoptotic process. This inactive proenzyme is activated by proteolytic cleavage. 7,46This study showed that radiation increased caspase-3 and AA-ORS treatment reduced cleaved caspase-3 in villous epithelial cells of 0Gy and 5Gy mice. Bcl-2, a downstream target of Erk1 / 2, is known to inhibit Bax in the intrinsic pro-apoptotic pathway. Increased Bcl-2 protein levels in the presence of AA-ORS suggest a protective mechanism against apoptosis. However, increased Bcl-2 protein levels in tissues from irradiated mice may suggest a radiation protective mechanism. A similar increase in Bcl-2 protein levels after irradiation has been reported and is consistent with previous findings (Ezekwudo, D. et al. Inhibition of expression of anti-apoptotic protein Bcl-2 and induction of cell death in radioresistant human prostate adenocarcinoma cell line (PC-3) by methyl jasmonate. Cancer Lett 270, 277-285, doi: 10.1016 / j.canlet.2008.05.022 (2008)). However, Bax protein did not show significant changes in radiation or treatment, indicating an effect of AA-ORS on apoptosis at a step upstream of Bax26. Increased p-Akt in AA-ORS-treated mice suggests that its effects may be achieved by activating proliferation or inhibiting apoptosis (Figure 5). Combined with the effects seen on caspase-3 and Bcl-2, these results may explain the pro-survival effects and increased proliferation observed in the case of AA-ORS treatment. However, further studies are needed to characterize the mechanism by which AA-ORS activate Erk1 / 2 and Akt, PCNA, caspase-3, Bcl-2, or Bax.

[0100] p53

[0101] Since Akt can also play a prominent role in malignant transformation, 10 AA-ORS was therefore used to study the role of the known tumor suppressor protein p53. Changes in the p53 protein could indicate that AA-ORS has a tumor suppressor effect. Mutations in p53 (tumor suppressor 53 gene) are the most common genetic lesions in human cancer. Mice homozygous for the null allele appear normal but are prone to spontaneously develop a variety of tumors. 11 p53 has also been shown to play an important role in the radiation response; in fact, the level of p53 accumulation in response to irradiation is primarily derived from the intensity of DNA damage. 12 Studies have shown that stem cell loss plays an important role in radiation-induced acute intestinal injury and mortality and is regulated by the p53 pathway and its transcriptional targets PUMA and p21.23,34,37 PUMA-dependent apoptosis rapidly reduces intestinal stem cells (ISCs) and their progenitors within hours after high-dose irradiation, and PUMA deficiency leads to improved animal survival and crypt regeneration by enhancing p21-dependent DNA repair and is essential for radiation-induced intestinal injury. 24,38 Together with Lgr5, p-Erk, and p-Akt, changes in cleaved caspase-3 indicated that AA-ORS increased villus height in intestinal tissues of both non-irradiated and irradiated mice not only through proliferation but also through reduced apoptosis and increased cell survival. To assess whether the villous epithelial cells derived from increased proliferation and reduced apoptosis were mature, differentiated, and functionally active, Na + Absorption capacity and glucose-stimulated Na + Absorption. NHE3 (Na in the small intestine + Both AA-ORS (the main transporter for glucose absorption) and SGLT1 (the transporter for sodium-coupled glucose absorption) are only found in mature and differentiated villus cells; they have enhanced function (Figures 3 and 4) and increased mRNA and protein levels. These studies show that AA-ORS treatment after irradiation increases electrolyte and glucose absorption (Figures 3, 4 and 7).

[0102] In one embodiment, the compositions described herein can be used to prevent DNA damage and / or repair damaged DNA. In another embodiment, the compositions promote the proliferation and / or development of stem cells and / or progenitor cells by preventing or reducing damage to DNA and / or repairing damaged DNA.

[0103] Irradiation, crypt counts, villus length, and immunohistochemistry

[0104] The increased weight gain and survival were found to be likely secondary to increases in crypt number and villus height, which in turn increase the surface area for absorption. The results suggest that crypt number and villus height increased with AA-ORS treatment starting 6 days after irradiation. Using a single-hit, multi-target model for crypt survival, the number of crypt progenitor units (N) per ileal circumference was significantly increased (P<0.001) without a change in DO (4.8±0.1Gy)( Figure 1A ). In the case of AA-ORS treatment, the Dq values ​​improved to an equivalent of 1.7 Gy with increased radiation tolerance, indicating improved crypt survival. Crypt survival studies have shown an increase in progenitor units or stem cells per crypt. Therefore, the effects of irradiation and AA-ORS on stem cell numbers were examined using antibodies specific for intestinal stem cell markers and the migration of daughter cells into the villi where they proliferate secondary to EdU incorporation. 2,4,36 At least three distinct crypt cell types are postulated to represent intestinal stem cells (ISCs). 2Each member of the population has different proliferation kinetics and sensitivity to radiation; therefore, each member is believed to have a unique function. 31 It is thought to dynamically switch from one type to the other in response to inhibitory and stimulatory signals caused by cytokines, hormones, or growth factors. 25 In contrast, slowly cycling intestinal epithelial stem cells (IESCs) [label-retaining cells (LRCs)] at the '+4 crypt position' contribute to homeostatic regenerative capacity, particularly during recovery from injury. 33 These LRCs express various markers, such as Bmi1, HopX, Lrig1 and / or Dclk1, and can change into rapidly cycling IESCs in response to injury. 34 Lgr5 can mark both types of cells, whereas Bmi1 and HopX are reported to preferentially mark +4 cells. 15 Lgr5+ ISCs are required for intestinal regeneration after radiation injury. 35 Lgr5- and Bmi1 are considered reserve cells that initiate a regenerative response following injury or radiation-induced damage. Studies have shown that the loss of Lgr5+ cells can be tolerated due to the activation of a stem cell pool expressing Bmi1. 2,30

[0105] The amino acid formulation AA-ORS that increases villus height has important implications for a variety of disease conditions beyond radiation- or chemotherapy-induced toxicity, which are characterized by reduced villus height, such as Crohn's disease, celiac disease, malnutrition, and environmental enteropathy. This study demonstrates that the systematic selection of certain nutrients based on their beneficial effects on gastrointestinal function helps improve in situ intestinal stem cell proliferation, maturation, and differentiation, leading to functional and highly villous epithelial cells that were initially damaged by irradiation ( Figure 7). This study also supports the observations of Leibowitz et al. that bone marrow-derived stem cells have no significant effect on the reconstruction of intestinal mucosa after high-dose radiation (Leibowitz, BJ et al. Ionizing irradiation induces acute haematopoietic syndrome and gastrointestinal syndrome independently in mice. Nat Commun 5, 3494, doi: 10.1038 / ncomms4494 (2014)). Future studies should seek to determine the mechanisms by which these amino acids increase stem cell populations, promote their proliferation, reduce cell apoptosis, and exclude malignant transformation. This work emphasizes the importance of carefully selecting different nutrients or individual amino acids to affect various stem cell populations, including hematopoietic stem cells.

[0106] Stem cells and / or progenitor cells

[0107] The compositions and methods described herein can be used to increase the survival, proliferation and / or development of stem and / or progenitor cells.The cell can be, for example, an embryonic cell, a pluripotent cell or a totipotent cell, and can be an in vivo or in vitro cell.

[0108] Stem cells are generally capable of differentiating into ectoderm, mesoderm, and endoderm cells. Pluripotent stem cells are undifferentiated cells that have the ability to differentiate into multiple cell types. Totipotent stem cells are undifferentiated cells that have the ability to differentiate into all cell types and by definition imply germline transmission.

[0109] In one embodiment, the stem cells are mesenchymal stem cells, which have the potential to differentiate into, for example, osteoblasts, chondrocytes, adipocytes, fibroblasts, smooth muscle cells, stromal cells, tenocytes, epithelial cells, neural cells, and vascular endothelial cells.

[0110] In one embodiment, the cell is an embryonic stem (ES) cell, which can proliferate indefinitely in an undifferentiated state. In addition, ES cells are omnipotent cells, which means that they can generate all cells present in the body (bone, muscle, brain cells, etc.). ES cells have been isolated from the inner cell mass (ICM) of the developing murine blastocyst (Evans et al., Nature 292: 154-156, 1981; Martin et al., Proc. Natl. Acad. Sci. 78: 7634-7636, 1981; Robertson et al., Nature 323: 445-448, 1986). In addition, human cells with ES properties have been isolated from endoderm cell masses (Thomson et al., Science 282: 1145-1147, 1998) and developing germ cells (Shamblott et al., Proc. Natl. Acad. Sci. USA 95: 13726-13731, 1998). Human and non-human primate embryonic stem cells have been produced (see U.S. Pat. No. 6,200,806, which is incorporated herein by reference).

[0111] In one embodiment, the cell is an adult stem cell, which self-renews and generates differentiated cells. Adult stem cells, also known as somatic stem cells, are stem cells that maintain and repair the tissue in which they reside. These cells can, for example, be bone marrow stem cells.

[0112] Precursor cells can also be used with the methods disclosed herein. Precursor cells can be isolated from various sources using methods known to those skilled in the art. Precursor cells can be of ectoderm, mesoderm or endoderm origin. Any precursor cells that can be obtained and maintained in vitro can be used according to the present method. The cells include cells of epithelial tissues (e.g., skin and intestinal lining), embryonic cardiomyocytes and neural precursor cells (Stemple and Anderson, 1992, Cell 71: 973-985). The cells also include pancreatic stem cells, cord blood stem cells, peripheral blood stem cells and stem cells derived from adipose tissue.

[0113] In one embodiment, stem cell also includes the pluripotent stem cell obtained by reprogramming somatic cells.Somatic cell reprogramming is the process of converting the epigenetic state of differentiated somatic cells into a pluripotent state that can produce any cell type.Somatic cell reprogramming can be realized, for example, by transferring the somatic cell nucleus into a donor oocyte, which is referred to as somatic cell nuclear transfer (SCNT).Somatic cell reprogramming can also be realized by direct reprogramming, referred to as induced pluripotent stem cells (iPSC), such as by retroviral expression of four transcription factors Oct4, Sox2, Klf4 and C-myc at the same time to achieve.These iPSCs enjoy all the key features of ES cells.

[0114] In another embodiment, other post-embryonic stem cells can be obtained from fetal liver, perinatal umbilical cord blood (UCB), human bone marrow, or G-CSF-stimulated peripheral blood starting from week 12 of gestation.

[0115] In certain embodiments, the stem cell is a neural stem cell (NSC), a skin stem cell, a hematopoietic stem cell (HSC), a mesenchymal stem cell (MSC), a tissue stem cell (e.g., a muscle stem cell), a mesodermal stem cell, an organ stem cell (e.g., a pancreatic stem cell and a liver stem cell), or an intestinal stem cell. In certain embodiments, the stem cell is an adult stem cell, an embryonic stem cell, a cancer stem cell, a neural stem cell (NSC), a skin stem cell, a hematopoietic stem cell (HSC), a mesenchymal stem cell (MSC), a tissue stem cell (e.g., a muscle stem cell), a mesodermal stem cell, an organ stem cell (e.g., a pancreatic stem cell and a liver stem cell), or an intestinal stem cell.

[0116] In one embodiment, the cell is a neuronal stem cell. In a non-limiting example, the cell is a neuronal precursor cell and / or a glial precursor cell. Undifferentiated neural stem cells differentiate into neuroblasts and glial cells, which give rise to neurons and glial cells.

[0117] Neural stem and progenitor cells can participate in various aspects of normal development, including migration along established migratory pathways to disseminated CNS regions, differentiation into a variety of developmentally appropriate and regionally appropriate cell types in response to microenvironmental cues, and non-destructive, non-tumorigenic dissemination with host progenitors and their progeny.

[0118] Human NSCs are able to express exogenous transgenes in vivo in these disseminated locations. Therefore, these cells can be used to treat a variety of conditions, including traumatic injuries of the spinal cord, brain, and peripheral nervous system; treatment of degenerative disorders, including Alzheimer's disease, Huntington's disease, and Parkinson's disease; affective disorders, including major depression; stroke, and many more.

[0119] In one embodiment, stem cell is muscle stem cell.The muscle tissue of adult vertebrate is regenerated in the reserve myoblast called satellite cell.Satellite cells are distributed in the whole muscle tissue, and are mitotically static in the absence of injury or disease.After recovering from the damage caused by injury or disease or in response to the stimulation of growth or overgrowth (hypertrophy), satellite cells reenter the cell cycle, propagate and differentiate into multinucleated myotubes, and form new muscle fibers.Myoblasts eventually produce alternative muscle fibers or fuse into existing muscle fibers, thereby increasing fiber perimeter by synthesizing contractile organ components.Myogenic standards include the expression of myogenic proteins (including intermediate filament protein knot protein) and myogenic transcription factors MyoD, Myf-5 and Pax-7.

[0120] In one embodiment, the stem cells are hair follicle stem cells. The bulge region of the hair follicle is a rich and readily accessible source of actively growing multipotent adult stem cells. Nestin is a protein marker for neural stem cells and is also expressed in hair follicle stem cells and their direct differentiated progeny. Hair follicle stem cells expressing nestin differentiate in vitro into, for example, neurons, glial cells, keratinocytes, and smooth muscle cells.

[0121] In one embodiment, stem cells are pancreatic stem cells and pancreatic multipotent progenitor (PMP) cells. These cells can be separated from tissues derived from islets and pancreatic ducts, and further developed into, for example, other PMP cells or nerves or pancreatic cells. Pancreatic cells optionally include alpha cells, delta cells, beta cells, pancreatic exocrine cells and pancreatic stellate cells. Alpha cells are mature glucagon-producing cells. In vivo, these cells are present in the islets of Langerhans. Beta cells are mature insulin-producing cells. In vivo, these cells are also present in the islets of Langerhans. Pancreatic stem cells are very important in the treatment of diabetes, particularly type I diabetes, and can be used to provide beta cells.

[0122] In one embodiment, stem cell is bone marrow stem cell. Bone marrow stem cell is a cell generated in the bone marrow, which can differentiate into cells of various body tissues. Bone marrow stem cells can also restore the function of tissue loss by differentiating into tissue cells under the influence of differentiation inducers. The example of bone marrow stem cell includes bone marrow mesenchymal stem cells that can, for example, differentiate into osteocytes, chondrocytes, adipocytes, muscle cells, tendon cells or bone marrow stromal cells, and hematopoietic stem cells that can differentiate into blood cells (such as red blood cells and white blood cells).

[0123] In one embodiment, stem cells are gastrointestinal stem cells. Epithelial cells line the gastrointestinal tract. The turnover of these cells is a constant process under normal homeostasis and increases after injury. Multipotent stem cells regulate this process by generating all gastrointestinal epithelial cell lineages and even the entire intestinal crypts and gastric glands. These stem cells are located in the lower part of the intestinal crypts, including the more static "+4" cells above the rapidly circulating crypt base columnar cells (CBC) and Paneth cells in mammals.

[0124] At least three distinct crypt cell types are postulated to represent intestinal stem cells (ISCs). Each member of the population has different proliferation kinetics and sensitivity to radiation; therefore, each is thought to have a unique function. They are thought to dynamically switch from one type to another in response to inhibitory and stimulatory signals caused by cytokines, hormones, or growth factors. In contrast, slowly cycling intestinal epithelial stem cells (IESCs) [marked by retention cells (LRCs)] in the "+4 crypt position" contribute to homeostatic regenerative capacity, especially during recovery from injury. These LRCs express various markers, such as BMI1, HOPX, LRIG1, and / or DCLK1, and can change to rapidly cycling IESCs in response to injury. Lgr5 can mark both cells, while Bmi1 and HopX are reported to preferentially mark +4 cells. Lgr5 + ISCs are required for intestinal regeneration after radiation- and / or chemotherapy-induced gastrointestinal injury. - and BMI1 are considered reserve cells that initiate a regenerative response following injury or radiation-induced damage. Similarly, the preparation can be used in subclinical disease conditions such as environmental enteropathy associated with increased intestinal permeability, increased local and systemic inflammation, and decreased villus height. Subjects with environmental enteropathy experience chronic malnutrition secondary to malabsorption of nutrients and minerals.

[0125] Compositions for promoting proliferation and / or development of stem cells and / or progenitor cells, and for wound healing, treating skin disorders, lung disorders, mucosal barrier disorders, and diseases or disorders associated with mucosal barrier function, and treating GI mucosal injuries

[0126] In one aspect, provided herein are therapeutic compositions for promoting the survival, proliferation and / or development of stem cells and / or progenitor cells.Compositions and methods are described herein for treating diseases or conditions associated with mucosal barrier function in subjects in need, such as wound healing, treating skin disorders (e.g., atopic dermatitis, psoriasis, bedsores or conditions associated with skin aging), treating lung disorders (e.g., asthma), improving mucosal barrier function and / or treating GI mucosal damage.

[0127] In one embodiment, the therapeutic composition comprises, consists essentially of, or consists of: one or more free amino acids selected from the group consisting of: threonine, valine, tyrosine, tryptophan, aspartic acid, serine, and derivatives thereof; and optionally a pharmaceutically acceptable carrier, adjuvant, and / or other active ingredients. In one embodiment, the therapeutic composition comprises, consists essentially of, or consists of: one or more free amino acids selected from the group consisting of: threonine, valine, tyrosine, tryptophan, aspartic acid, and serine; and optionally a pharmaceutically acceptable carrier, adjuvant, and / or other active ingredients. In certain embodiments, the composition is sterile.

[0128] In one embodiment, the total molar osmotic pressure concentration of the composition is about 100mosm to about 280mosm, or any value therebetween. Preferably, the total molar osmotic pressure concentration is about 150 to about 260mosm. In another embodiment, the total molar osmotic pressure concentration of the composition is any value below about 280mosm.

[0129] The composition may have a pH of, for example, 2.5 to 8.5. In certain embodiments, the composition has a pH of about 2.5 to about 6.5, about 3.0 to about 6.0, about 3.5 to about 5.5, about 3.9 to about 5.0, or about 4.2 to about 4.6. In other embodiments, the pH is about 6.5 to about 8.5, about 7.0 to about 8.0, or about 7.2 to about 7.8.

[0130] In certain embodiments, the composition comprises one or more free amino acids selected from the group consisting of threonine, valine, tyrosine, tryptophan, aspartic acid and serine. In certain embodiments, the composition comprises one or more free amino acids selected from the group consisting of threonine, valine, tyrosine, tryptophan, aspartic acid, serine and derivatives thereof. In certain embodiments, the composition comprises one or more free amino acids selected from the group consisting of threonine, valine, tyrosine, tryptophan and aspartic acid. The composition preferably comprises one or more free amino acids selected from the group consisting of threonine, valine, serine, tyrosine and tryptophan. In certain embodiments, the composition comprises two or more free amino acids selected from the group consisting of threonine, valine, tyrosine, tryptophan, aspartic acid and serine. In certain embodiments, the composition comprises three or more free amino acids selected from the group consisting of threonine, valine, tyrosine, tryptophan, aspartic acid and serine. In certain embodiments, the composition comprises four or more free amino acids selected from the group consisting of threonine, valine, tyrosine, tryptophan, aspartic acid and serine. In certain embodiments, the composition comprises threonine, valine, tyrosine, tryptophan and aspartic acid. In certain embodiments, the composition comprises the free amino acids of threonine, valine, tyrosine, tryptophan and serine.

[0131] If present in the composition, these amino acids can be present, for example, at the following concentrations: threonine is about 0.4 to about 1.5, about 0.7 to about 1.3, or about 0.9 to about 1.1 g / L; valine is about 0.7 to about 1.7, about 0.9 to about 1.5, or about 1.1 to about 1.3 g / L; serine is about 0.6 to about 1.6, about 0.8 to about 1.4, about 1.0 to about 1.2 g / L; tyrosine is about 0.05 to about 0.4, or about 0.1 to about 0.3 g / L; and tryptophan is about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 g / L. In a certain embodiment, the therapeutic composition includes threonine (about 1.0 g / L), valine (about 1.2 g / L), serine (about 1.1 g / L), tyrosine (about 0.2 g / L), and tryptophan (about 1.6 g / L). In one embodiment, the composition does not include serine.

[0132] In another embodiment, the composition comprises only one free amino acid selected from the group consisting of threonine, valine, tyrosine and tryptophan and / or its derivatives, or consists essentially of only one free amino acid selected from the group consisting of threonine, valine, tyrosine and tryptophan and / or its derivatives. In another embodiment, the therapeutic composition comprises threonine as a free amino acid, or consists essentially of threonine as a free amino acid. The therapeutic composition may also comprise valine as a free amino acid, or consists essentially of valine as a free amino acid. In addition, the therapeutic composition comprises tyrosine as a free amino acid, or consists essentially of tyrosine as a free amino acid. The therapeutic composition comprises tryptophan as a free amino acid, or consists essentially of tryptophan as a free amino acid. In addition, the therapeutic composition comprises aspartic acid as a free amino acid, or consists essentially of aspartic acid as a free amino acid. In certain embodiments, the composition comprises serine as a free amino acid.

[0133] In another embodiment, the composition may also comprise or consist essentially of two free amino acids selected from the group consisting of threonine, valine, serine, tyrosine, tryptophan and aspartic acid, including a combination of threonine and valine, a combination of threonine and serine, a combination of threonine and tyrosine, a combination of threonine and tryptophan, a combination of valine and serine, a combination of valine and tyrosine, a combination of valine and tryptophan, a combination of serine and tyrosine, a combination of serine and tryptophan, a combination of tyrosine and aspartic acid, a combination of serine and aspartic acid, a combination of valine and aspartic acid, a combination of threonine and aspartic acid, a combination of tryptophan and aspartic acid, and a combination of tyrosine and tryptophan.

[0134] In another embodiment, the composition may comprise or consist essentially of three free amino acids selected from the group consisting of threonine, valine, serine, tyrosine, tryptophan, and aspartic acid, including a combination of threonine, valine, and serine; a combination of threonine, valine, and tyrosine; a combination of threonine, valine, and tryptophan; a combination of threonine, serine, and tyrosine; a combination of threonine, serine, and tryptophan; a combination of threonine, tyrosine, and tryptophan; a combination of valine, serine, and tyrosine; and tryptophan; a combination of valine, tyrosine and tryptophan; and a combination of serine, tyrosine and tryptophan; a combination of threonine, valine and aspartic acid; a combination of threonine, serine and aspartic acid; a combination of threonine, tyrosine and aspartic acid; a combination of threonine, tryptophan and aspartic acid; a combination of valine, serine and aspartic acid; a combination of valine, tyrosine and aspartic acid; a combination of valine, tryptophan and aspartic acid; a combination of serine, tyrosine and aspartic acid; a combination of serine, tryptophan and aspartic acid; a combination of tyrosine, tryptophan and aspartic acid.

[0135] In another embodiment, the composition may comprise or consist essentially of four free amino acids selected from the group consisting of threonine, valine, serine, tyrosine, tryptophan, and aspartic acid, including a combination of threonine, valine, serine, and tyrosine; a combination of threonine, valine, serine, and tryptophan; a combination of threonine, valine, tyrosine, and tryptophan; a combination of threonine, serine, tyrosine, and tryptophan; and a combination of valine, serine, tyrosine, and tryptophan; The present invention relates to a combination of threonine, valine, tyrosine and aspartic acid; a combination of threonine, valine, tryptophan and aspartic acid; a combination of threonine, serine, tyrosine and aspartic acid; a combination of threonine, valine, tryptophan and aspartic acid; a combination of threonine, serine, tyrosine and aspartic acid; a combination of threonine, serine, tryptophan and aspartic acid; a combination of valine, serine, tyrosine and aspartic acid; a combination of valine, serine, tryptophan and aspartic acid; a combination of valine, tyrosine, tryptophan and aspartic acid; a combination of serine, tyrosine, tryptophan and aspartic acid.

[0136] In another embodiment, the composition may comprise or consist essentially of five free amino acids selected from the group consisting of threonine, valine, serine, tyrosine, tryptophan and aspartic acid, including a combination of threonine, valine, serine, tyrosine and tryptophan; a combination of threonine, valine, serine, tyrosine and aspartic acid; a combination of threonine, valine, serine, tryptophan and aspartic acid; a combination of threonine, valine, serine, tryptophan and aspartic acid; a combination of threonine, valine, tyrosine, tryptophan and aspartic acid; a combination of threonine, serine, tyrosine, tryptophan and aspartic acid.

[0137] In another embodiment, the composition may comprise or consist essentially of threonine, valine, serine, tyrosine, tryptophan, and aspartic acid as free amino acids.

[0138] In certain embodiments, the composition may include natural amino acids or derivatives thereof, which maintain substantially the same or better activity in terms of enhancing the survival, proliferation and / or development of stem cells and / or progenitor cells. In certain embodiments, the composition may include natural amino acids or derivatives thereof, which maintain substantially the same or better activity in terms of: wound healing in a subject in need, treatment of skin disorders (e.g., atopic dermatitis, psoriasis, bedsores or disorders associated with skin aging), treatment of pulmonary disorders (e.g., asthma), improvement of mucosal barrier function and / or treatment of GI mucosal damage. Derivatives may, for example, be enantiomers, and include the D and L forms of amino acids. Derivatives may, for example, be iodotyrosine or norvaline. Other amino acid derivatives include, for example, norleucine, ornithine, penicillamine, pyroglutamine derivatives, or other derivatives of alanine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, isoleucine, leucine, lysine, methionine, proline, phenylalanine, serine, threonine, tryptophan or valine. In certain embodiments, the amino acid derivative is a derivative of threonine, valine, tyrosine, tryptophan, aspartic acid or serine. Other amino acid derivatives include, but are not limited to, those synthesized by, for example, acylation, methylation and / or halogenation of amino acids. These derivatives include, for example, β-methyl amino acids, C-methyl amino acids and N-methyl amino acids.

[0139] In some specific embodiments, the compositions of the present disclosure do not include one or more amino acids selected from the group consisting of serine, lysine, glycine, isoleucine and asparagine. Alternatively, in certain embodiments, even if these amino acids are present in the composition, they are present in an amount that does not inhibit stem cell and / or progenitor cell proliferation and / or development.

[0140] In certain specific embodiments, the compositions of the present disclosure do not include a free amino acid selected from the group consisting of serine, lysine, glycine, isoleucine and asparagine, or contain only a negligible amount of a free amino acid selected from the group consisting of serine, lysine, glycine, isoleucine and asparagine. In other embodiments, the therapeutic composition does not include lysine as a free amino acid, or the therapeutic composition does not include glycine as a free amino acid; or the therapeutic composition does not include aspartic acid as a free amino acid; or the therapeutic composition does not include isoleucine as a free amino acid; or the therapeutic composition does not include asparagine as a free amino acid.

[0141] In some embodiments, the therapeutic composition does not include any two free amino acids selected from the group consisting of serine, lysine, glycine, isoleucine, and asparagine, including a combination of lysine and glycine, a combination of lysine and aspartic acid, a combination of lysine and isoleucine, a combination of lysine and asparagine, a combination of glycine and aspartic acid, a combination of glycine and isoleucine, a combination of glycine and asparagine, a combination of aspartic acid and isoleucine, a combination of aspartic acid and asparagine, and a combination of isoleucine and asparagine.

[0142] In other embodiments, the composition does not include any three free amino acids selected from the group consisting of: serine, lysine, glycine, isoleucine and asparagine, including a combination of lysine, glycine and aspartic acid; a combination of lysine, glycine and isoleucine; a combination of lysine, glycine and asparagine; a combination of lysine, aspartic acid and isoleucine; a combination of lysine, aspartic acid and asparagine; a combination of lysine, isoleucine and asparagine; a combination of glycine, aspartic acid and isoleucine; a combination of glycine, aspartic acid and asparagine; a combination of glycine, isoleucine and asparagine; and a combination of aspartic acid, isoleucine and asparagine.

[0143] In further embodiments, the composition does not include any four free amino acids selected from the group consisting of: serine, lysine, glycine, aspartic acid, isoleucine and asparagine, including a combination of lysine, glycine, aspartic acid and isoleucine; a combination of lysine, glycine, aspartic acid and asparagine; a combination of lysine, aspartic acid, isoleucine and asparagine; a combination of lysine, glycine, isoleucine and asparagine; and a combination of glycine, aspartic acid, isoleucine and asparagine.

[0144] In a specific embodiment, the composition of the present disclosure does not include lysine, glycine, aspartic acid, isoleucine and asparagine. In another specific embodiment, the composition does not include serine, lysine, glycine, aspartic acid, isoleucine and asparagine, or only includes negligible amounts of serine, lysine, glycine, aspartic acid, isoleucine and asparagine. In one embodiment, the composition does not include glutamine and / or methionine; and any dipeptide, oligopeptide or polypeptide or protein that can be hydrolyzed into glutamine and / or methionine.

[0145] In certain specific embodiments, the therapeutic composition may include lysine, wherein the total concentration of lysine is less than 300 mg / l, 100 mg / l, 50 mg / l, 10 mg / l, 5 mg / l, 1 mg / l, 0.5 mg / l, or 0.01 mg / l. The therapeutic composition may also include aspartic acid, wherein the total concentration of aspartic acid is less than 300 mg / l, 100 mg / l, 50 mg / l, 10 mg / l, 5 mg / l, 1 mg / l, 0.5 mg / l, or 0.01 mg / l. The therapeutic composition may also include glycine, wherein the total concentration of glycine is less than 300 mg / l, 100 mg / l, 50 mg / l, 10 mg / l, 5 mg / l, 1 mg / l, 0.5 mg / l, or 0.01 mg / l. The therapeutic composition may further include isoleucine, wherein the total concentration of isoleucine is less than 300 mg / l, 100 mg / l, 50 mg / l, 10 mg / l, 5 mg / l, 1 mg / l, 0.5 mg / l or 0.01 mg / l. The therapeutic composition may also include asparagine, wherein the total concentration of asparagine is less than 10 mg / l, 5 mg / l, 1 mg / l, 0.5 mg / l or 0.01 mg / l.

[0146] In an optional embodiment, the composition may comprise the free amino acid glutamine, and optionally one or more dipeptides containing glutamine, wherein the total concentration of the free amino acid glutamine and the dipeptides containing glutamine is less than 300 mg / l, or any concentration below 300 mg / l, such as 100 mg / l, 50 mg / l, 10 mg / l, 5 mg / l, 1 mg / l, 0.5 mg / l or 0.01 mg / l. In certain embodiments, the composition may comprise the free amino acid glutamine, and optionally one or more peptides containing glutamine, wherein the total concentration of the free amino acid glutamine and the peptides containing glutamine is less than 300 mg / l, or any concentration below 300 mg / l, such as 100 mg / l, 50 mg / l, 10 mg / l, 5 mg / l, 1 mg / l, 0.5 mg / l or 0.01 mg / l.

[0147] In another alternative embodiment, the therapeutic composition may comprise the free amino acid methionine, and optionally one or more methionine-containing dipeptides, wherein the total concentration of the free amino acid methionine and the methionine-containing dipeptides is less than 300 mg / l, or any concentration below 300 mg / l, such as 100 mg / l, 50 mg / l, 10 mg / l, 5 mg / l, 1 mg / l, 0.5 mg / l, or 0.01 mg / l. In certain embodiments, the composition may comprise the free amino acid methionine, and optionally one or more methionine-containing dipeptides, wherein the total concentration of the free amino acid methionine and the methionine-containing peptides is less than 300 mg / l, or any concentration below 300 mg / l, such as 100 mg / l, 50 mg / l, 10 mg / l, 5 mg / l, 1 mg / l, 0.5 mg / l, or 0.01 mg / l.

[0148] In certain embodiments, the composition further comprises additives (e.g., nutrients, electrolytes, vitamins, minerals, etc.). In certain embodiments, the composition comprises iron or zinc. In certain embodiments, the therapeutic composition comprises one or more electrolytes selected from the following: for example, Na + ; K + ;HCO 3 - ;CO 3 2- ;Ca 2+ Mg 2+ ;Fe 2 ;CI - ; Phosphate ions, such as H 2 PO 4 - , HPO 4 2- and PO 4 3- ; zinc; iodine; copper; iron; selenium; chromium; and molybdenum. In an alternative embodiment, the composition does not contain HCO 3 - or CO 3 2- In another alternative embodiment, the composition comprises HCO at a total concentration of less than 5 mg / l or at a concentration below 5 mg / l. 3 - and CO 3 2- In some embodiments, the composition does not contain electrolytes. For example, in some embodiments, the composition does not include one or more or any of the following: Na + ; K + ;HCO 3 - ;CO3 2-;Ca 2+ Mg 2+ ;Fe 2 ;Cl - ; Phosphate ions, such as H 2 PO 4 - , HPO 4 2- and PO 4 3- ; zinc; iodine; copper; iron; selenium; chromium; and molybdenum.

[0149] In certain embodiments, the composition does not contain one or more ingredients selected from the following: oligosaccharides, polysaccharides and carbohydrates; oligopeptides or polypeptides or proteins; lipids; short-chain, medium-chain and / or long-chain fatty acids; and / or foods containing one or more of the above nutrients. In certain embodiments, the composition does not include glucose or sucrose.

[0150] In one embodiment, phosphate ions such as H 2 PO 4 - , HPO 4 2- and PO 4 3- For buffering the compositions of the present disclosure. In one embodiment, the therapeutic composition uses HCO 3 - or CO 3 2- In another embodiment, the therapeutic composition does not use HCO 3 - or CO 3 2- As a buffer.

[0151] In certain embodiments, the composition comprises: valine, threonine, tyrosine, electrolytes, Na+ (about 10 mmol to 60 mmol) and K+ (about 1 mmol to 20 mmol). In certain embodiments, the composition includes a buffer.

[0152] Stem cell and / or progenitor cell therapy and therapies for wound healing, treatment of skin conditions, lung disorders, improvement of mucosal barrier function and / or treatment of GI mucosal damage

[0153] Amino acid compositions as therapies for treating GI, lung and skin disorders are described herein. The present disclosure provides compositions and methods for enhancing the survival, proliferation and / or development of stem cells and / or progenitor cells. The present disclosure provides compositions and methods for treating diseases or conditions associated with mucosal barrier function in subjects in need, such as wound healing, treating skin disorders (e.g., atopic dermatitis, psoriasis, bedsores or conditions associated with skin aging), treating lung disorders (e.g., asthma), improving mucosal barrier function and / or treating GI mucosal damage. The number of cells can be increased by increasing the survival, proliferation and / or development of stem cells and / or progenitor cells. In one embodiment, the method includes exposing stem cells and / or progenitor cells to compositions of the present disclosure. Stem cells and / or progenitor cells can be exposed to compositions in culture, in vitro, in situ or in vivo, including exposure after administration, implantation and / or delivery to a subject.

[0154] The subject can be, for example, a human being in need of promoting the survival, proliferation and / or development of stem cells and / or progenitor cells. The subject can be, for example, a human subject suffering from a disease or condition in need of treatment. In addition to humans, the animal can be any species, including but not limited to mammalian species, including but not limited to domestic and experimental animals, such as dogs, cats, mice, rats, guinea pigs and hamsters; livestock, such as horses, cattle, pigs, sheep, goats, ducks, geese and chickens; other primates, such as apes, chimpanzees, orangutans and monkeys; fish; amphibians, such as frogs and salamanders; reptiles, such as snakes and lizards; and other animals, such as foxes, camels, bears, antelopes, llamas, weasels, rabbits, minks, beavers, minks, otters, sables, seals, coyotes, chinchillas, deer, muskrats and opossums.

[0155] In one embodiment, the method results in an increase in the survival, proliferation and / or development of stem cells and / or progenitor cells. In certain embodiments, the method results in an improvement in the condition of a subject suffering from a disease or condition associated with mucosal barrier function such as a wound, a skin disorder (e.g., atopic dermatitis, psoriasis, bedsores or a condition associated with skin aging), a pulmonary disorder (e.g., asthma), a mucosal barrier function and / or GI mucosal damage.

[0156] In one embodiment, the method comprises introducing a composition according to the invention into stem cells and / or progenitor cells in culture to promote survival, proliferation and / or development. Thus, the composition can be used to obtain an increased number of cells for the treatment of various diseases and disorders.

[0157] In one embodiment, the present disclosure provides a method for improving the treatment outcome of implanted stem cells, the method comprising administering a composition of the present disclosure in conjunction with stem cell implantation. The administration of the composition can be at or near the target stem cell implantation site in humans or non-human animals. In certain embodiments, there is provided herein a method for treating a disease or condition such as wound healing associated with mucosal barrier function of a subject in need, treating a skin disorder (e.g., atopic dermatitis, psoriasis, bedsores or a condition associated with skin aging), treating a lung disorder (e.g., asthma), improving mucosal barrier function and / or treating GI mucosal damage, the method comprising administering a composition as described herein to the subject in need.

[0158] In one embodiment, the recipient of the administered stem and / or progenitor cells may be immunosuppressed by the use of immunosuppressive drugs such as cyclosporine, or by implementing a local immunosuppressive strategy using topically applied immunosuppressants, although such immunosuppression is not necessarily a prerequisite in certain immunoprivileged tissues (e.g., brain and eye tissues).

[0159] In certain embodiments, the stem cells and / or progenitor cells administered are autologous in nature, i.e., prepared from the recipient's own tissue. In this case, the progeny of the stem cells can be generated from the dissociated or separated tissue and propagated in vitro using the compositions disclosed herein. After the cell number is appropriately expanded, the cells can be harvested and prepared for administration to the affected tissue of the recipient.

[0160] In one embodiment, the present disclosure provides a method for promoting stem cell proliferation and differentiation in a subject in need thereof, wherein the method comprises: identifying a subject in need thereof, and administering to the subject an effective amount of a composition comprising, consisting essentially of, or consisting of: one or more free amino acids selected from the group consisting of threonine, valine, tyrosine, tryptophan, aspartic acid, and serine; and optionally, one or more pharmaceutically acceptable carriers, adjuvants, and / or other active agents, wherein the composition has a total osmotic pressure concentration of about 100 to about 280 mosm and a pH of about 2.5 to about 6.5. In certain embodiments, provided herein are methods for treating a disease or condition associated with mucosal barrier function, such as wound healing, treating a skin condition (e.g., atopic dermatitis, psoriasis, bedsores, or a condition associated with skin aging), treating a lung disorder (e.g., asthma), improving mucosal barrier function, and / or treating GI mucosal damage, wherein the method comprises: identifying a subject in need thereof, and administering to the subject an effective amount of a composition comprising, consisting essentially of, or consisting of: one or more free amino acids selected from the group consisting of threonine, valine, tyrosine, tryptophan, aspartic acid, and serine; and optionally, one or more pharmaceutically acceptable carriers, adjuvants, and / or other active agents, wherein the composition has a total osmotic pressure concentration of about 100 to about 280 mosm and a pH of about 2.5 to about 6.5.

[0161] In another embodiment, the compositions and methods of the present disclosure can be used to overcome myelosuppression secondary to drugs, chemicals, and unexplained viral or bacterial infections.

[0162] In certain embodiments, the present disclosure can be used to promote the proliferation and development of stem and / or progenitor cells in diseases and conditions, including, but not limited to, malignancies; Paneth cell deficiency; hypopituitarism; celiac disease, such as celiac disease that does not respond to a gluten-free diet; tropical sprue; radiation-related ischemia; drug-induced villous atrophy, such as neomycin and azathioprine-induced villous atrophy, severe food intolerance; congenital Crohn's disease; autoimmune bowel disease; enterocolitis; hepatitis; intestinal cancer; intestinal lymphoma; type 1 diabetes; allergies; eye conditions, such as corneal tears; eosinophilic gastroenteritis; viral gastroenteritis, and immunodeficiency syndrome.

[0163] In some embodiments, the present disclosure can be used to promote the proliferation and differentiation of stem cells in conditions and diseases induced by viral, fungal or bacterial infections, such as bone marrow suppression induced by viral, fungal or bacterial infections. For example, the compositions and methods can be used to treat patients with low platelet counts caused by, for example, dengue virus.

[0164] The compositions described herein can also be used to treat or ameliorate symptoms of deficits caused, for example, by neurodegenerative diseases, traumatic injury, neurotoxic injury, ischemia, developmental disorders, disorders affecting vision, spinal injury or disease, demyelinating diseases, autoimmune diseases, infections, inflammatory diseases, or corporal diseases.

[0165] In certain embodiments, the implanted stem cells are capable of proliferating, migrating to areas of tissue damage, and / or differentiating in a tissue-specific manner and functioning in a manner that reduces the defect.

[0166] In one embodiment, methods and compositions according to the present disclosure are particularly useful for patients exposed to radiation or receiving radiation, chemotherapy, and / or proton therapy.

[0167] In another aspect, the present disclosure provides a method for treating a subject in need of a gastrointestinal tract (e.g., small intestinal mucosa, esophagus, stomach, large intestine, etc.), a urogenital tract, or an organ with a mucosal lining, the method comprising administering a composition as described herein to the subject. In another aspect, the present disclosure provides a method for treating a condition associated with a mucosal barrier in a subject in need thereof, the method comprising administering a composition as described herein to the subject.

[0168] The compositions of the present disclosure can be used to treat or improve any disease or condition requiring proliferation and / or development of stem cells and / or progenitor cells. In a specific embodiment, the compositions and methods of the present disclosure can be used to treat or improve radiation-induced small intestinal damage by promoting the proliferation of stem cells. In another specific embodiment, the present disclosure can be used to treat or improve small intestinal damage caused by radiation therapy, particularly pelvic and abdominal radiation therapy. In a specific embodiment, radiation therapy is used for cancer treatment.

[0169] In addition, the present disclosure can be used to promote the proliferation of stem cells to treat or improve small intestinal damage caused by chemotherapeutic agents, including but not limited to cisplatin, 5-fluorouracil (5-FU), hydroxyurea, etoposide, arabinoside, 6-mercaptopurine, 6-thioguanine, fludarabine, methotrexate, steroids and / or combinations thereof. Exemplary chemotherapeutic agents include but are not limited to antiestrogens (e.g., tamoxifen, raloxifene and megestrol), LHRH agonists (e.g., goserelin (goscrclin) and leuprorelin), antiandrogens (e.g., flutamide and bicalutamide), photodynamic therapy (e.g., verteporfin (BPD-MA), phthalocyanine, photosensitizer Pc4 and demethoxy-hypocrellin A (demethoxy-hypocrellin A), and / or combinations thereof. A, 2BA-2-DMHA)), nitrogen mustards (e.g., cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas (e.g., carmustine (BCNU) and lomustine (CCNU)), alkyl sulfonates (e.g., busulfan and treoxavan), triazenes (e.g., dacarbazine, temozolomide), platinum compounds (e.g., cisplatin, carboplatin, oxaliplatin), vinca alkaloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), taxanes (e.g., paclitaxel or paclitaxel equivalents, such as nanoparticle albumin-bound paclitaxel ( ABRAXANE), docosahexaenoic acid-conjugated paclitaxel (DHA-paclitaxel, Taxoprexin), polyglutamic acid-conjugated paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX), tumor-activated prodrug (TAP) ANG1005 (brain-targeted angiopep-2 conjugated to three paclitaxel molecules), paclitaxel-EC-1 (paclitaxel conjugated to the erbB2 recognition peptide EC-1), and glucose-conjugated paclitaxel, e.g., 2'-paclitaxel methyl 2-pyranosyl glucopyranosyl succinate;), epipodophyllins (e.g., etoposide, etoposide phosphate, teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan, irinotecan, crisnatol, mitomycin C), antimetabolites, DHFR inhibitors (e.g., methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMP dehydrogenase inhibitors (e.g., mycophenolic acid, tiazofurin, ribavirin, and EICAR), ribonucleotide reductase inhibitors (e.g., hydroxyurea and deferoxamine), uracil analogs (e.g., 5-fluorouracil (5-FU), floxuridine, doxifluridine, ratitrexed, tegafur-uracil, capecitabine), cytosine analogs (e.g., cytarabine), C), cytosine arabinoside and fludarabine), purine analogs (e.g., mercaptopurine and thioguanine), vitamin D3 analogs (e.g., EB1089, CB 1093, and KH 1060), prenylation inhibitors (e.g., lovastatin), dopaminergic neurotoxins (e.g., 1-methyl-4-phenylpuridinium ion), cell cycle inhibitors (e.g., staurosporine), actinomycins (e.g., actinomycin D, dactinomycin), bleomycins (e.g., bleomycin A2, bleomycin B2, pelocycin), anthracyclines (e.g., daunorubicin, doxorubicin, pegylated liposomal doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDR inhibitors (e.g., verapamil), Ca; 2+ ATPase inhibitors (e.g., thapsigargin), imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g., axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN TM , AZD2171), dasatinib ( BMS-354825), erlotinib Gefitinib Imatinib ( CGP57148B, STI-571), lapatinib ( ), neratinib (CEP-701), neratinib (HKI-272), nilotinib Semaxinib (SU5416), sunitinib ( SU11248), toceranib, )、Vandetanib( ZD6474), vatalanib (PTK787, PTK / ZK), trastuzumab Bevacizumab Rituximab Cetuximab Panitumumab Ranibizumab ), Nilotinib Sorafenib (NEXAVARC), everolimus Alemtuzumab Gemtuzumab ozogamicin Temsirolimus )、ENMD-2076、PCI-32765、AC220、Dovitinib Lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK TM ), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647 and / or XL228), proteasome inhibitors (e.g., bortezomib (VELCADE)), mTOR inhibitors (e.g., rapamycin, temsirolimus (CCI-779), everolimus (RAD-001), ridaforolimus), AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235 (Novartis), BGT226 (Norvartis), XL765 (Sanofi

[00136] The invention relates to the treatment of leukemia and leukemia with the use of the following agents: Aventis), PF-4691502 (Pfizer), GDC0980 (Genetech), SF1126 (Semafoe), and OSI-027 (OSI), oblimersen, gemcitabine, carminomycin, leucovorin, pemetrexed, cyclophosphamide, dacarbazine, procarbizine, prednisolone, dexamethasone, camptothecin, plicamycin, asparaginase, aminopterin, methopterin, porphyrin, melphalan, isovinblastine, leurosine, chlorambucil, trabectedin, procarbazine, discodermolide, carminomycin, aminopterin, and hexamethonium.

[0170] In certain embodiments, the present disclosure can be used to promote the survival and proliferation of stem cells to treat or improve diseases involving small intestinal damage, including but not limited to inflammatory bowel disease (IBD), ulcerative colitis, duodenal ulcer, Crohn's disease and / or celiac disease (also known as celiac disease). The present disclosure can be used to treat or improve small intestinal damage caused by pathogenic infection such as virus, bacteria, fungus or other microbial infection.

[0171] In one embodiment, stem cells and / or progenitor cells have been subjected to radiation before being treated with the compositions disclosed herein. In another embodiment, stem cells and / or progenitor cells will be subjected to radiation after being treated with the compositions disclosed herein. Radiation can be applied to cells, for example, 1 minute, 5 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 1 day, 5 days, 7 days, 14 days, 30 days, 60 days, 3 months, 6 months, 1 year, 2 years or 3 years or longer before or after treating cells with the compositions described herein. The dosage of radiation can be, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 120 or 150 Gy.

[0172] In one embodiment, the present disclosure can be used in the context of bone marrow transplantation. Bone marrow stem cells and / or progenitor cells can be treated with the compositions of the present disclosure in vivo or ex vivo. The treatment enhances cell survival, proliferation and / or development.

[0173] In another embodiment, the present disclosure can be used in the context of Prochymal, which is used to manage acute graft-versus-host disease in children. It is an allogeneic stem cell therapy based on mesenchymal stem cells (MSCs) derived from the bone marrow of an adult donor. The survival, proliferation and / or development of MSCs can be enhanced by contacting them in vivo or ex vivo with a composition of the present disclosure.

[0174] In another embodiment, the compositions and methods of the present disclosure can be used for cardiac treatment. Stem cell therapy for the treatment of myocardial infarction generally utilizes autologous bone marrow stem cells; however, other types of adult stem cells, such as adipose tissue-derived stem cells, can be used. In one embodiment, the use of stem cell therapy results in the regeneration of cardiac tissue to reverse tissue loss that leads to the development of heart failure after cardiac injury.

[0175] In another embodiment, the compositions and methods of the present disclosure can be used for blood cell formation and amplification. Fully mature human red blood cells can be produced in vitro by hematopoietic stem cells (HSC) as red blood cell precursors. In this process, HSC can grow together with stromal cells to produce an environment that simulates the conditions of bone marrow (the natural growth site of red blood cells). In addition to using the compositions of the present disclosure, growth factor erythropoietin can also be added to induce stem cells to complete terminal differentiation into red blood cells. The compositions and methods can also be used to expand the population of red blood cells, white blood cells and / or platelets, to improve, for example, oxygen carrying capacity (for example, for athletes), the immune system (including for treating immunocompromised subjects), and for improving blood coagulation.

[0176] In another embodiment, embryonic stem cells treated with the compositions of the present disclosure can be used to regrow cochlear hair.

[0177] In another embodiment, stem cells processed according to the present disclosure can be used to treat blindness and visual impairment.In a specific embodiment, the compositions and methods are used to treat corneal lacerations.

[0178] In another embodiment, the present disclosure can be used to enhance the success of tissue transplants, including transplants of pancreatic beta cells that produce insulin. These cells can be prepared, for example, from embryonic stem cells that have been caused to differentiate into beta cells. These cells can be treated with the compositions of the present disclosure in vivo or ex vivo.

[0179] In another aspect, the present disclosure provides a method for treating a wound and / or promoting wound healing in a subject in need, the method comprising administering a composition as described herein to the subject. In certain embodiments, the present disclosure provides a method for treating a wound in a subject in need, the method comprising administering a composition as described herein to the subject. In certain embodiments, the present disclosure provides a method for treating a wound or burn in a subject in need, the method comprising administering a composition as described herein to the subject. In certain embodiments, the present disclosure provides a method for treating a burn in a subject in need, the method comprising administering a composition as described herein to the subject. In certain embodiments, the wound is a partial thickness wound or a full thickness wound. In certain embodiments, the burn is a partial thickness burn or a full thickness burn.

[0180] The present disclosure can also be used in the case of wound healing. In adults, injured tissue is most often replaced by scar tissue, which is characterized by disordered collagen structure, hair follicle loss and irregular vascular structure. In one embodiment, stem cell "seeds" are placed in a tissue bed (tissue bed) in a wound bed (wound bed), and the stem cells are allowed to stimulate differentiation in the tissue bed cells. This method can be significantly enhanced by contacting the wound (with or without adding stem cells) with a composition of the present disclosure. In certain embodiments, the composition is applied to the skin. In certain embodiments, the composition is applied to stem cells and / or progenitor cells.

[0181] In other embodiments, the compositions and methods described herein can be used for cosmetic applications, where, for example, the application requires rejuvenation of the skin layers and / or underlying tissue. This rejuvenation can be assisted by, for example, the survival, proliferation, and / or development of enhanced stem cells and / or progenitor cells. For example, this rejuvenation can be assisted by, for example, treating a disease or condition associated with mucosal barrier function, such as a wound, a skin condition (e.g., atopic dermatitis, psoriasis, bedsores, or a condition associated with skin aging), a pulmonary disorder (e.g., asthma), a mucosal barrier function, and / or GI mucosal damage.

[0182] In this embodiment, the methods of the present disclosure generally include the step of topically applying the composition to the skin (eg, epidermis) of a patient in need of such treatment, wherein a therapeutically effective amount of such composition is applied. In one embodiment, the composition is applied to the face.

[0183] Advantageously, the present invention provides compositions and methods for combating skin aging, wherein combating skin aging can include, for example, treating the appearance of wrinkles, fine lines, and other forms of undesirable skin texture. By presenting the composition to the dermis and / or epidermis of the skin, the form, strength, and function of the skin are enhanced. In certain embodiments, the compositions and methods described herein can be used in cosmetic applications, where, for example, rejuvenation of the skin layers and / or underlying tissue is desired.

[0184] In another aspect, the present disclosure provides a method for treating and / or preventing a skin disorder (e.g., atopic dermatitis, psoriasis, or a disorder associated with skin aging) in a subject in need thereof, the method comprising administering a composition as described herein to the subject. In certain embodiments, the skin disorder is atopic dermatitis, psoriasis, skin aging, a disorder associated with skin aging, or a bedsore. In some embodiments, the skin disorder is pruritus (scabies), psoriasis, eczema, burns, or dermatitis. In certain embodiments, the skin disorder is psoriasis. In certain embodiments, the skin disorder is pruritus.

[0185] In certain embodiments, in addition to amino acids, the compositions of the present disclosure include agents that can be used to delay, minimize, or eliminate skin aging, wrinkles, and / or other histological changes typically associated with intrinsic conditions (e.g., aging, menopause, acne, etc.) and extrinsic conditions (e.g., environmental pollution, wind, heat, sunlight, radiation, low humidity, harsh surfactants, etc.).

[0186] The present invention can be used to therapeutically and / or prophylactically improve visible and / or tactile characteristics in the skin. For example, in one embodiment, the length, depth and / or other dimensions of fine lines and / or wrinkles are reduced.

[0187] In another aspect, the disclosure provides a method for treating a lung disorder in a subject in need, the method comprising administering a composition as described herein to the subject. In certain embodiments, the composition is administered systemically or via inhalation. In another aspect, the disclosure provides a method for improving lung function, lung healing, reducing pneumonia, reducing airway resistance and / or improving lung function in a subject in need, the method comprising administering a composition as described herein to the subject. In certain embodiments, a pulmonary disorder is lung injury, pneumonia, a disorder associated with airway resistance, asthma or an inflammatory disorder of the lung. In certain embodiments, the composition is administered systemically or via inhalation.

[0188] In one embodiment, the composition applied to the skin or other tissue may further comprise collagen and / or hyaluronic acid (HA). In one embodiment, the HA is cross-linked HA. The composition may further comprise, for example but not limited to, the following components: a dermatologically acceptable carrier, an exfoliating agent, an anti-acne agent, an anti-wrinkle agent / anti-atrophy agent, vitamin B12, 3 Compounds, retinoids, hydroxy acids, antioxidants / free radical scavengers, chelating agents, flavonoids, anti-inflammatory agents, anti-cellulite agents, topical anesthetics, tanning agents, skin whitening agents, emollients and skin repair agents, antimicrobial and antifungal agents, sunscreens, conditioning agents, structuring agents, thickeners (including thickeners and gelling agents), composition preparations and preservatives. In this regard, International PCT Application Publication No. WO 2008 / 089408 is incorporated herein by reference in its entirety.

[0189] The compositions of the present disclosure may also be administered at surgical sites, including at minimally invasive surgical sites, to improve healing and surgical outcomes.

[0190] Stem cells can also be used to treat infertility according to the present disclosure. In certain embodiments, a person suffering from a condition for which survival, proliferation and / or development of stem cells would be beneficial is first diagnosed. For example, a subject can be diagnosed with the condition, and then the composition of the application is administered by a pathway and amount that results in survival, proliferation and / or development of stem cells. Preferably, the administration then results in treatment (e.g., improvement) of the condition.

[0191] Use of the composition to promote proliferation and / or development of intestinal stem cells and / or progenitor cells, and to treat diseases or disorders associated with mucosal barrier function, skin disorders, lung disorders, improve mucosal barrier function and GI mucosal damage

[0192] The purposes of amino acid compositions for treating GI, lung and skin disorders are described herein. In a specific embodiment, the compositions of the present invention can be used to induce intestinal epithelial cell proliferation, which results in an increase in villus height, wherein the villus includes mature differentiated epithelial cells that result in increased electrolyte and nutrient absorption. In one embodiment, compositions according to the present disclosure stimulate the proliferation and differentiation of stem cells and / or progenitor cells, as demonstrated by increasing the expression levels of NHE3 and SGLT1 in the brush border membrane. Therefore, the compositions increase villus height, and also increase the expression of key transporters for electrolyte and nutrient absorption.

[0193] Therefore, in one embodiment, the present disclosure provides a pharmaceutical composition for preventing or treating gastrointestinal damage associated with loss of small intestinal epithelial cells, particularly in the villus region and brush border, and / or treating or ameliorating diseases or conditions associated with changes in the absorptive capacity of the small intestine by promoting differentiation or proliferation of stem cells. These stem cells are located in the lower part of the intestinal crypts and include more quiescent "+4" cells above the rapidly cycling crypt basal columnar cells (CBCs) and Paneth cells in mammals.

[0194] The present disclosure also provides methods, the method is used to treat or improve diseases or conditions associated with loss of small intestinal epithelial cells, especially in the villus area and brush border, by promoting differentiation and proliferation of stem cells, and diseases or conditions associated with changes in transporter function in the small intestinal epithelium. The method includes applying an effective amount of the composition of the present disclosure to a subject in need of such treatment. In another aspect, the present disclosure provides methods, the method is used to treat diseases or conditions associated with mucosal barrier function such as wound healing of a subject in need, treat skin disorders (e.g., atopic dermatitis, psoriasis, bedsores or conditions associated with skin aging), treat lung disorders (e.g., asthma), conditions associated with improving mucosal barrier function, and / or treat GI mucosal damage.

[0195] Preparations and kits

[0196] The present disclosure provides treatment or pharmaceutical compositions, which include a therapeutically effective amount of the subject composition and optionally one or more pharmaceutically acceptable carriers. The present disclosure provides treatment, medicine, beauty or nutritional compositions, which include a therapeutically effective amount of the subject composition and optionally one or more pharmaceutically acceptable carriers. This pharmaceutical carrier can be a liquid, such as water. The therapeutic composition can also include excipients, adjuvants, flavoring agents, etc., which help to process the active compound into a preparation that can be used pharmaceutically. Suitable preparations depend on the selected route of administration. In an embodiment, the therapeutic composition and all ingredients contained therein are sterile. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EWMartin. Such compositions contain a therapeutically effective amount of the therapeutic composition, and a suitable amount of carrier, so as to provide a form suitable for administration to the patient. The preparation should be suitable for enteral administration mode.

[0197] In one embodiment, administration of the composition can be systemic. Oral, intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular, intraventricular, intranasal, transmucosal, subcutaneous, topical, rectal, and other modes of administration are contemplated.

[0198] In one embodiment, for injection, the active ingredient can be formulated in an aqueous solution, preferably in a physiologically compatible buffer. For transmucosal administration, a permeabilizing agent suitable for the barrier to be penetrated is used in the preparation. Such permeabilizing agents are generally known in the art. For oral administration, the active ingredient can be combined with a carrier suitable for inclusion in tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. The preparation can also be prepared for inhalation therapy. For administration by inhalation, the composition can use a suitable propellant, delivered in an aerosol spray presentation form from a pressurized package or atomizer. The composition can also be used as a powder by inhalation or other routes.

[0199] The therapeutically effective dose of the composition described at present can be determined by those skilled in the art, and the goal is to obtain the desired number of stem cells and / or precursor cells. The increase of stem cell and precursor cell number can be assessed using the markers of these cells or by determining the increase of the number of differentiated offspring of these cells. The method for measuring the increase number of differentiated cells is known in the art. For example, immunohistochemistry, behavioral assessment or electrophysiological techniques can also be used. Those skilled in the art can easily detect the increase of the number of cells of a specific phenotype.

[0200] In a specific embodiment, the method according to the present disclosure includes administering therapeutic compositions by a sustained release system. Suitable examples of sustained release systems include suitable polymeric materials (e.g., semipermeable polymer matrix in the form of shaped articles, such as films or microcapsules), suitable hydrophobic materials (e.g., emulsions in acceptable oils) or ion exchange resins and sparingly soluble derivatives (e.g., sparingly soluble salts). Sustained release compositions can be administered orally, parenterally, intracisternal, intraperitoneally, externally (e.g., by powders, ointments, gels, drops, or transdermal patches), or administered as oral or nasal sprays. Sustained release matrices include polylactide (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556, 1983, poly(2-hydroxyethyl methacrylate)); (Langer et al., J. Biomed. Mater. Res. 15:167-277, 1981; Langer, Chem. Tech. 12:98-105, 1982, ethylene vinyl acetate (Langer et al., Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988).

[0201] In one embodiment, an implantable drug infusion device can be used to provide a patient with a constant long-term dose or infusion of a therapeutic composition. Such devices can be classified as active or passive.

[0202] In one embodiment, polymers can be used for controlled release of ions. Various degradable and non-degradable polymer matrices for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537, 1993). For example, block copolymers, poloxamer 407, hydroxyapatite, and liposomes.

[0203] The pharmaceutical composition of the present invention can be used alone or in combination with one or more known effective drugs for treating diseases. The composition can also be formulated in combination with at least one other agent, such as a stabilizing or buffering compound, which can be administered in any sterile biocompatible pharmaceutical carrier (including but not limited to saline, buffered saline, dextrose and water). In addition to the key component cells or influencing factors in the composition discussed herein, the composition may also contain a suitable pharmaceutically acceptable carrier, including excipients and adjuvants, which help to process the active compound into a pharmaceutically usable preparation. The composition can be prepared into a single dosage form using a pharmaceutically acceptable carrier or excipient, or can be contained in a multi-dose container.

[0204] In one embodiment, the composition may further contain other proliferation and / or differentiation inducers. The proliferation or differentiation inducer may be any agent known as a proliferation or differentiation inducer. Examples include fibroblast growth factor (FGF), epidermal growth factor (EGF) and retinoic acid.

[0205] The composition may further contain other commonly used additives, such as antioxidants, buffers, antibacterial agents, etc., and may be formulated into injectable preparations (such as aqueous solutions, suspensions, emulsions, etc.), pills, capsules, granules, tablets, etc. by further adding diluents, dispersants, surfactants, binders, lubricants, etc.

[0206] The food composition of the present invention can be contained in health functional food.

[0207] The health functional food of the present invention can be prepared according to the commonly used methods in the art, and commonly used raw materials and ingredients can be added when preparing the health functional food.

[0208] When the composition of the present invention is included in a health functional food, the composition can be added alone or together with another health functional food or other food ingredients according to a common method. The amount of active ingredients can be appropriately determined according to the purpose of use (e.g., prevention, health improvement or therapeutic intervention). The food composition can further include, for example, prebiotic or probiotic substances.

[0209] The type of food is not limited. Examples of foods to which the composition can be added include meat, sausages, bread, chocolate, candy, snacks, cakes, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, and the like.

[0210] The present disclosure also encompasses kits (e.g., packs, treatment packs, cosmetic packs, or nutritional packs). The kits provided may include a pharmaceutical composition or compound described herein and a container (e.g., a vial, an ampoule, a bottle, a syringe, and / or a dispenser package, or other suitable container). In some embodiments, the kit provided may optionally further include a second container containing a pharmaceutical excipient for diluting or suspending the pharmaceutical composition or compound described herein. In some embodiments, the pharmaceutical composition or compound described herein provided in combination in the first container and the second container is combined to form a unit dosage form.

[0211] Thus, in one aspect, a kit is provided comprising a first container comprising a composition as described herein. In certain embodiments, the kit can be used to treat a disorder (e.g., GI, lung, and skin disorders) in a subject in need thereof. In certain embodiments, the kit can be used to prevent a disorder (e.g., GI, lung, and skin disorders) in a subject in need thereof.

[0212] In certain embodiments, the kit described herein also includes instructions for using the compositions included in the kit. The kit described herein may also include information required by regulatory agencies (e.g., the U.S. Food and Drug Administration (FDA)). In certain embodiments, the information included in the kit is prescription information. In certain embodiments, the kit and instructions provide for treating and / or preventing obstacles (e.g., GI, lung and skin disorders) in subjects in need. The kit described herein may include one or more additional pharmaceutical agents described herein or other agents as separate compositions.

[0213] Dosage

[0214] In one embodiment, the present disclosure relates to administering a composition according to the present disclosure to a subject, and further administering stem cells and / or progenitor cells to the subject. The composition is administered at a site of the subject to allow contact with the cells. This site can be in the same location as the stem cell administration site, near the stem cell administration site, or away from the stem cell administration site.

[0215] Stem cells and / or progenitor cells can be administered, for example, by injecting one or more cells with a syringe, inserting stem cells with a catheter, or surgically implanting stem cells. In certain embodiments, stem cells are administered into a body cavity connected to the target tissue fluid. In certain preferred embodiments, the body cavity is a cerebral ventricle. In other embodiments, cells are inserted using a syringe or catheter, or are surgically implanted directly at the target tissue site. In other embodiments, stem cells and / or progenitor cells are administered parenterally. Parenteral administration is defined as administration by a route that bypasses the gastrointestinal tract. Parenteral administration includes intraventricular administration.

[0216] Typically, the composition can be administered by any of a variety of routes, including but not limited to oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, external, sublingual or rectal. Various factors can be administered in the same location as the administration of stem cells. The administration of the influencing factors and stem cells can be performed simultaneously, or sequentially, and in the same or different locations, as long as the relative position and timing allow the factors to affect stem cells and / or progenitor cells.

[0217] For example, by using "consisting essentially of...", the therapeutic composition does not contain any unspecified ingredients, including but not limited to free amino acids, dipeptides, oligopeptides or polypeptides or proteins that have a direct beneficial or adverse therapeutic effect on promoting stem cell development; as well as monosaccharides, disaccharides, oligosaccharides, polysaccharides and carbohydrates. In addition, by using the term "consisting essentially of...", the composition may contain substances that have no therapeutic effect on promoting stem cell development; these ingredients include carriers, excipients, adjuvants, flavoring agents, etc. that do not affect the promotion and / or development of stem cells.

[0218] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that those skilled in the art may propose various modifications or changes based on the examples and embodiments, and that the modifications or changes should be included in the spirit and scope of the present application.

[0219] Example

[0220] Materials and methods

[0221] Animal Model: Eight-week-old male NIH Swiss mice were fed a normal diet and housed 4 mice per cage. Mice were irradiated using a Gammacell 40 Exactor low-dose research irradiator (Best Theratronics, Ottawa, Ontario) housing two cesium-137 sources in a parallel and opposing geometry, providing isotropic irradiation with a dose uniformity within ±3%. Mice received a single fraction of TBI at a dose rate of 0.9 Gy / min. Mice were secured with a plastic clamp in the middle of the irradiation chamber, which allowed 5 mice to be irradiated simultaneously. Mice treated with the formulation were given AA-ORS once daily by gastric gavage (0.3 ml per mouse). A control group was given normal saline. This amino acid formulation was given as supportive therapy and was not part of replacement therapy. Mice were fasted for 8 hours prior to gavage. At peak anion secretion, which occurred 6 days after irradiation, CO was administered. 2 Animals were humanely euthanized by inhalation followed by cervical dislocation (according to AVMA animal euthanasia guidelines).Toxicity was primarily due to acute GI syndrome and was only minimally disturbed by bone marrow syndrome. 18 After exsanguination, ileal mucosa was obtained as described previously. 18,20 All experiments were approved by the University of Florida Institutional Animal Care and Use Committee (IACUC) and performed according to IACUC protocol #3875.

[0222] Crypt counts and villus length measurements: Paraffin sections (5 μm) were obtained from intestinal segments oriented so that the sections were cut perpendicular to the long axis of the intestine. Crypts were counted per circumference and villus length was calculated from 10 sections obtained from the ileum. To determine cell survival curve parameters, crypt counts were normalized and analyzed using classical methods. 21 AA-ORS treatment was continued for 6 days. Normal saline was used as a control.

[0223] Cell proliferation and crypt to villus migration assay: 5-ethynyl-2'-deoxyuridine (EdU, thymidine analog) is incorporated into cellular DNA, and the subsequent reaction of EdU with fluorescent azide in a copper-catalyzed reaction is used to study cell proliferation in the crypt cell region. Mice were injected with 0.5mg EdU (16.7mg / Kg) in 150ml PBS to assess mitotic activity in crypt cells (these studies reveal the S phase in the crypts) and euthanized at 24, 48 and 72 hours after injection. Paraffin sections from mouse ileum were prepared and the incorporated EdU (Thermo Fisher Scientific catalog number A10044) was visualized according to the manufacturer's instructions (Alexa 647 imaging kit, catalog number C10340). The sections were then mounted in an anti-fluorescence quenching mounting medium (fluorescent mounting media) with DAPI (VectaShield, catalog number H-1200). Cells were scored for the entire crypt and villus unit. At least 60 crypts and corresponding villi were analyzed per mouse. EdU-labeled cells were normalized to the total number of cells per crypt or villus. Positively stained enterocytes were shown to migrate from the base of the crypt to the tip of the villus.

[0224] Flux studies of sodium and chloride absorption: The stripped ileal sheets were mounted on a 0.3 cm 2 The exposed surface area was placed between the two halves of an Ussing chamber (P2304, Physiologic Instruments, San Diego, CA, USA). -1 )Na + 140、Cl - 119.8, K + 5.2 HPO 4 - 2.4 H 2 PO 4 - 0.4, Mg 2+ 1.2 Ca 2+ 1.2 and HCO 3 -25% Ringer's solution with 95% O2 and 5% CO 2 The cells were bubbled bilaterally and maintained at 37° C. After allowing the tissue to stabilize for 45 min, the voltage / current clamp apparatus (VCC MC-8, Physiologic Instruments) was used to record the activity expressed as μeq·h as previously described. -1 cm -2 The basic short-circuit current (I sc ) and expressed as mS.cm -2 The electrical conductivity (G). 18,20 For flux studies, sodium ( 22 Na) and chlorine ( 36 C1) to study sodium and chloride fluxes across the ileal mucosa. 18,20, 22 Na activity was measured using a gamma counter (Wizard 2,2480 automatic gamma particle counter, Perkin Elmer, USA), while 36 C1 was measured using a liquid scintillation counter (LS 6500 Multipurpose Scintillation Counter, Beckman Coulter, Inc., Brea, CA, USA).

[0225] Real-time quantitative polymerase chain reaction (PCR): RNA was extracted from intestinal tissue samples of non-irradiated and irradiated mice (0 Gy, 5 Gy, and 7 Gy) using the TRIZOL method. TM Select cDNA synthesis kit, Bio-Rad, Hercules, CA) to prepare c-DNA; semi-quantitative and real-time PCR were performed using specific oligonucleotide primers for caspase 3, Lgr5, sglt1 and BMI1. c-DNA (2 μL) was added to 25 μL of PCR mixture for semi-quantitative PCR, and 20 μL of SyBr green mixture was added for quantitative PCR; 30 PCR cycles (or more, as shown in the results) were performed using a Veriti thermal cycler (Thermo Fisher Scientific, Waltham, MA) for semi-quantitative PCR and a CFXConnect real-time system cycler (Bio-Rad) for real-time PCR. One cycle consisted of denaturation at 94°C for 30 sec, annealing for 60 sec and extension at 72°C for 90 sec. Amplicons were resolved by agarose gel electrophoresis and detected by ethidium bromide staining. The expression of proteins was detected using CFX Manager TMReal-time analysis was performed using the ELISA software (Bio-Rad). Normalization used the Δ-ΔCt (DDCt) method. In short, DCt = Ct (target gene treated) - Ct (reference gene treated) and DCt = Ct (target gene control) - Ct (reference gene control). Therefore, DDCt = DCt (treated) - Ct (control). From Equation 2 (-DDQ) Fold changes were calculated.

[0226] Western Blot: Total cell lysates from non-irradiated and irradiated AA-ORS-treated or saline-treated mice were prepared in ice-cold RIPA buffer [50 mmol / L Tris-HCl (pH 7.4), 150 mmol / L NaCl, 1% IGEP AL, 1 mmol / L EDTA, 0.25% sodium deoxycholate, 1 mmol / L sodium fluoride, 1 mmol / L sodium orthovanadate, 0.5 mmol / L PMSF, 10 μg / mL aprotinin, 10 μg / mL leupeptin]. The protein concentration in each extract was determined by BCA assay (Sigma, St. Louis, MO). Cell extracts were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); proteins were transferred to polyvinylidene difluoride (PVDF) membranes and probed with primary antibodies detecting Lgr5, SGL T1, Bmi 1, caspase 3, p-ERK, and total ERK. Signals were detected using the Odyssey CLX from LI-COR. Equal gel loadings were checked using a reversible Ponceau S stain (Cat. No. 786-575, G Biosciences) according to the manufacturer's instructions. The abundance of the protein of interest was normalized to the total amount of protein in each lane. This technique minimizes the changes associated with comparing protein density to a single protein.

[0227] Immunohistochemical identification of nuclear proteins PCNA and Ki-67 and stem cell-specific membrane protein Lgr5 was performed using polyclonal rabbit anti-mouse GPCR (Lgr5) (Abcam catalog number ab75732), Ki-67 (Abcam catalog number ab15580) and PCNA (Abeam catalog number ab 18197) antibodies. The expression of proteins was visualized using a rabbit-specific ABC detection kit (Abcam catalog number ab64261) according to the manufacturer's instructions. In brief, formalin-fixed, paraffin-embedded full-thickness ileum samples were cut into 4 μm thick cross-sections, mounted on Superfrost Plus slides, deparaffinized and rehydrated. For antigen retrieval, a pressure cooker (125°C for 30sec and 90°C for 10sec) and a repair buffer, Deloaker RTU buffer (Biocare Medical catalog number RV1000MMRTU) at pH 6.0, were used for heat pretreatment. After quenching endogenous peroxidase and blocking nonspecific binding, sections were incubated with primary antibodies diluted in PBS (Lgr5–1:100, Ki-67-1:1000, PCNA-1:4000, for 2 h; 15 min; and 2 h, respectively, at room temperature). PBS was used as a negative control. Tissues were then incubated with biotinylated goat anti-rabbit secondary antibodies for 10 min. After incubation with streptavidin peroxidase, diaminobenzidine was used to obtain the desired staining intensity by visualization under a microscope. Sections were counterstained with Mayer's hematoxylin (Electron Microscopic Sciences (EMS) catalog number 26043-05), dehydrated, and mounted in Permount mounting medium (Fisher Scientific catalog number SP15-100). Slides for PCNA and Ki-67 were evaluated by light microscopy using a 20x objective, and for Lgr5 by oil immersion using a 40x objective. The number of positive brown cells was counted from 50 crypts in each group and analyzed.

[0228] Statistics: Results are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed in 2 steps: 1) global differences were tested using analysis of variance (ANOVA) (or its nonparametric equivalent Kruskal-Wallis); 2) Bonferroni-adjusted P values ​​were calculated for all pairwise comparisons.

[0229] Example 1 - AA-ORS increases crypt counts and villus length in intestinal tissue from irradiated mice

[0230] Intestinal tissues of irradiated mice (5-15 Gy) treated with AA-ORS showed a significant increase in crypt counts (N) per circumference. Since crypts are formed by regenerative units, these results suggest an increase in the number of epithelial progenitor cells. Similarly, villus length was significantly increased at all radiation doses with AA-ORS treatment compared to saline treatment ( Figure 1B ). This is consistent with increased progenitor cell proliferation and / or longer epithelial cell survival prior to spontaneous shedding.

[0231] Crypt survival curves were modeled using a single-hit multi-target cell survival model to assess biological effects. Without limiting constant cell sensitivity, N values ​​were 10.4 ± 0.2 and 5.3 ± 0.1 (P < 0.001), indicating that the number of progenitor units per circumference was almost doubled compared to the control. When limiting a constant Do (4.8 ± 0.1 Gy), the difference remained significant at 8.8 ± 0.4 to 6.1 ± 0.3 (P < 0.001). The quasi-threshold dose (Dq) value, a comprehensive measure of crypt radiation tolerance, was 10.5 ± 0.5 Gy for mice treated with AA-ORS and 8.8 ± 0.4 Gy for mice treated with saline (P < 0.01).

[0232] Compared with the saline group, the AA-ORS group had a "broad shoulder" in the low radiation dose area, indicating an increase in the number of progenitor cells (less senescence) per intestinal circumference ( Figure 1A ).

[0233] As expected, the terminal part followed an exponential relationship. Because 5 Gy was the lowest radiation dose at which both crypt counts and villus height increased significantly, all subsequent studies were performed in 0- and 5-Gy irradiated mice.

[0234] The increased weight gain and survival were found to be likely secondary to an increase in crypt number and villus height, which in turn increases the surface area for absorption. Crypt number and villus height have been shown to increase with AA-ORS treatment starting 6 days after irradiation. Using a single-hit, multi-target model for crypt survival, the number of crypt progenitor units (N) per ileal circumference was found to be significantly increased (P<0.001) with no change in Do (4.8±0.1 Gy) ( Figure 1A ). In the case of AA-ORS treatment, the Dq values ​​improved to an equivalent of 1.7 Gy with increased radiation tolerance, suggesting improved crypt survival. Crypt survival studies have shown an increase in progenitor units or stem cells / crypt. Therefore, the effects of irradiation and AA-ORS on stem cell numbers were examined using antibodies specific for intestinal stem cell markers and the migration of daughter cells into the villi for proliferation secondary to EdU incorporation. 14-16

[0235] Example 2-AA-ORS promotes intestinal epithelial cell migration

[0236] Ileal sections from irradiated mice treated with AA-ORS showed an increase in crypt counts. As the villus epithelium continues to turn over, cells also continuously migrate along the crypt-villus axis. 23 As the cells move along the villus, the epithelial cells undergo further maturation and differentiation before migrating to the tip of the villus where they are shed by a process called anoikis. 24 EdU incorporation was used to study cell proliferation.

[0237] Cells were found to migrate from the base of the crypt to the tip of the villus in approximately 72 hours. Ileal sections from non-irradiated mice showed that cells (incorporating EdU) reached the tip of the villus at different times (75±0.9 hours; n=10 mice), with an average of 76.5 hours. AA-ORS treatment for 6 days did not result in a significant difference in the length covered by migrating cells between 0 and 5 Gy irradiated mice (153.5±6 mm vs. 158.1±7 mm; n=10 mice). However, AA-ORS treatment for 6 days resulted in a significant difference in 5 Gy irradiated mice (158.1±7 mm vs. 183.1±4 mm; p<0.01, n=10 mice) ( Figure 2A and Figure 2B ). These studies showed that AA-ORS increased proliferation and was associated with the length covered.

[0238] Example 3 - AA-ORS Increases Sodium and Chloride Absorption

[0239] AA-ORS increases villous height and proliferation in the crypt cell region.To determine whether increased villous height results in functionally mature and differentiated villous epithelial cells, isotope flux studies were performed to measure sodium and chloride absorption.

[0240] Non-irradiated mice had a 1.9 ± 0.6 ueq.cm 2 .h- 1 Net sodium absorption (J Net Na) and 1.9±0.4ueq.cm 2 .h- 1 Net chlorine absorption (J Net C1)( FIG. 3A to FIG. 3B ). J of ileum of 5Gy irradiated mice Net Na(1.9±0.6μeq.cm 2 .h- 1 Relative to 0.1±0.0μeq.cm 2 .h- 1 ; P<0.001, n=8) and J NetC1(1.9+0.4μeq.cm 2 .h- 1 Relative to -0.9±0.4μeq.cm 2 .h- 1 AA-ORS treatment resulted in a decrease in 0Gy (3.9±0.7ueq.cm 2 .h- 1 ; p<0.05, n=8) and 5Gy (3.4±0.7ueq.cm 2 .h- 1 ; p<0.001, n=8) mice had significantly increased net sodium absorption in the ileum ( Figure 3A Similarly, AA-ORS treatment resulted in 0 Gy (4.1 ± 0.6 ueq.cm 2 .h- 1 ; p<0.05, n=8) and 5Gy (3.6±0.6ueq.cm 2 .h- 1 ; p<0.001, n=8) mice with increased chloride absorption in the ileum ( Figure 3B ).

[0241] These studies suggest that the increase in villous height induced by AA-ORS is functional by showing an increase in electrolyte absorption capacity and sodium-coupled glucose absorption, which are functions of mature and differentiated villous epithelial cells.

[0242] Immunohistochemistry of NHE3 protein showed NHE3 antibody recognition along the brush border region of villous epithelial cells ( Figure 3C Chorionic cells from 5 Gy irradiated animals showed little or no expression along the brush border membrane. Treatment with AA-ORS increased NHE protein expression along the marginal zone in epithelial cells of 0 Gy and 5 Gy irradiated mice ( Figure 3C Western blot analysis showed increased NHE3 protein expression in the intestinal tissues of AA-ORS-treated mice irradiated at 0 Gy (3.5-fold) and 5 Gy (15.5-fold) compared with saline-treated irradiated mice ( FIG. 3D to FIG. 3E ). These studies showed an increase in NHE3 protein in the brush border membrane of villous epithelial cells. To determine whether the increase in NHE3 protein was caused by an increase in NHE3 mRNA, its levels in intestinal tissue were measured using qPCR ( Figure 3F ). Unlike NHE3 protein levels, NHE3 mRNA levels were significantly different only at 5 Gy when compared to saline-treated 5 Gy irradiated mice. Protein levels did not correlate with changes in NHE3 mRNA levels, and similar observations have been reported previously.

[0243] Example 4 - AA-ORS increases glucose-stimulated sodium absorption

[0244] Glucose stimulates sodium absorption via specific transporters in the apical membrane of mature and differentiated epithelial cells located in the villi. To determine whether the increase in villus height induced by AA-ORS results in improved glucose absorption, we used 22 Na flux studies assess glucose-stimulated sodium absorption.

[0245] Ileal tissue from 5 Gy irradiated mice showed a significant decrease in glucose-stimulated sodium absorption (4.8 ± 0.5 ueq.cm 2 .h- 1 Relative to 0.3±0.1ueq.cm 2 .h- 1 ; p<0.001, n=6). Ileal tissue from 5 Gy irradiated mice treated with AA-ORS showed a significant increase in glucose-stimulated sodium absorption (0.3±0.1 ueq.cm 2 .h- 1 Relative to 3.1±0.3ueq.cm 2 .h- 1 ; p<0.001, n=6), while 0 Gy irradiated mice did not show such an increase (4.8±0.5ueq.cm 2 .h-1 relative to 5.9±0.7ueq.cm 2 .h- 1 ; p = ns, n = 6)( Figure 4A AA-ORS treatment enhanced SGLT1 expression at both transcriptional and translational levels in 0- and 5-Gy irradiated mice compared with saline-treated mice ( FIG. 4B to FIG. 4D These studies have shown that the interdigestive period (NHE3-mediated Na + absorption) and digestive phase (glucose stimulated Na + Absorption) in the villous epithelium, both of which are functions of mature and differentiated villous epithelial cells, indicating that the increase in villous height induced by AA-ORS is functional.

[0246] β-galactosidase (lactoprotein) levels in isolated villous cells were measured using Western blot analysis. Mainly, β-galactosidase expression occurs in mature and differentiated villous epithelial cells. AA-ORS treatment increased β-galactosidase protein levels in villous cells of 0 Gy and 5 Gy irradiated mice ( Figure 4B ).

[0247] Example 5 - Effects of AA-ORS on Intestinal Stem Cells and Proliferation Markers

[0248] At least three distinct crypt cell types are postulated to represent intestinal stem cells (ISCs). 14 Each member of the population has different proliferation kinetics and sensitivity to radiation; therefore, each member is thought to have a unique function. 28 It is believed to dynamically switch from one type to the other in response to inhibitory and stimulatory signals caused by cytokines, hormones or growth factors. 29 In contrast, slowly cycling intestinal epithelial stem cells (IESCs) at the “+4 crypt position” [labeled resident cells (LRCs)] contribute to homeostatic regenerative capacity, particularly during recovery from injury. 30 These LRCs express various markers, such as BMI1, HOPX, LRIG1 and / or DCLK1, and can change into rapidly cycling IESCs in response to injury. 31 Lgr5 can mark both types of cells, whereas Bmi1 and HopX are reported to preferentially mark +4 cells. 14 Lgr5 + ISCs are required for intestinal regeneration after radiation injury. 32 Lgr5 - and BMI1 are considered reserve cells that initiate a regenerative response following injury or radiation-induced damage. Studies have shown that Lgr5 + Loss of cells was tolerated. 14,32

[0249] To determine whether the increase in stem cell number and proliferation was associated with the increased villus height observed with AA-ORS, the effects of AA-ORS on stem cell markers and proliferation were investigated.

[0250] Irradiation resulted in a significant decrease in Lgr5 protein levels ( Figure 5A ). AA-ORS increased Lgr5 protein levels in 0Gy and 5Gy irradiated mice compared to saline-treated controls. However, intestinal tissue from 5Gy irradiated mice showed no significant changes in BMI1 protein levels when compared to 0Gy. Similarly, AA-ORS did not induce changes in BMI1 protein levels in 0Gy and 5Gy irradiated mice ( Figure 5A ). In 0Gy and 5Gy mice treated with AA-ORS, Lgr5 transcript levels, but not BMI1 levels, were significantly increased ( FIG. 5C to FIG. 5D ).

[0251] It was found that 5Gy resulted in a significant decrease in Lgr5 transcript and protein levels when compared to 0Gy, while Bmi1 levels did not change much. It was found that Lgr5 mRNA and protein levels were significantly enhanced in the case of AA-ORS treatment, while Bmi1 did not change much, indicating an increase in Lgr5-positive stem cells. These results also showed that the length covered by migrating cells in intestinal sections of AA-ORS-treated irradiated mice was significantly greater when compared to saline-treated mice. Since transcription stops in cells undergoing apoptosis, increased cell survival preferentially increases short-lived transcripts such as Lgr5, rather than long-lived transcripts. BMI1 protein levels in intestinal tissue did not change in response to radiation or AA-ORS, indicating that the reserve population of ISCs was not affected at the radiation doses studied. In addition, the increase in Lgr5 protein levels in the case of AA-ORS supports these observations in the crypt count study, that is, when suitable as a single-hit multi-target model, AA-ORS treatment after increasing radiation doses leads to an increase in the number of ISCs.

[0252] Western blot analysis of ERK1 / 2 and pERK1 / 2 was studied using whole cell fractions to evaluate the effects of radiation and AA-ORS on proliferation. To determine transcript levels of ERK and AKT, qPCR studies were performed in epithelial cells isolated from 0Gy and 5Gy irradiated tissues in the absence and presence of treatment. ERK1 / 2 and AKT are phosphorylated upon activation. Western blot analysis showed significant differences in p-ERK protein levels at 0Gy and 5Gy ( Figure 5A ). There were no significant differences in total ERK protein levels between AA-ORS-treated and saline-treated mice at either 0 Gy or 5 Gy. Similarly, in the case of ERK, 0 and 5 Gy mice treated with AA-ORS did not show significant differences in AKT levels, whereas they did show an increase in p-AKT protein levels when compared to the corresponding saline-treated irradiated groups. Intestinal tissue from 5 Gy mice showed a significant decrease in p-AKT compared to 0 Gy mice.

[0253] These studies showed that phosphorylation levels of the protein increased in the case of AA-ORS treatment without changes in total protein expression. Since the action of MAPK depends on its downstream effector, activating transcription factor 4 (Atf4), Atf4 protein levels were measured using western blot analysis. 5Gy irradiation reduced the protein level of Atf4, but AA-ORS treatment increased Atf4 protein levels in both 0Gy and 5Gy irradiated mice. Study of ERK and AKT transcript levels using qPCR showed that 5Gy irradiation resulted in a significant decrease in mRNA levels compared to 0Gy. AA-ORS treatment resulted in a significant increase ( FIG. 5E to FIG. 5F). Early changes in normal cell proliferation in the intestine serve as an indicator of deviation from normal gastrointestinal function. Changes in the expression of proliferating cell nuclear antigen (PCNA), a 36 kD protein, are considered a marker of intestinal changes. AA-ORS increased PCNA protein levels in 0 Gy and 5 Gy irradiated mice ( Figure 5A ).

[0254] The effect of AA-ORS on B-cell lymphoma-2 protein (Bcl-2), a downstream target of Erk1 / 2, was also investigated. Bcl-2 prevents cell death rather than promoting cell proliferation by regulating the expression levels of the pro-apoptotic Bcl-2-associated X protein (Bax) in the intrinsic caspase cascade. In 0 Gy mice, Bcl-2 levels increased in the presence of AA-ORS treatment. Irradiation resulted in a significant increase in Bcl-2 protein levels. Treatment with AA-ORS did not show a further increase in Bcl2 protein levels in 5 Gy irradiated mice ( Figure 5B ). The increase in Bcl-2 protein levels following irradiation may indicate a protective mechanism against apoptosis. However, Western blot analysis using a Bax-specific antibody did not reveal significant differences in protein levels ( Figure 5B ). These studies are consistent with previous observations that interventions targeting Bcl-2 do not necessarily alter the protein levels of Bax4.

[0255] Since AA-ORS increased p-ERK, this study suggests that amino acids help maintain mitogenic stimulation until late G1 to facilitate successful entry into S phase. 38

[0256] This study showed that radiation increases caspase-3 and AA-ORS treatment reduces cleaved caspase-3 in villous epithelial cells of 0Gy and 5Gy mice. The increase in pAKT in mice treated with AA-ORS suggests that its effect may be through activation of proliferation or inhibition of apoptosis (Figure 5). Combined with the effects seen on caspase-3, these results may explain the pro-survival effects and increased proliferation observed with AA-ORS treatment. However, further studies will be critical to characterize the mechanism by which AA-ORS activates ERK1 / 2 and AKT or caspase-3.

[0257] Example 6 - AA-ORS reduces cleaved caspase-3 and caspase-3 transcript levels

[0258] Activation of caspase-3 results in the formation of 19kD cleaved caspase-3. Cleaved caspase-3 increases with apoptosis. Western blot analysis showed no significant differences in total caspase-3 after irradiation and treatment compared to controls. Intestinal tissue from 5Gy mice showed a significant increase in cleaved caspase-3 compared to tissue from 0Gy mice. However, cleaved caspase-3 was decreased in the case of AA-ORS when compared to the corresponding irradiated controls ( Figure 5B ). Caspase 3 mRNA levels measured using qPCR showed a significant increase in caspase 3 transcript levels after 5 Gy irradiation when compared to 0 Gy. Treatment with AA-ORS resulted in a significant decrease in caspase 3 transcript levels in intestinal tissues from 0 Gy and 5 Gy irradiated mice ( Figure 5G ).

[0259] Combined with the changes in Lgr5, p-ERK and p-AKT, the changes in cleaved caspase-3 indicated that AA-ORS increased villus height in the intestinal tissues of non-irradiated and irradiated mice not only through proliferation but also through reduced apoptosis and increased cell survival.

[0260] To assess whether the villous epithelial cells were mature, differentiated, and functionally active due to increased proliferation and / or decreased apoptosis, sodium absorption capacity and glucose-stimulated sodium absorption were measured. NHE3 (the major transporter for sodium absorption in the small intestine) and SGLT1 (the transporter for sodium-coupled sodium absorption) were only present in mature and differentiated villous cells and were shown to have increased function ( FIG. 3A to FIG. 3F and FIG. 4A to FIG. 4D ), mRNA, and protein levels. Thus, these studies suggest that treatment with AA-ORS after irradiation increases electrolyte and glucose absorption ( FIG. 5A to FIG. 5G ).

[0261] Example 7 - Western blot analysis of total p53

[0262] p53 is a tumor suppressor protein, and its activity prevents the formation of tumors. Mutations in the p53 tumor suppressor gene are the most common genetic lesions in human cancer. Mice homozygous for the null allele appear normal but are prone to spontaneously develop various tumors. p53 has been shown to play an important role in the response to irradiation. The level of p53 accumulation in response to irradiation is mainly derived from the intensity of DNA damage. It has been demonstrated that the loss of stem cells plays an important role in radiation-induced acute intestinal injury and lethality and is regulated by the p53 pathway and its transcriptional targets PUMA and p21. PUMA-dependent apoptosis rapidly reduces ISCs and their progenitors within hours after high-dose radiation, and the lack of PUMA leads to improved animal survival and crypt regeneration by enhancing p21-dependent DNA repair, and is critical for radiation-induced intestinal damage.

[0263] Western blot analysis showed no significant difference in p53 (tumor suppressor protein) levels in intestinal tissues of mice treated with 0Gy and 5Gy. AA-ORS showed a small but consistent increase in p53 protein levels ( Figure 5B ).

[0264] These studies suggest that the proliferative effects associated with AA-ORS may not be related to tumorigenesis.

[0265] Example 8. Immunohistochemistry

[0266] Using a polyclonal antibody against Lgr5+, avidin-biotin detection of intestinal stem cells showed a decrease in Lgr5+ cells in the presence of radiation (3.0±0.2 vs. 1.6±0.2; P<0.001, n=50 crypts). Treatment with AA-ORS did not show a significant difference in Lgr5+ in 0 Gy irradiated mice. However, in 5 Gy irradiated mice, AA-ORS increased Lgr5+ cells (1.6±0.2 vs. 3.4±0.3; P<0.001, n=50 crypts) ( FIG. 6A to FIG. 6B ).

[0267] Ki-67 is a nuclear protein that is a cellular marker for proliferation and is associated with ribosomal RNA transcription. 1 , S, G 2 Ki-67 is also present in cells during mitosis and division, but not in resting cells (G 0 ). Ileal sections showed Ki-67 expression along the crypts (except at their lower poles), and expression extended to the lower 1 / 3 of the villi. There was no significant difference in Ki-67 expression in the presence of radiation, but treatment with AA-ORS resulted in a significant increase in Ki-67-expressing cells (50.7 ± 1.3 vs. 54.6 ± 1.4; P < 0.05, n = 50 crypts ( FIG. 6C to FIG. 6D ).

[0268] Immunostaining of PCNA in ileal sections showed its expression along the crypts (except at their lower pole). Irradiation resulted in a significant decrease in PCNA expression in the crypts and lower villus regions (31.1 ± 0.8 vs. 26.6 ± 0.6; P < 0.001, n = 50 crypts). Treatment with AA-ORS showed a significant increase in 5 Gy mice (26.6 ± 0.6 vs. 34.2 ± 0.5; P < 0.001, n = 50 crypts), but not in 0 Gy irradiated mice ( FIG. 6E to FIG. 6F ).

[0269] Western blot analysis showed that cell homogenates from the small intestine of mice irradiated at 5 Gy showed a significant increase compared with 0 Gy. Mice treated with AA-ORS increased Ki67 protein levels at 0 Gy, but no further increase at 5 Gy. These studies suggest that epithelial cell proliferation induced by AA-ORS at 0 Gy, but not at 5 Gy, is mediated via Ki67.

[0270] This study demonstrates that systematic selection of certain nutrients based on their beneficial effects on GI function helps improve intestinal stem cell proliferation, maturation, and differentiation, leading to functionally active villous epithelial cells, whose function and height are initially impaired by irradiation ( Figure 7 ).

[0271] Example 9-AA-ORS improves gastrointestinal function in mice

[0272] Using electrophysiological techniques, we showed that radiation-induced Cl − secretion can occur at radiation doses that are too low to cause overt histopathological changes. 55 Radiation-induced intestinal dysfunction is characterized by: (1) increased Cl- secretion, which is associated with increased fluid secretion; (2) decreased Na+ absorption, which leads to reduced fluid absorption; and (3) increased paracellular permeability, which leads to increased translocation of luminal antigenic material to systemic compartments, thereby generating local and systemic immune responses. Increased penetration of luminal contents into systemic compartments increases plasma endotoxins and proinflammatory cytokines (e.g., IL1β). 51

[0273] As outlined in previous studies, amino acid-based oral rehydration solutions (AA-ORS) were previously developed. 51 Specific amino acids were selected based on the following findings in intestinal tissue of irradiated mice: the selected amino acids: (1) increase Na+ absorption via amino acid-coupled Na+ absorption; (2) do not stimulate Cl- secretion and therefore do not stimulate fluid secretion; and (3) reduce paracellular permeability or tighten the mucosal barrier. Treatment with AA-ORS for 14 days improved electrolyte absorption, reduced paracellular permeability and plasma endotoxin and proinflammatory cytokine levels, better maintained body weight, and improved survival of mice exposed to an otherwise lethal dose of whole-body irradiation (8.5 Gy TBI) 51 Subsequent studies have shown that these improvements occur as early as 7 days after AA-ORS treatment; however, the exact mechanism of these effects is unclear.

[0274] In conclusion, AA-ORS comprising a selected set of amino acids were identified as intestinal radiation mitigators in mice by selectively enhancing stem cell markers such as Lgr5 and BMI1 in intestinal stem and progenitor cells and by blocking caspase-3-mediated apoptosis. Thus, this study demonstrates the effects of simple amino acids on intestinal stem cell proliferation, maturation and differentiation leading to functionally active villous epithelial cells whose function and height are impaired by irradiation.

[0275] The examples and embodiments described herein are for illustrative purposes only, and suggest that various modifications or changes may be made thereto by those skilled in the art under their inspiration, and these modifications or changes are included in the spirit and scope of the present application. In addition, any element or limitation of any invention disclosed herein or its embodiment may be combined with any and / or all other elements or limitations disclosed herein (alone or in any combination) or any other invention or embodiment, and all such combinations are contemplated by the scope of the present invention, but are not limited thereto.

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Claims

1. A preparation, wherein the preparation comprises: A therapeutically effective free amino acid combination, wherein the therapeutically effective free amino acid combination consists of: a therapeutically effective amount of the free amino acids threonine, valine, tyrosine, serine, and aspartic acid; Wherein the formulation is prepared for oral administration.

2. A preparation, wherein the preparation comprises: A therapeutically effective free amino acid combination, wherein the therapeutically effective free amino acid combination consists of therapeutically effective amounts of the free amino acids threonine, valine, tyrosine, serine and tryptophan: wherein the formulation is prepared for inhalation therapy.

3. The preparation according to claim 1 or 2, wherein (i) the therapeutically effective amount of threonine has a concentration range of 0.4 g / L to 1.5 g / L or 0.7 g / L to 1.3 g / L; (ii) the therapeutically effective amount of valine has a concentration range of 0.7 g / L to 1.7 g / L or 0.9 g / L to 1.5 g / L; (iii) the therapeutically effective amount of tyrosine has a concentration range of 0.05 g / L to 0.4 g / L; (iv) the therapeutically effective amount of serine has a concentration range of 0.6 g / L to 1.6 g / L or 0.8 g / L to 1.4 g / L; (v) when present, the therapeutically effective amount of tryptophan is at a concentration ranging from 1.1 g / L to 2.1 g / L or from 1.3 g / L to 1.9 g / L; or Any combination of (i) to (v).

4. The preparation according to claim 1 or 2, wherein (i) the therapeutically effective amount of threonine has a concentration range of 0.9 g / L to 1.1 g / L; (ii) the therapeutically effective amount of valine has a concentration range of 1.1 g / L to 1.3 g / L; (iii) the therapeutically effective amount of tyrosine has a concentration range of 0.1 g / L to 0.3 g / L; (iv) the therapeutically effective amount of serine has a concentration range of 1.0 g / L to 1.2 g / L; (v) when present, the therapeutically effective amount of tryptophan is at a concentration ranging from 1.5 g / L to 1.7 g / L; or Any combination of (i) to (v).

5. The preparation according to claim 1 or 2, wherein (i) the therapeutically effective amount of threonine has a concentration range of 1.0 g / L; (ii) the therapeutically effective amount of valine has a concentration range of 1.2 g / L; (iii) the therapeutically effective amount of tyrosine has a concentration range of 0.2 g / L; or (iv) when present, the therapeutically effective amount of tryptophan is at a concentration in the range of 1.6 g / L; or Any combination of (i) to (iv).

6. The preparation according to claim 1 or 2, wherein The therapeutically effective amount of threonine is 8 mM, The therapeutically effective amount of valine is 10 mM, The therapeutically effective amount of tyrosine is 1.2 mM, When present, the therapeutically effective amount of tryptophan is 8 mM, When present, the therapeutically effective amount of aspartic acid is 8 mM, and The therapeutically effective amount of serine is 10 mM.

7. The preparation according to claim 1 or 2, wherein At least one of the free amino acids or each of the free amino acids is an L-amino acid.

8. The preparation according to claim 1 or 2, wherein The formulation further comprises a pharmaceutically acceptable carrier, buffer, adjuvant or excipient.

9. The preparation according to claim 1 or 2, wherein The formulation also contains water.

10. The preparation according to claim 1 or 2, wherein The formulation is sterile.

11. The preparation according to claim 1 or 2, wherein The preparation is a pharmaceutical preparation.

12. The preparation according to claim 2, wherein The formulation is formulated for pulmonary administration.

13. The preparation according to claim 2, wherein The formulation is formulated for intranasal administration.

14. The preparation according to claim 1 or 2, wherein The formulation is for treating asthma in a subject in need thereof, and the subject is a mammal.

15. The preparation according to claim 14, wherein The subject is a human, cat, dog, pig, horse, cow, sheep or goat.

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