HK1224577A1 - Materials and methods for improving gastrointestinal function - Google Patents

Description

Materials and methods for improving gastrointestinal function

The application is a divisional application of Chinese patent application with the application date of 2011, 9 and 26, and the application number of 201180045566.0, and the name of the invention is 'material and method for improving gastrointestinal function'.

Cross Reference to Related Applications

Priority of united states provisional application serial No. 61/386,317 filed 24/2010 and united states provisional application serial No. 61/431,629 filed 11/2011, which are claimed herein, are incorporated by reference in their entirety.

Government support

The invention was made with government support under fund No. rc2-AI-087580 awarded by the National Institute of Health (NIH). The government has certain rights in the invention.

Technical Field

The present invention relates to materials and methods for improving gastrointestinal function.

Background

Radiation (radiation) is a common treatment for malignant tumors in the abdomen and pelvis, which can cause severe damage to the lining of the Gastrointestinal (GI) tract, which is composed of rapidly dividing intestinal epithelial cells. The toxic effects of radiation on the gastrointestinal system cause symptoms such as nausea, vomiting, diarrhea, electrolyte imbalance (electrolyte imbalance) and dehydration, and adversely affect the recovery of the patient during cancer treatment. Even at low doses, a continuous loss of small intestinal villi and brush border is observed within days after irradiation (irradiation). While crypt cells (cryptcell) can rapidly refill (repopulate) this region after a mild to moderate dose of (irradiation) IR, they become lost at a logarithmic rate after high doses of irradiation.

Irradiation is particularly destructive to the villous epithelium where nutrient and electrolyte absorption occurs. The villous epithelium undergoes a continuous process of cell loss and regeneration in which a continuous supply of immature intestinal cells (derived from progenitor cells located within the lower pole (lowerpole) of the siberian crypt (crypts lieberkuhn) migrate from the proliferative compartment at the base of the crypt (proliferative component) to the top of the villus. During their short life span, these intestinal cells gradually mature into villous cells along the crypt-villus axis (crypt-villous). Radiation treatment of the abdominal and pelvic regions destroys not only existing villous hair cells, but also the intestinal cells that form new villous cells, and thus, almost the entire villous epithelium can be depleted even at moderate doses.

Radiation therapy is complicated by its acute GI toxicity due to the increasing use of high total radiation doses and cytotoxic agents. Damage to the GI tract not only results in malabsorption and loss of nutrients and fluids, but also disrupts intestinal barrier function. Enterorrhea (leakygut) allows pathogens to easily enter across mucosal barriers, causing inflammation, bacteremia and endotoxemia. For example, although GI toxicity usually occurs at higher doses, acute radiation enteritis, diarrhea, and abdominal pain can occur within days after irradiation, even at doses as low as 5-12 Gy (1.8-2 Gy per fraction is used for conventional fractionation procedures of radiation). Chronic radiation enteritis can occur 18 months to 6 years after radiation therapy, but it can also occur even 15 years later^27-29。

Treatment options for radiation enteritis are limited. Conventional treatment regimens include: applying an anti-diarrheal agent to prevent fluid loss; administering montmorillonite (smectite) as an adsorbent for bile salts; opioids are administered to reduce gastric or rectal pain, and steroids are administered to reduce inflammation. Clinical trials have also investigated the efficacy of lactobacillus acidophilus (l. acidophilus), montmorillonite or sucralfate (sucralfate) in the prevention of diarrhea, but only achieved moderate relief of acute GI symptoms³⁰。

A common method of treating radiation enteritis is the use of Total Parenteral Nutrition (TPN) to provide intestinal rest. However, there is still a need to determine whether parenteral nutrition meets the nutritional needs of the patient, or indeed has a therapeutic effect on radiation enteritis. Although TPN can correct nutritional imbalances in certain patients, severe radiation enteritis can still occur³⁷. TPN also causes intestinal atrophy, usually within 48 hours of administration. TPN also weakens the mechanical and immunological barriers³⁸。

The exact biological mechanism (not yet established) thought to lead to mucosal atrophy during TPN involves localized nutrient-sensing cellular signaling³⁹And body fluid signals (e.g. gastrointestinal hormones)^40，41Both of which are described below. TPN has been shown to induce a rapid (< 8h) decrease in intestinal blood flow (this precedes villous atrophy) and to inhibit protein synthesis at 24 hours and cell proliferation and survival at 48 hours⁴². In contrast, oral feeding rapidly increased intestinal blood flow in neonatal and mature animals^43，44. Similarly, in neonatal piglets enteral feeding (entertaining) almost immediately (within 1 to 3 hours) increased portal venous blood flow (PBF) to 50% higher than in food deprived piglets⁴⁵. Thus, as shown in several studies, enteral feeding is far superior to parenteral feeding^7，8。

Currently, there is a lack of nutritional treatments that can effectively alleviate radiation enteritis. Although early studies showed that an elemental diet (elementary diet) or a specific exclusion diet (specific exclusion diet) could be beneficial in selected circumstances^2，31，32However, the efficacy of this method was not demonstrated later. Current dietary treatments only provide nutritional support for malnourished patients with chronic radiation enteritis.

Animal studies demonstrated that glutamine protects the mucosa of both the upper and lower GI tract from damage caused by chemotherapy or Radiation Therapy (RT)^33-35. However, clinical trials have failed to show that oral glutamine feeding can prevent or alleviate urgency in patients receiving pelvic radiation therapySexual diarrhea³⁶. Therefore, there is a need to develop improved feeding compositions for the treatment of radiation-induced GI damage. These and other benefits are provided by the present invention, as will be apparent from the following disclosure.

Disclosure of Invention

The present invention provides therapeutic compositions and methods for improving small bowel function. The compositions of the present invention are useful for treating or ameliorating gastrointestinal damage associated with the loss of small intestine epithelial cells, particularly in the villous region and brush border, and/or for treating or ameliorating diseases or conditions associated with altered absorptive capacity of the small intestine.

Advantageously, the therapeutic compositions of the present invention can be tailored to the unbalanced absorption state of the gastrointestinal system caused by loss of small intestinal epithelial cells and altered function of transport proteins in the small intestine. In a preferred embodiment, the composition of the invention is formulated for oral administration.

In one embodiment, the therapeutic composition comprises, consists essentially of, or consists of: a free amino acid selected from one or more of lysine, glycine, threonine, valine, tyrosine, aspartic acid, isoleucine, tryptophan, asparagine, and serine; and optionally pharmaceutically acceptable carriers, electrolytes, vitamins, buffers and flavoring agents. The therapeutic composition is administered by enteral route. In one embodiment, the total osmolarity (osmolarity) of the composition is from about 230mosm to 280mosm, or preferably from about 250mosm to 260 mosm. In one embodiment, the pH of the composition is about 7.1 to 7.9, preferably about 7.4.

In a particular embodiment, the composition of the invention does not comprise glucose, glutamine, methionine and/or lactose.

Also provided are methods for treating or ameliorating diseases or conditions associated with loss of small intestinal epithelial cells, particularly in the villus region and brush border, and diseases or conditions associated with altered function of transport proteins in small intestinal epithelium. The method comprises administering to a subject in need of such treatment an effective amount of a composition of the invention by enteral route. Preferably, the composition of the invention is administered orally and reaches the intestine of the subject.

The invention also provides methods for preparing therapeutic compositions, and methods for screening for nutrients or electrolytes for inclusion in the therapeutic/dietary compositions of the invention by selecting those that retain or acquire substantial absorptive capacity following small intestinal epithelial cell destruction. These methods may be applied to individual patients, thereby facilitating the development of compositions and methods specifically designed to meet the needs of individual patients.

Drawings

Figure 1 shows the effect of Irradiation (IR) on net anion secretion (a) and conductance (B). (A) Study 12GyIR tissue on days 1, 3 and 4. Day 2 see I_scIs increased to the maximum. Arrows indicate the time points when forskolin (forskolin) was added. (B) Effect of increasing dose of IR on net anion secretion. All these tissues were studied on day 6, and n ═ 12. The results show an IR dose-dependent increase in conductance.

FIG. 2 shows that as the irradiation dose increases, I_scA change in (c). All these values are derived from n-24 tissues. Experiments were performed in conventional ringer's solution (ringer solution) on day 4 post irradiation, with a total osmolarity of 296mosm across the room. Histopathological sections showed minimal villus and crypt damage at 3Gy and extensive villus and crypt damage at 7Gy compared to 0 Gy.

FIG. 3A shows I in mouse epithelial cells after irradiation with 3Gy_scChange over time. Values represent mean ± s.e.m, n ═ 6 tissues. I is seen on day 6 after radiation_scIs increased to the maximum. No significant differences were seen between days 5, 6 and 7. With time > 7 days after irradiation, phase 5, 6 or 7 daysRatio, I_scSlightly decreased. Day 5, 6 and 7I_scThe values are similar. Fig. 3B shows ion transport by small intestine epithelial cells. FIG. 3C shows the I of the basis of bumetanide (bumetanide) in non-irradiated tissue and 3Gy irradiated tissue_scAnd cAMP-stimulated I_scThe influence of (c). FIG. 3D shows HCO₃ ^-Contribution to net anion secretion. By replacing Cl in ringer's solution with an equimolar amount of isethionate^-To determine that forskolin stimulates I in 0Gy (＊: p < 0.02) tissue_scBut not in 3Gy tissue. FIG. 3E shows bath (bath) Na⁺For HCO₃ ^-The effect of secretion. All results shown in fig. 3 are from n-6 tissues. Error bars represent SEM.

Figure 4A shows the change in plasma endotoxin levels after IR. On day 6 post-IR, plasma endotoxin levels were measured. FIG. 4B shows Cl plotted against membrane voltage change (diluted potential)^-With Na⁺Change in permeability ratio (permeabilityratio). Irradiation at 7Gy resulted in a complete loss of selectivity.

FIG. 5 shows that irradiation increases the levels of inflammatory mediators, including IL-1 β, TNF α, and MIP- α.

FIG. 6 shows HCO induced by irradiation₃ ^-Changes in secretion and HCO₃ ^-Immunostaining of secretory machinery. (A) Showing irradiation vs. bath Na⁺For HCO₃ ^-The effect of secretion. Experiments in A) with 140mM Na⁺Or B) is Na-free⁺In a Cl-containing solution. Tissue was stimulated with forskolin. Comparison of HCO in mice irradiated with 0Gy and 3Gy₃ ^-And (4) secreting. Significantly higher bath Na was observed in mice irradiated with 0Gy compared to mice irradiated with 3Gy⁺Dependent HCO₃ ^-Secretion (p < 0.001). Results were obtained from n-6 tissues. Error bars represent s.e.m. (B-E) shows immunostaining of jejunal tissue of mice that received 0Gy irradiation and 3Gy irradiation using the NBCe1a/B antibody.

Figure 7 shows the IR dose-dependent changes in glucose transport and kinetics. (A) Shows that irradiation results in glucose-stimulated Na measured in Uussingchamber⁺I_scDose-dependently decreased. (B) It is shown that the SGLT1 affinity for glucose decreases with increasing irradiation dose.

Fig. 8 shows that irradiation reduced the glucose stimulated current in a dose dependent manner starting from irradiation with 1 Gy. Irradiation at 7Gy almost completely inactivated glucose transport.

Fig. 9 shows the short circuit current, showing saturation kinetics with increasing glucose concentration. In particular, glucose transport is saturated at a concentration of 4 mM. Fig. 9B shows the irradiation dose-dependent increase in Km value. K was observed at 7Gy_mIs increased to the maximum. This indicates that irradiation causes a decrease in the affinity of SGLT-1 to glucose.

FIG. 10 shows V_maxDecreasing with increasing radiation dose. V observed at 7Gy_maxA minimum reduction in. This indicates that irradiation causes a decrease in functional SGLT-1 for glucose transport.

Fig. 11 shows the change in Km with the passage of time after irradiation. Km increased immediately after irradiation and returned to normal (control value) about 14 days after irradiation.

Fig. 12A and B show the results of the mouse survival study after 9Gy and 15.6Gy irradiation. Glucose-treated mice began to die on days 5 and 7, while control mice did not die until day 10 post-irradiation. On day 20, 30% of the control mice were alive, whereas none of the glucose-treated mice survived on day 20.

FIG. 13 shows Western blot analysis of SGLT-1 protein levels in whole cell lysates. The results show that irradiation increases SGLT-1 expression.

FIG. 14 shows Western blot analysis of SGLT-1 protein levels in brush border membrane vesicles of jejunal tissue. Irradiation increased SGLT-1 protein levels in a dose-dependent manner. SGLT-1 protein was not detected in colon tissue.

FIG. 15 shows irradiation induced Glutamine stimulated I_scThe dose-dependently increased.

FIG. 16 shows irradiation induced lysine stimulation of I_scDose-dependently decreased.

Fig. 17A and B show mouse survival after lysine (a) or glucose (B) treatment after IR. Administration of lysine resulted in increased survival, while administration of glucose resulted in decreased survival.

Figure 18 shows Western blot analysis of various transporters. Western blot analysis showed NKCC1(A), CFTR (C) and NBCe1-A/B (B) protein levels in the jejunum of mice. From left to right, lanes represent 0Gy, 1Gy, 3Gy, 5Gy and 7 Gy. Irradiation increased NKCC1 protein levels from 1Gy to 5Gy, such elevation decreased at 7Gy (a). After irradiation, NBCe1-A/B protein levels were significantly reduced. CFTR protein levels were significantly elevated in jejunal tissue after irradiation with 3Gy compared to 0Gy (C). The jejunum has the highest NBCe1-A/B protein levels (D) compared to the duodenum, ileum, or colon. Tissues were harvested for Western blot at day 6 post irradiation.

FIGS. 19A and B are graphical patterns of cAMP-stimulated (A) and irradiation-induced (B) anion secretion.

FIG. 20 shows lesions of small intestinal mucosa in mice treated with 5-fluorouracil (5-FU) (FIG. 20A) and cisplatin (FIG. 20B). (A) Isc changes in 5-FU injected mice are shown. (B) Changes in Isc in cisplatin-injected mice are shown.

FIG. 21 shows that administration of a therapeutic composition of the invention increases the function of the small intestine in mice that have received 5-FU.

Detailed Description

The present invention provides therapeutic compositions and methods for improving small bowel function. The composition is formulated for enteral administration (entera dministration). The compositions and methods of the invention are particularly useful for treating or ameliorating gastrointestinal damage associated with loss of small intestine epithelial cells, particularly in the villus region and brush border, and/or for treating diseases or conditions associated with altered function of transporters in the small intestine epithelium.

Advantageously, the therapeutic compositions of the present invention can be tailored to the unbalanced absorption state of the gastrointestinal system caused by loss of small intestinal epithelial cells, particularly in the villous region and brush border of the small intestine, and alteration of transporter function. In particular, the present invention may improve small intestinal mucosal healing, restore small intestinal function, enhance fluid retention, prevent or alleviate small intestinal atrophy, and/or restore or enhance small intestinal barrier function in patients with small intestinal mucosal damage.

In one embodiment, the therapeutic composition comprises, consists essentially of, or consists of: one or more free amino acids selected from lysine, glycine, threonine, valine, tyrosine, aspartic acid, isoleucine, tryptophan, asparagine, and serine; and optionally, pharmaceutically acceptable carriers, electrolytes, vitamins, buffers and flavoring agents. The therapeutic composition is administered by the enteral route. In one embodiment, the total osmolality of the composition is from about 230mosm to 280mosm, or preferably from about 250mosm to 260 mosm. In one embodiment, the pH of the composition is from about 4.0 to 8.5, preferably from 5.0 to 8.2, more preferably from 6.0 to 8.0, more preferably from 7.1 to 7.9, and most preferably about 7.4.

In a particular embodiment, the composition of the invention does not comprise glucose, glutamine, methionine and/or lactose.

Also provided are methods for treating or ameliorating diseases or disorders associated with loss of small intestine epithelial cells, particularly in the villus region and brush border, and diseases or disorders associated with altered function of transport proteins in small intestine epithelium. The method comprises administering to a subject in need of such treatment an effective amount of a composition of the invention via the enteral route.

The present invention is based, at least in part, on the discovery that: after injury to the small intestine mucosa, enteral feeding to the subject with only nutrients that maintain or acquire sufficient absorptive capacity improves mucosal healing, restores small intestine function, enhances fluid retention, and alleviates a range of associated disease symptoms including, but not limited to, malabsorption, diarrhea, nausea, vomiting, electrolyte imbalance, and dehydration.

According to the invention, it has been determined that, after irradiation and chemotherapy, reference is made to, for example, glucose, glutamine and lysine, and electrolytes (such as Na)⁺、HCO₃ ^-And Cl^-) An alteration in transporter function was observed. Furthermore, irradiation causes an elevated net anion secretion. Changes in nutrient and electrolyte absorption capacity occur immediately after radiation and chemotherapy, but absorption capacity can be restored to normal (about 8 to 14 days after irradiation in a mouse model).

Specifically, irradiation causes a dose-dependent decrease in glucose absorption by irradiation due to a decrease in the affinity of the sodium-dependent glucose transport system (SGLT-1) for glucose. Functional studies on glucose stimulation showed that irradiation caused a dose-dependent decrease in glucose transport activity and a decrease in the affinity of SGLT-1 for glucose.

The presence of unabsorbed nutrients or solutes in the intestinal lumen is known to cause osmotic diarrhea. According to the present invention, it has been found that oral feeding of glucose and/or glutamine to irradiated subjects results in osmotic diarrhea and reduced survival, while oral feeding of each amino acid or combination thereof selected from the group consisting of: lysine, glycine, threonine, valine, tyrosine, aspartic acid, isoleucine, tryptophan, asparagine and/or serine.

Therapeutic composition for increasing small intestine function

In one aspect, the present invention provides therapeutic compositions for improving small bowel function. In one embodiment, the therapeutic composition comprises, consists essentially of, or consists of: one or more free amino acids selected from lysine, glycine, threonine, valine, tyrosine, aspartic acid, isoleucine, tryptophan, asparagine, and serine; and optionally, pharmaceutically acceptable carriers, electrolytes, vitamins, buffers and flavoring agents. The therapeutic composition is administered by the enteral route.

Preferably, the composition is weakly basic and is hypotonic compared to the osmolarity of small intestine epithelial cells, such as villous and crypt cells of the small intestine. Preferably, the composition of the present invention comprises water. Preferably, the composition is formulated as an oral rehydration drink (oralrehydrogenation dry) for enhancing small intestine function that is disrupted by loss or damage to villous epithelial cells.

In one embodiment, the total osmolality of the composition is from about 230mosm to 280mosm, or any value therebetween. Preferably, the total osmolarity is from about 250mosm to 260 mosm. In another embodiment, the total osmolality of the composition is any value below 280 mosm.

In one embodiment, the pH of the composition is about 7.1 to 7.9, or any value therebetween. Preferably, the pH of the composition is about 7.3 to 7.5, more preferably about 7.4.

In certain embodiments, each free amino acid may be present at a concentration of 4mM to 40mM, or any value therebetween, wherein the total osmolality of the composition is from about 230mosm to 280 mosm. Alternatively, if the amino acid concentration is calculated on a mg/L basis, each free amino acid may be present at a concentration of 300mg/L to 8000mg/L or any value therebetween, wherein the total osmolality of the composition is from about 240mosm to 280 mosm.

In certain specific embodiments, the therapeutic composition comprises one or more free amino acids present at each of the following concentrations: lysine at a concentration of about 730mg/l to 6575mg/l or any value therebetween; aspartic acid at a concentration of about 532mg/l to 4792mg/l or any value therebetween; glycine at a concentration of about 300mg/l to 2703mg/l or any value therebetween; isoleucine at a concentration of about 525mg/l to 4722mg/l or any value therebetween; threonine at a concentration of about 476mg/l to 4288mg/l or any value therebetween; tyrosine in a concentration of about 725mg/l to 6523mg/l or any value therebetween; valine at a concentration of about 469mg/l to 4217mg/l or any value therebetween; tryptophan at a concentration of about 817mg/l to 7352mg/l or any value therebetween; asparagine at a concentration of about 528mg/l to 4756mg/l or any value therebetween; and/or serine at a concentration of about 420mg/l to 3784mg/l or any value therebetween; wherein the total osmolality of the composition is from about 240mosm to about 280 mosm.

In one embodiment, the present invention provides a beverage comprising the following components: lysine (11mosm to 21mosm), aspartic acid (3mosm to 13mosm), glycine (19mosm to 29mosm), isoleucine (19mosm to 29mosm), threonine (19mosm to 29mosm), tyrosine (0.5mosm to 5mosm), valine (19mosm to 29mosm), tryptophan (5mosm to 20mosm), asparagine (3mosm to 13mosm) and serine (3mosm to 8mosm) or a subset of these (subset).

In a particular embodiment, the composition comprises lysine, glycine, threonine, valine, and tyrosine in free amino acid form. In another embodiment, the composition comprises lysine, glycine, threonine, valine, tyrosine, aspartic acid, isoleucine, tryptophan, asparagine and serine in free amino acid form.

In another embodiment, the composition comprises one or more dipeptides made of the same or different amino acids selected from the group consisting of: lysine, glycine, threonine, valine, tyrosine, aspartic acid, isoleucine, tryptophan, asparagine, or serine.

In one embodiment, the composition does not comprise glutamine and/or methionine; and any dipeptide, oligopeptide or polypeptide or protein that can be hydrolyzed to glutamine and/or methionine.

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

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 dipeptide is less than 300mg/l, or any concentration below 300mg/l, such as 100mg/l, 50mg/l, 10mg/l, 5mg/l, 1mg/l, 0.5mg/l, or 0.01 mg/l.

In one embodiment, the therapeutic composition does not comprise any saccharide including any monosaccharide, disaccharide, oligosaccharide, polysaccharide and carbohydrate. In a particular embodiment, the therapeutic composition does not comprise glucose, and/or any disaccharides, oligosaccharides, polysaccharides and carbohydrates that can be hydrolyzed to glucose. In a particular embodiment, the composition does not comprise lactose. In another specific embodiment, the therapeutic composition does not comprise fructose and/or galactose, and/or any di-, oligo-, poly-, and carbohydrates hydrolysable to fructose and/or galactose.

In an alternative embodiment, the therapeutic composition may comprise the monosaccharide glucose, and optionally, one or more disaccharides comprising glucose (except lactose), wherein the total concentration of the monosaccharide glucose and the disaccharide comprising glucose is less than 3g/l, or any concentration below 3g/l, such as 1g/l, 500mg/l, 300mg/l, 100mg/l, 50mg/l, 10mg/l, 5mg/l, 1mg/l, 0.5mg/l, or 0.01 mg/l.

In certain embodiments, the therapeutic composition comprises one or more electrolytes selected from, for example: na (Na)⁺；K⁺；HCO₃ ^-；CO₃ ^2-；Ca²⁺；Mg²⁺；Fe²；Cl^-(ii) a Phosphate ions, e.g. H₂PO₄ ^-、HPO₄ ^2-And PO₄ ^3-(ii) a Zinc; iodine; copper; iron; selenium(ii) a Chromium; and molybdenum. In an alternative embodiment, the composition does not comprise HCO₃ ^-Or CO₃ ^2-. In another alternative embodiment, the composition comprises HCO at a total concentration of less than 5mg/l or at a concentration of less than 5mg/l₃ ^-And CO₃ ^2-。

In another embodiment, the therapeutic composition comprises one or more vitamins, including but not limited to vitamin a, vitamin C, vitamin D (e.g., vitamin D)₁、D₂、D₃、D₄And/or D₅) Vitamin E, vitamin B₁(thiamine), vitamin B₂(e.g., riboflavin), vitamin B₃(e.g., niacin or niacinamide), vitamin B₅(pantothenic acid), vitamin B₆(pyridol), vitamin B₇(Biotin) and vitamin B₉(e.g., folic acid), vitamin B₁₂(Cobalamin) and vitamin K (e.g., vitamin K)₁、K₂、K₃、K₄And K₅) And choline.

In certain embodiments, the composition does not comprise one or more ingredients selected from the group consisting of: oligosaccharides, polysaccharides and carbohydrates; an oligopeptide or polypeptide or protein; a lipid; small, medium and/or long chain fatty acids; and/or a food comprising one or more of the above nutrients.

In one embodiment, phosphate ions (e.g., H) are used₂PO₄ ^-、HPO₄ ^2-And PO₄ ^3-) To buffer the composition of the invention. In one embodiment, the therapeutic composition uses HCO₃ ^-Or CO₃ ^2-As a buffer. In another embodiment, the therapeutic composition does not use HCO₃ ^-Or CO₃ ^2-As a buffer.

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 the basic and novel characteristics of the invention, i.e., compositions and methods for treating lesions in the small intestinal epithelium, particularly in the villous area and brush border. For example, by using "consisting essentially of," a therapeutic composition does not contain any unspecified ingredients, including, but not limited to, free amino acids, dipeptides, oligopeptides or polypeptides or proteins, as well as mono-, di-, oligo-, polysaccharides, and carbohydrates, that have a direct beneficial or adverse therapeutic effect on the treatment of lesions in the small intestinal epithelium (particularly in the villous region and brush border). Furthermore, by using the term "consisting essentially of", the composition may comprise a substance that has no therapeutic effect on the treatment of small intestinal epithelial lesions; such ingredients include carriers, excipients, adjuvants (adjuvant), flavoring agents, etc., which do not affect the health or function of the injured small intestine epithelium, particularly in the villous region and brush border.

The term "oligopeptide" as used herein refers to a peptide consisting of three to twenty amino acids. The term "oligosaccharide" as used herein refers to a saccharide consisting of three to twenty monosaccharides.

In one embodiment, the compositions of the invention comprise nutrients (e.g., free amino acids) and/or electrolytes that maintain or achieve increased absorptive capacity in a subject with small intestinal epithelial cell damage compared to the absorptive capacity of a normal control without small intestinal epithelial cell (e.g., villous cells, crypt cells, intestinal cells, and intestinal progenitor cells) damage.

In another embodiment, the compositions of the invention do not comprise nutrients (e.g., amino acids) and/or electrolytes that are not absorbed or have reduced absorption in a subject with small intestine epithelial cell damage as compared to the absorptive capacity of a normal control without small intestine epithelial cell (e.g., villous cells, crypt cells, intestinal cells, and pro-intestinal progenitor (projentior) cell damage. Advantageously, the compositions of the present invention facilitate easy absorption of nutrients by the small intestine to reduce undue energy expenditure, thereby providing intestinal rest immediately after mucosal injury.

Therapeutic methods for improving small bowel function

Another aspect of the invention provides methods for treating or ameliorating a disease or disorder associated with loss or damage to small intestine epithelial cells, particularly in the villus region and brush border. In one embodiment, the loss or damage of small intestine epithelial cells causes a change in the absorptive capacity of nutrients, electrolytes and/or fluids. Advantageously, the present invention improves small intestine epithelial mucosal healing in patients with small intestine epithelial cell loss or damage, particularly in patients with small intestine villous atrophy; improving the function of the small intestine; enhancing nutrient absorption and fluid retention in the small intestine; preventing or relieving small intestine atrophy; relief of abdominal pain; preventing and/or treating diarrhea; restoring or enhancing small intestine barrier function; and/or reducing intestinal mucositis, bacteremia and/or endotoxemia.

Thus, the present invention is particularly advantageous for improving gastrointestinal health in: a subject receiving cytotoxic chemotherapeutic agents, pelvic or abdominal radiation, proton therapy, and abdominal surgery; a subject having an infection or an autoimmune disease associated with acute or chronic small intestine inflammation; objects that are regularly or occasionally exposed to radiation, such as astronauts and pilots that are regularly exposed to spatial radiation; and objects exposed to radiation due to nuclear accidents, war acts, or terrorism.

In one embodiment, the method comprises administering to a patient or subject in need of such treatment an effective amount of a composition of the invention via the enteral route. The composition may be administered to the patient or subject immediately before, during, and/or after the damage to small intestine epithelial cells, and may be administered one or more times per day.

The term "subject" or "patient" as used herein describes an organism (including mammals such as primates) to which treatment with a composition according to the present invention can be provided. Mammalian species that may benefit from the disclosed treatment methods include, but are not limited to: apes (apes), chimpanzees, orangutans, humans, monkeys; domestic animals (domestic animals) such as dogs, cats; livestock and poultry (livestock) such as horses, cattle, pigs, sheep, goats, chickens; and animals such as mice, rats, guinea pigs, and hamsters.

In a specific embodiment, the subject in need of treatment is a patient having an injury to mucosal epithelial cells of the small intestine (including the mucosal layers of the duodenum, jejunum, and ileum). In particular, subjects in need of treatment are patients with lesions in the villous region of the small intestine and brush border. For example, subjects in need of treatment have villous atrophy (e.g., partly or completely decaying villous regions and brush border) compared to normal; a reduction in villus cells in the small intestine of at least 5% (e.g., at least 10%, 20%, 30%, or 50%); at least 5% (e.g., at least 10%, 20%, 30%, or 50%) fluff height is lost; loss of function of one or more transporters (transporters) in the villus region and brush border of the small intestine, wherein transporters include, but are not limited to, the SGLT-1 transporter, the AE2 transporter, the NHE1 transporter, and the NBCe1-a/B transporter, wherein the loss of transporter function is at least 5% (e.g., at least 10%, 20%, 30%, or 50%); and/or a change in the absorptive capacity of one or more nutrients in the small intestine, wherein the nutrients are selected from: isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, histidine, tyrosine, alanine, arginine, glutamine, aspartic acid, asparagine, cysteine, glycine, proline, serine, asparagine, glucose, fructose, and/or lactose, wherein the change in absorptive capacity is at least 5% (e.g., at least 10%, 20%, 30%, or 50%).

For example, the absorptive capacity of the small intestine can be determined by using the ewings chamber as described in the materials and methods section herein. For example, by measuring an index (including, e.g., K)_m、V_maxAnd I_sc) To determine the change in absorption state. Lesions in the villous and other regions of the small intestine may be determined, for example, by examination of a biopsy sample of the small intestine mucosa.

The skilled practitioner can readily determine the disease and treatment that causes damage to small intestinal mucosal epithelial cells, such as small intestinal villus cells. As is well known in the medical profession, patients suffering from certain diseases such as Inflammatory Bowel Disease (IBD), ulcerative colitis, duodenal ulcers and Crohn's disease suffer from chronic destruction of the small intestinal mucosa. Radiation therapy, chemotherapy, and proton therapy also cause damage to small intestinal cells.

The term "treating" or any grammatical variation thereof as used herein includes, but is not limited to, alleviating the symptoms of a disease or disorder; and/or reduce, suppress, inhibit, reduce, or affect the course, severity, and/or extent of a disease or disorder.

The term "ameliorating," or any grammatical variation thereof, as used herein includes, but is not limited to, delaying onset, or reducing the severity of a disease or disorder (e.g., diarrhea, bacteremia, and/or endotoxemia). As used herein, improvement does not require the complete absence of symptoms.

The term "effective amount" as used herein refers to an amount that is capable of treating or ameliorating a disease or condition or is capable of producing the desired therapeutic effect.

In a specific embodiment, the present invention provides a method for promoting intestinal health in a subject having a small intestine epithelial cell injury, wherein the method comprises: identifying a subject that has, or will suffer from, small intestine epithelial cell damage and that requires treatment or amelioration, and administering to the subject by enteral route an effective amount of a composition comprising, consisting essentially of, or consisting of: one or more free amino acids selected from lysine, glycine, threonine, valine, tyrosine, aspartic acid, isoleucine, tryptophan, asparagine, and serine; water; and optionally, a therapeutically acceptable carrier, electrolyte, vitamin, buffer and flavoring agent, wherein the total osmolality of the composition is from 240mosm to 280mosm, and the pH is from about 7.1 to 7.9.

In one embodiment, a subject having (or about to have) small intestine epithelial cell damage is not administered one or more of the following nutrients by the enteral route, wherein the nutrients are selected from the group consisting of: glutamine, methionine, and any dipeptide, oligopeptide or polypeptide or protein that can be hydrolyzed to glutamine and/or methionine; glucose and any disaccharides, oligosaccharides, polysaccharides, and carbohydrates hydrolyzable to glucose; and/or foods that require the absorption of any of the above nutrients in the small intestine after digestion.

In another embodiment, the following nutrients are not administered via the enteral route to a subject having (or about to have) a small intestine epithelial cell injury, wherein the nutrients are selected from the group consisting of: carbohydrates, lipids, fatty acids and/or foods that require absorption of any of the above nutrients in the small intestine after digestion. For patients exposed to radiation or receiving radiotherapy, chemotherapy, and proton therapy, damage to small intestinal epithelial cells typically lasts at least 3, 7, 14, 21, 30 days, or any time between 1 and 30 days.

In another embodiment, after any time between 1 and 30 days (e.g., 3, 7, 14, 21, 30 days), one or more of the following nutrients are administered via the enteral route to enhance mucosal healing as a result of exposure of the subject to radiation or to radiation therapy, chemotherapy, and/or proton therapy, wherein the nutrients are selected from the group consisting of: glutamine, methionine and any dipeptide, oligopeptide or polypeptide or protein that can be hydrolyzed to glutamine and/or methionine; glucose and any disaccharides, oligosaccharides, polysaccharides, and carbohydrates hydrolyzable to glucose; and/or foods that require the absorption of any of the above nutrients in the small intestine after digestion.

In a specific embodiment, the composition of the invention is administered orally and reaches the small intestine of the subject. Optionally, the method further comprises administering by parenteral route the desired nutrients and electrolytes that are not administered in sufficient amounts by enteral route.

In one embodiment, the present invention is not used to provide significant amounts or all of the necessary nutrients to a subject, but is used to improve small intestinal mucosal healing, restore small intestinal function, enhance fluid retention, prevent or alleviate villous atrophy of the small intestine, prevent and/or treat diarrhea, and/or restore or enhance intestinal barrier function. In a specific embodiment, the drink composition is also based on an increase in barrier function. Barrier function can be determined using a variety of techniques, including: a) the conductance measurements on tissues mounted in ews' chamber are elevated, b) the dilution potential used to measure the relative permeability (PCl/PNa) of Cl and Na (only an intact and functional barrier can maintain ion selectivity; when barrier function is lost, the ion selectivity ratio is close to 1), and c) measuring plasma endotoxin levels. When the mucosal barrier function is lost, commensal intestinal bacteria can find their way into the systemic circulation, resulting in elevated plasma endotoxin levels. Endotoxin levels in a patient blood sample can be measured. Plasma endotoxin levels can also be used as an indicator to measure treatment improvement.

The compositions of the invention may be used to treat or ameliorate any disease or condition associated with the loss, destruction or reduction of small intestine epithelial cells, particularly the loss, destruction or reduction of function or number of villous, intestinal and/or intestinal progenitor cells of the small intestine. The invention is particularly useful for treating or ameliorating any disease or disorder associated with loss, inactivation, or altered function of a transporter in small intestinal epithelial cells, particularly a transporter in small intestinal villus cells.

In one embodiment, the compositions and methods of the invention are useful for treating or ameliorating a disease or condition arising from or associated with: a decrease in the affinity of the sodium-dependent glucose transport system (SGLT-1) for glucose; NH (NH)₂Terminal electrogenesis Na + -HCO₃Loss or reduced activity of (-) cotransporter (NBCe 1-A/B); top Cl^--HCO₃ ^-Loss or decreased activity of the exchange transporter (AE 1); and/or an elevated level or activity of the CFTR and/or NKCC-1 transporter system.

In a particularly preferred embodiment, the compositions and methods of the present invention are useful for treating or ameliorating small bowel injury caused by radiation. In a particular embodiment, the present invention can be used to treat or ameliorate small bowel injury caused by radiation therapy, particularly pelvic and abdominal radiation therapy. In a specific embodiment, the radiation therapy is for cancer therapy.

In addition, the present invention may be used to treat or ameliorate small bowel injury caused by frequent radiation exposure (e.g., exposure of astronauts and pilots to space radiation, such as radiation exposure caused by radioactive weapons and accidental nuclear leaks). In particular, the invention is useful for treating or ameliorating acute and/or chronic radiation enteritis.

In certain specific embodiments, the compositions and methods of the present invention are useful for treating or ameliorating small bowel injury, wherein the patient has received the following radiation: 1.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 Gy. In another embodiment, the subject has received a dose of radiation greater than 20 Gy.

In addition, the present invention may be used to treat or ameliorate small bowel injury caused by chemotherapeutic agents, including but not limited to: cisplatin, 5-fluorouracil (5-FU), hydroxyurea, etoposide, cytarabine (arabinoside), 6-mercaptopurine, 6-thioguanine, fludarabine, methotrexate, steroids, and/or combinations thereof.

In addition, the present invention can be used to treat or ameliorate small intestine damage caused by proton therapy.

In certain embodiments, the invention may be used to treat or ameliorate diseases involving small bowel injury, including but not limited to Inflammatory Bowel Disease (IBD), ulcerative colitis, duodenal ulcers, crohn's disease, and/or celiac disease (also known as celiac disease). The present invention is useful for treating or ameliorating small intestine damage caused by pathogenic infections (e.g., viral, bacterial, fungal, or other microbial infections).

In a particular embodiment, the invention is useful for treating or ameliorating small intestine villous atrophy, i.e., partial or complete, progressive attenuation of villous regions and brush border, and diseases and conditions resulting from, associated with, and/or caused by small intestine villous atrophy.

In certain embodiments, the present invention may be used to treat or ameliorate focal villous atrophy and/or diffuse villous atrophy; proliferative villous atrophy and/or dysplastic villous atrophy; and/or villous atrophy with and without mucosal inflammation.

In certain embodiments, the invention is useful for treating or ameliorating proliferative villous atrophy (with crypt hyperplasia) and related diseases and disorders, including but not limited to celiac (with gluten sensitive bowel disease); chronic wounds; small intestine transplantation; urinary ileal catheters; intestinal mucositis; intestinal ulcers; performing intestinal anastomosis; a glucagonoma; extensive small bowel resection; primary ileal villous atrophy; microscopic colitis (microscopical colitis) atrophy; atrophy of intestinal microvilli; and mitochondrial cell disease (mitochondrial respiratory chain abnormalities).

In certain embodiments, the invention may be used to treat or ameliorate dysplastic villous atrophy (without crypt hyperplasia) and related diseases or conditions, including but not limited to: a malignant tumor; paneth cell defect (pantethcellldeffence); hypofunction of the pituitary; celiac disease that is unresponsive to a gluten-free diet; tropical sprue (tropicalesprue); radiation-related ischemia; drug-induced villous atrophy, such as villous atrophy induced by neomycin and azathioprine.

In certain embodiments, the invention may be used to treat or ameliorate villous atrophy with mucosal inflammation and related diseases and disorders, including but not limited to: celiac disease; severe food intolerance (alimentaryintolerance); congenital crohn's disease; autoimmune bowel disease; enterocolitis; and immunodeficiency syndrome.

In certain embodiments, the present invention may be used to treat or ameliorate villous atrophy caused by diseases including, but not limited to: hepatitis; intestinal cancer; intestinal lymphoma; type1 diabetes mellitus; (ii) an allergic reaction; eosinophilic gastroenteritis (eosinophilic gastroenteritis); viral gastroenteritis; and autoimmune bowel disease.

In certain embodiments, the invention may be used to treat or ameliorate villous atrophy associated with celiac disease in the small intestine, including, but not limited to, villous atrophy of Marsh3a type (> 40 intraepithelial lymphocytes per 100 intestinal cells), Marsh3b type villous atrophy (> 40 intraepithelial lymphocytes per 100 intestinal cells; significant villous atrophy), Marsh3c type villous atrophy (absence or near absence of villous regions per 100 intestinal cells > 40 epithelial lymphocytes) (modified Marsh classification based on celiac disease and intestinal villous atrophy).

The present invention may also be used to treat or ameliorate symptoms associated with small bowel injury, including but not limited to malabsorption, diarrhea, nausea, vomiting, electrolyte imbalance, malabsorption, and dehydration.

Preparation of therapeutic composition for improving small intestine function

In another aspect, methods for preparing the therapeutic compositions of the present invention are provided. In one embodiment, the method comprises preparing a composition for promoting intestinal health in a subject having a loss or damage of small intestinal epithelial cells, wherein the composition comprises, consists essentially of, or consists of: an effective amount of one or more ingredients, wherein the ingredients are absorbed by the small intestine of a subject having a loss or injury of small intestine epithelial cells, wherein the total osmolality of the composition is from 230mosm to 280mosm, or any value therebetween (preferably from about 250mosm to 260mosm), wherein the pH of the composition is from about 7.1 to 7.9, or any value therebetween (preferably about 7.4), wherein the composition is formulated for enteral administration.

In one embodiment, the ingredient is selected from: free amino acids, dipeptides, monosaccharides, disaccharides, or combinations thereof, and optionally, electrolytes, vitamins, flavorants, and/or carriers.

In one embodiment, the invention provides a method of screening for nutrients or electrolytes for inclusion in a therapeutic composition of the invention by screening for nutrients or electrolytes that maintain or gain absorptive capacity following disruption of small intestinal epithelial cells in the villus and crypt region.

The screening methods of the invention can be used to identify therapeutic nutrients and/or electrolytes that can be used to treat or ameliorate diseases or conditions associated with the loss, destruction, or reduction of small intestine epithelial cells, particularly the loss, destruction, or reduction of villous cells, intestinal cells, and/or intestinal progenitor cells. In some specific embodiments, the methods can be used to design compositions and methods to meet the needs of a particular patient or patient population. In a particular embodiment, the composition of the invention may be used for treating or ameliorating small bowel injury following radiation therapy, chemotherapy, proton therapy or caused by acute or chronic inflammation of the small bowel.

In one embodiment, the screening method of the present invention comprises:

a) contacting the mucosal damaged small intestine epithelial tissue with a candidate nutrient or electrolyte;

b) determining the level of capacity of the small intestine epithelial tissue to absorb the nutrients or electrolytes;

c) comparing the level to a predetermined level (e.g., in normal tissue); and

d) a candidate nutrient or electrolyte is selected if its absorptive capacity is at least, e.g., 50%, 60%, 70%, 80%, or 90% of a predetermined level.

In one embodiment, the screening method of the present invention comprises:

a) administering a candidate nutrient or electrolyte to a subject having a small intestinal mucosal lesion via an enteral route;

b) determining the level of intestinal absorption capacity of the nutrient or electrolyte;

c) comparing the level to a predetermined level (as in a normal subject); and

d) a candidate nutrient or electrolyte is selected if its absorption level is at least, e.g., 50%, 60%, 70%, 80%, or 90% of a predetermined level.

Can be based on an index (including, for example, K)_m、V_maxAnd I_sc) To determine the level of absorbent capacity.

The predetermined reference value can be established by a person skilled in the art. For example, the predetermined reference value may be established by measuring the level of absorptive capacity of said nutrients or electrolytes in normal small intestine epithelial tissue without damage to the mucosa (e.g. villous cells, crypt cells, intestinal cells and intestinal progenitor cells). In another case, the predetermined reference value may be established by measuring the level of absorptive capacity of said nutrients or electrolytes in a normal population without damage to small intestinal epithelial cells (such as villi cells, crypt cells, intestinal cells and intestinal progenitor cells).

In another embodiment, the screening method of the invention comprises:

a) determining the function of small intestine tissue with mucosal lesion;

b) contacting the candidate nutrient or electrolyte with small intestine tissue;

c) determining the function of the small intestine tissue after contacting the small intestine tissue with the candidate nutrient or electrolyte; and

d) selecting the candidate nutrient or electrolyte if it improves small intestine function.

In another embodiment, the screening method of the invention comprises:

a) determining small intestine function of a subject with small intestine mucosa injury;

b) administering to the subject a candidate nutrient or electrolyte by enteral route;

c) determining small bowel function in the subject following administration of the candidate nutrient; and

d) selecting the candidate nutrient or electrolyte if it improves small intestine function.

In certain embodiments, small intestine function is increased if administration of the candidate nutrient or electrolyte decreases the paracellular permeability (permeability), enhancing small intestine barrier function. In addition, small bowel function is enhanced if enteral administration of the candidate nutrient or electrolyte prevents or treats diarrhea and/or prolongs survival.

In certain embodiments, the methods as described in the examples (particularly examples 15 to 17) can be used to select nutrients and electrolytes that enhance small intestine function in subjects with small intestine mucosal lesions.

Suitable candidate electrolytes include, for example, Na⁺、K⁺、HCO₃ ^-、Cl^-、Mg²⁺、Ca²⁺、Fe²⁺And/or Zn²⁺。

Suitable candidate nutrients include essential and nonessential amino acids selected from, for example, the following: isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, histidine, tyrosine, selenocysteine (selenocysteine), alanine, arginine, aspartic acid, cysteine, glycine, proline, serine, asparagine, and pyrrolysine. Suitable candidate nutrients may also include fatty acids, sugars (e.g., monosaccharides, disaccharides, and oligosaccharides), electrolytes, and vitamins.

Candidate nutrients may also include unnatural amino acids such as ornithine, citrulline, hydroxyproline, homoserine (homoserine), phenylglycine, taurine, tyrosine iodide, 2, 4-diaminobutyric acid, α -aminoisobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, γ -aminobutyric acid, -aminocaproic acid, 6-aminocaproic acid, 2-aminoisobutyric acid, 3-aminopropionic acid, norleucine (norleucine), norvaline (norvaline), sarcosine (sarcosine), homocitrulline (homocitrulline), cysteic acid (cysteine), τ -butylglycine, τ -butylalanine, phenylglycine, cyclohexylalanine, and β -alanine.

In another embodiment, the selection of nutrients and electrolytes also depends, at least in part, on the IR dose received by the subject, the radiation source, the body part being irradiated, and/or the time after irradiation; the type, dosage, and/or time after chemotherapy of the chemotherapeutic agent; and the dose of proton therapy received by the subject, and/or the time after proton therapy.

Screening assays of the invention can be performed using a combination of techniques well known in the art, including, but not limited to, ewings cell studies, cytology, immunohistochemistry, Western blotting, enzyme-linked immunosorbent assays (ELISA), Polymerase Chain Reaction (PCR), ion flux assays (ionfluxexperient), immunoprecipitation, immunofluorescence, radioimmunoassays, and immunocytochemistry.

In particular, the components may be selected based on their ability to be absorbed by the patient's small intestinal mucosa (as determined by in situ or isolated intestinal preparations), and the ability of the small intestine to absorb such components is measured using techniques such as ewings' chamber.

Formulation and administration

The present invention provides a therapeutic or pharmaceutical composition comprising a therapeutically effective amount of a composition of the invention and optionally a pharmaceutically acceptable carrier. Such a pharmaceutical carrier may be a sterile liquid, such as water. The therapeutic compositions may also contain excipients, adjuvants, flavoring agents, etc. that do not affect the health or function of the injured small intestine epithelium, particularly in the villous region and brush border. In one embodiment, the therapeutic composition and all of the ingredients contained therein are sterile.

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" of e.w. martin. Such compositions comprise a therapeutically effective amount of the therapeutic composition, together with a suitable amount of carrier to provide the appropriate form of administration to the patient. The formulation should be suitable for enteral modes of administration.

The invention also provides a pharmaceutical pack (kit) comprising one or more containers filled with one or more ingredients (e.g. a compound, carrier or pharmaceutical composition of the invention).

In one embodiment, the pharmaceutical pack or kit further comprises instructions for administration, e.g., for an effective therapeutic dose, and/or for a time of administration with reference to, e.g., time after exposure to radiation, chemotherapy, or proton therapy. In one embodiment, the therapeutic dose of the composition is determined by the extent of damage to the mucosa of the small intestine. For example, for a subject who has received or is about to receive radiation, the therapeutic dose of the composition is determined based on the radiation source, the body part being irradiated, and/or the time after radiation. For subjects who have received or are about to receive chemotherapy, the therapeutic dose of the composition is determined based on the type of chemotherapeutic agent, the dose of the chemotherapeutic agent, and/or the time after chemotherapy. For a subject who has received or will receive proton therapy, the therapeutic dose of the composition is determined based on the dose of proton therapy received by the subject and/or the time after proton therapy.

Materials and methods

Laboratory animal

To study the active HCO₃ ^-Secreting, 8 week old, unirradiated and irradiated male BALB/c mice were obtained from the national cancer institute. Mice were randomly grouped and used to deliver gamma-irradiation at 1.84 Gy/min¹³⁷A Cs source, which irradiates the abdomen with ShepherdMark-I according to the gastroenteritic acute radiation syndrome (GIARS) model. The radiation is given in single fraction (single fraction). During radiation treatment of pelvic or abdominal tumors, the GIARS model will achieve maximal radiation damage to intestinal tissue and mimic intestinal damage.

Checking short-circuit current (I) as a function of both time after irradiation and radiation dose rise_sc) To determine that significant I is produced_scEarliest time and smallest spoke required for changeAnd (4) shooting dose. These studies were approved by the university of rochester committee on animal care and use (university of rochester animalcareandusecommitate).

Ion flux study

After exsanguination, a jejunal segment was obtained by excluding 12cm of the distal small intestine adjacent to the cecum. The segment was washed and rinsed in ice cold ringer's solution before peeling the mucosa from the underlying muscle layer (Zhang, Ameen et al 2007). Placing the mucosa at an area of 0.30cm²Between the two halves of a yuss type celluloid (Lucite) chamber (P2304, physiologins, san diego, CA92128USA), electrical parameters were recorded using a voltage/current clamp arrangement (VCCMC-8, physiologins, san diego, CA92128USA) (vidyagar et al 2005; vidyagar et al 2004; Zhang et al 2007; vidyagar and Ramakrishna 2002). In a medium containing 8mM glutamine and using 95% oxygen (O)₂) With 5% carbon dioxide (CO)₂) The mixture was used to bathe both sides of (bathed) bowel preparation in aerated conventional ringer's solution (table 1).

TABLE 1 composition of the solution

Note that: values are in mM. Ionic solutions were used for ion exchange experiments. The pH of all solutions was 7.4.

In the absence of Cl^-In the solution of (1), using H₂SO₄The pH was adjusted to 7.4 and,

while in all other solutions, HCl was used for conditioning.

Abbreviations: UB, unbuffered solution

HCO₃ ^-Measurement of motion

Measurement of stripped empty Using a double-burette TIM856(radiometer analytical SAS, Villeurbannen, France)HCO in intestinal pieces₃ ^-Secretion (Vidyasagar et al 2005; Vidyasagar et al 2004; Zhang et al 2007). By adding 0.01. mu.l of 0.025M sulfuric acid (H)₂SO₄) The automated pump keeps the pH of the chamber solution constant. By adding HCO to the solution containing elevated concentrations₃ ^-Adding a known amount of H to the weak buffer solution of (2)₂SO₄To establish a standard-to-steady (standard-to-stat) pH calibration to generate a linear titration curve.

The bath side (serosal side) of the jejunal tissue was exposed to the buffer solution, while the luminal side was exposed to the HCO-free₃ ^-Low buffer solution (0.1-mh epes (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer, ph 7.4). HCO₃ ^-The secretion corresponds to the amount of acid added to the luminal solution to maintain the pH at 7.4 (or to stabilize the pH). All these experiments were performed under voltage clamp conditions. Free of HCO₃ ^-Using 100% O₂Aerated, containing HCO₃ ^-Using 95% O₂And 5% CO₂And (6) inflating. HCO₃ ^-The secretion is expressed as μ eq · h^-1·cm^-2(Vidyasagar et al 2005; Vidyasagar et al 2004; Zhang et al 2007).

After the tissue is mounted, bath HCO is initially absent₃ ^-When there is HCO₃ ^-Secreted but rapidly dropped to 0 within 20 to 30 minutes. If bath HCO is not present during titration₃ ^-Then HCO₃ ^-The secretion is still close to 0. HCO in bath solution₃ ^-Result in HCO₃ ^-Secretion rises rapidly, which remains constant for at least 2 hours (Vidyasagar et al 2005; Vidyasagar et al 2004; Zhang et al 2007). When the inhibitor was added to the mucosal solution, the pH was adjusted and allowed to equilibrate for 30 minutes until a steady rate of HCO was observed₃ ^-And (4) secreting. When inhibitors were added to the bath side (bathside), the tissues were also equilibrated for 30 minutes to reach a steady rate of HCO₃ ^-Secretion (see table 3).

All experiments were performed during the initial 1 hour steady state. Each experiment was performed using 1 tissue from each animal; for each tissue sample, only 1 experimental condition was studied. All experiments were repeated at least 4 times.

Immunohistochemistry

Frozen tissue sections from both unirradiated and irradiated mice were immunofluorescent stained using an anti-NBCe 1-A/B antibody (Bevensee, Schmitt et al 2000). NBCe1-A/B is a polyclonal antibody raised against the carboxy terminus common to both sodium bicarbonate cotransporters (NBCe1-A and NBCe 1-B). Immunostaining was completed on day 6 post irradiation. Washing the separated tissue in ice-cold conventional ringer's solution, embedding in frozen section embedding medium, and placing in liquid nitrogen; 6- μm sections were made in a cryostat (cryostat).

Western blot study

Jejunal lysates were prepared from mucosal debris from unirradiated and irradiated mice. Tissues were analyzed by Western blotting (Bevensee et al. 2000) for NKCC1(Santa CruzCA, USA), NBCe1-A/B (Mark Daniel Parker, CaseWesternReserve university medical school, Cleveland, OH) and cystic fibrosis transmembrane conductance regulator (CFTR) (Santa CruzCA, USA) protein expression.

At pH7.4, containing 25-mM HEPES, 10% glycerol and a mixture containing protease inhibitors (10mM iodoacetamide, 1mM phenylmethylsulfonyl fluoride and 2. mu.g.ml)^-1Leupeptin) in 1% triton x-100 (polyethylene glycol p- (1, 1, 3, 3-tetramethylbutyl) -phenyl ether) triacylglycerol hydrolase buffer (all chemicals were obtained from Sigma-aldrich co., USA unless otherwise specified). Protein concentration was determined using the Bradford assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the equivalent load of proteins from irradiated and non-irradiated samples. The NKCC1, NBCe1-A/B and CFTR proteins were detected using affinity purified polyclonal antibodies.

Statistics of

Results are expressed as mean ± standard error of mean. Statistical analysis was performed in 2 steps: 1) analysis of variance (ANOVA) (or its nonparametric equivalent Kruskal-Wallis) was used to test for gross differences; and 2) calculate Bonferroni corrected P values for all pairwise comparisons.

Examples

The following are examples illustrating methods for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise specified.

Example 1 irradiation elevated Net anion secretion

This example shows that irradiation increases net anion secretion and causes greater loss of villous epithelial cells compared to crypt cells. Specifically, small intestinal epithelial tissue was obtained from mice that received 12Gy irradiation, and anion secretion was examined using the ews chamber study. Transepithelial I measurements on days 1, 2, 3 and 4_sc(index of anion secretion).

As shown in FIG. 1A, transepithelial I was observed 48 hours after irradiation compared to non-IR exposed tissue and IR exposed tissue at 24 hours and 72 hours after irradiation_scMaximum rise (fig. 1A). At the end of 48 hours I_scThe significant increase in (b) indicates that irradiation disrupts the delicate balance between absorption and secretion. In contrast, I recorded at the end of 48 hours and 72 hours_scLower than non-IR mouse tissue.

Histopathological sections also showed greater loss of villous epithelial cells compared to crypt cells due to irradiation. While histopathological sections taken 48 hours ago showed minimal villus damage and little or no crypt cell damage, histopathological sections taken on days 3 and 4 showed extensive damage of crypt and villus cells. In particular, after 3 days, the villous cells become almost completely depleted. Loss of crypt cells was also observed as evidenced by failure to stimulate anion secretion in response to secretory stimuli at 72 and 96 hours post-IR (fig. 1A). At high doses of IR, there are insufficient crypt stem cells that mature and differentiate to form villous epithelial cells.

Fig. 1B shows that irradiation increases transepithelial conductance (fig. 1B). Transepithelial conductance (S) (combined transcellular and paracellular conductance) was measured by ews' S laboratory experiment.

According to ohm' S law (Ohmslaw)1/S ═ R, an increase in transepithelial conductance indicates a decrease in transepithelial resistance (TER or R). Mouse small intestine has low epithelial resistance. Resistance of the paracellular pathway to transcellular resistance^65-67Much lower. E.g. 1/TER ═ 1/R_{Trans-cellular})+(1/R_{Paracyte cell}) As shown, the paracellular and transcellular pathways are parallel; thus, the TER measured substantially reflects the paracellular resistance.

Example 2 irradiation induced short-circuit Current (I)_sc) Dose-dependent elevation of

This example reveals a dose-dependent increase in the short-circuit current caused by irradiation, indicating increased secretion of the electrogenic anions. Briefly, mice receiving 0, 1, 3, 5, 7, 9, or 12Gy irradiation were sacrificed on day 4. FIG. 2 shows I in mouse tissues irradiated at 3, 5 and 7Gy compared to those irradiated at 0Gy and 1Gy_scSignificantly elevated (＊ p < 0.001). reduced I was observed in mouse tissues irradiated at 9 and 12Gy compared to mice irradiated at 3, 5 and 7Gy_sc(＊＊ p < 0.01, FIG. 2.) irradiation with 1 to 3Gy causes I_scHighest elevation and minimal histopathological changes.

Furthermore, irradiation causes I_scVarying with time. For mice sacrificed on days 0, 1, 2, 3, 4, 5, 6 or 7, I was observed on days 5 and 6 after IR_scMaximum lift ofHigh (fig. 3A). To determine I as a function of time_scThe mice were irradiated at 3Gy and sacrificed on days 0, 1, 2, 3, 4, 5, 6, 7, 9, 11 and 14 to record the electrical parameter Kruskal-Wallis (P < 0.001). Post hoc analysis on day 6 after irradiation showed I_scIs increased to the maximum.

As shown in FIG. 3A, I recorded on days 1 and 2 after irradiation_scShowing very little statistical difference. However, I recorded at a post-irradiation time of > 2 days_scSignificant differences (＊ P < 0.01) were shown when compared to day 0, I recorded on days 4, 5, 6 and 7_scIn (c), a small significant difference was observed. I recorded on days 9 and 10 after irradiation_scThere was also no significant difference from that recorded on day 7 after irradiation (＊＊ P ═_Ns). Although, it is shown to receive 3Gy (4.8. + -. 0.5. mu. eq.h)^-1.cm^-2) In IR mice, day 6 later I_scA significant increase was shown, but it continued to remain at elevated levels on day 14 post irradiation and even over 2 years. FIG. 3A shows that I occurs on day 6 in mice irradiated with 3Gy_scIs increased to the maximum.

Observed after irradiation I_scThe increase is primarily due to a net increase in the secretion of the electrogenic anions. I is_scThere are 3 possible mechanisms for the increase: 1) increased secretion of electrogenic anions (e.g., Cl)^-And/or HCO₃ ^-) (ii) a 2) Increased power generation Na⁺Absorption; or 3) increased electrogenesis K⁺And (4) absorbing. Irradiation is unlikely to cause increased electrogenesis in mouse small intestine⁺And (5) an absorption process. Furthermore, because irradiation causes diarrhea, it causes K⁺Loss rather than K⁺Absorb, so that I_scIs unlikely to be due to increased K⁺And (4) absorbing.

Example 3-Na⁺And Cl^-Reduction of absorption

This example shows irradiation with Na⁺And Cl^-The absorption is reduced. As shown in Table 2, use²²EusRoom flux studies of Na-replacement revealed Na in mice that did not receive IR (0Gy)⁺Net absorption of (table 2) due to mucosal/serosal flux (J)_ms) Exceeds serosal/mucosal flux (J)_sm). Irradiation of J_msDecreases in a dose-dependent manner and results in a net reduced Na⁺Absorption (J)_NetNa). At doses of 7Gy and 9Gy, J_smFar exceeding J_msResulting in a net secretion. In addition, under high dose irradiation, the mouse fecal sample became loose or poorly formed, further demonstrating reduced absorption and increased secretion of electrolytes. Similarly, neat Cl^-Absorption also decreases with increasing IR dose. Neat Cl observed at 9Gy^-And (4) secreting. Cl^-The decrease in absorption is due to J_msCl^-And decreases.

TABLE 2Na⁺And Cl^-Unidirectional net flux (J)_Net＝J_ms-J_sm)

Example 4 irradiation induced increased paracellular permeability

This example shows that irradiation causes loss of the small intestinal lining mucosa (lingmucosa), resulting in impaired small intestinal barrier function. This increased small intestine permeability facilitates the entry of enterosymbiotic bacteria, peptides and toxins into the systemic compartment (systemic component), causing endotoxemia. As shown in fig. 4A, irradiation increased plasma endotoxin levels as measured by a limulus polyphemus reagent (tachypleus amebocytate) kit.

Irradiation also elevated Cl as indicated by the change in dilution potential determined in the ewings study^-And Na⁺(PCl/PNA) Permeability. The use of the dilution potential as an indicator of membrane permeability is based on the following principle. In particular, an intact semi-permeable membrane (e.g., small intestinal mucosa) is maintained through the use of a variety of membranesIonic strength bathes the mucosal and serosal side solutions to artificially create an electrochemical potential gradient. However, a leaky membrane (leakymembrane) that readily diffuses through the membrane has a reduced membrane electrochemical potential. Thus, the higher the permeability across the membrane, the lower the potential gradient. Cl of free-permeating membrane^-And Na⁺The relative permeability of (PCl/PNA) is 1, which indicates a complete loss of selectivity.

In the non-IR mice, membrane selectivity was retained, with Cl^-In contrast, Na⁺More readily permeate through the membrane. Irradiation lowers the membrane dilution potential. In particular, at 7Gy, Na⁺And Cl^-Become equally permeable through the membrane, indicating a significant loss of selectivity (fig. 4B). The increase in electrolyte permeability caused by irradiation is consistent with the increase in plasma endotoxin levels shown in figure 4A. Monitoring changes in membrane permeability can be used as a sensitive tool to monitor the increase in mucosal barrier function caused by a subject's diet irradiated orally.

Example 5 increase in levels of inflammatory mediators by irradiation

Inflammatory mediator levels were measured in IR-exposed and non-IR-exposed mice using the LUMINEX multiple bead array technique (multiplexararytechnique). As shown in FIG. 5, irradiation increased the production of IL1- β, TNF- α, and MIP- α (FIG. 5).

Example 6-reduction of anion secretion by irradiation is NKCC 1-dependent and CFTR-dependent

This example shows that anion secretion under irradiation is NKCC1 dependent and CFTR dependent. To determine NKCC1 vs. basal I_scTo the bath solution, 100 μ M bumetanide (Sigma-aldrich co., USA) was added. FIG. 3C shows bumetanide-suppressible current (5.5. + -. 0.5. mu. eq.h) in irradiated tissue^-1.cm^-2For 0.6 +/-0.1 mu eq.h^-1.cm^-2) However, this is not the case in 0Gy mice (1.6)±0.2μeq.h^-1.cm^-2For 0.9 +/-0.1 mu eq.h^-1.cm^-2). In addition, cAMP stimulation results in mice irradiated with 0Gy (1.6. + -. 0.2. mu. eq.h)^-1.cm^-2For 6.9 +/-0.6 mu eq.h^-1.cm^-2P < 0.001) and 3Gy irradiated mice (5.5. + -. 0.5. mu. eq.h)^-1.cm^-2For 7.3 +/-0.5 mu eq.h^-1.cm^-2P < 0.05) of_scAnd (4) rising.

Furthermore, in 3Gy, bumetanide resulted in forskolin (Sigma-Aldrich Co., USA) stimulated I_scDecrease (7.3 +/-0.5 mu eq.h)^-1.cm^-2For 0.4 +/-0.1 mu eq.h^-1.cm^-2) However, this is not the case in 0Gy (6.9. + -. 0.6. mu. eq.h)^{- 1}.cm^-2For 1.3 +/-0.2 mu eq.h^-1.cm^-2). This indicates a larger NKCC 1-dependent anion secretion without irradiation (P < 0.05).

The results also show that anion secretion under irradiation is CFTR dependent. To determine I_scWhether the bumetanide insensitive moiety of (a) occurs through apical membrane anion channels, was determined using the nonspecific anion channel blocker 5-nitro-2- (3-phenylpropylamino) -benzoic acid (Sigma-aldrich co., USA) (10 μ MNPPB) and the specific cystic fibrosis transmembrane conductance regulator (CFTR) blocker (100 μ M glibenclamide, Sigma-aldrich co., USA). By nonspecific anion channel blocker (NPPB) (0.1. + -. 0.01. mu. eq.h)^-1.cm^-2) And glibenclamide (0.1 + -0.01 μ eq.h)^-1.cm^-2) Eliminates bumetanide insensitivity I in 0Gy mice_sc. This indicates that anion secretion occurs through the anion channel or CFTR (fig. 3B).

Example 7 HCO induced by irradiation₃ ^-Reduction of secretion

Infectious diarrhea (such as cholera) results in feces rich in HCO₃ ^-Loss of fluid and metabolic acidosis. This example shows that in the case of infectious diarrheaConversely, IR induced increased Cl^-Secreted and reduced HCO₃ ^-And (4) secreting.

To determine Cl^-Contribution to net anion secretion by Cl^-Blockers of uptake into cells (blockers of Na-K-2Cl co-transport). Addition of 10 μ M bumetanide eliminated almost all IR-related I_scIt was shown that IR-induced anion secretion was mainly due to elevated Cl^-Secreted, and the secretion was NKCC 1-dependent (fig. 3A-C).

The pH stabilization experiments confirmed IR-induced HCO₃ ^-Secreted (table 3). When Na in solution (bath) of serosa bath⁺When displaced by the non-penetrating cation NMDG, HCO₃ ^-Abolishing secretion of (A), indicating HCO₃ ^-The substrate-side membrane transported to the cells is bath Na⁺Is dependent. Similar experiments were repeated at 5Gy in mice on day 6 post IR. In-bath Na⁺In the presence of HCO₃ ^-Secretion was significantly reduced.

Immunofluorescent staining of frozen tissue sections from both non-IR and IR-passed mice was performed using the NBCe1a/B antibody (FIGS. 6B-E). Specific staining with NBCe1a/b antibody indicated that NBCe1a/b was expressed in villous epithelial cells, but not in crypt cells. Immunostaining from IR mouse tissue showed that NBCe1a/b antibody was not recognized in villi or crypts. Tissues from mice irradiated with 3gy (ir) did not express NBCe 1-a/B-specific staining patterns in villi or crypts. At high doses of IR, HCO₃ ^-The decreased secretory function is due to the loss of villous epithelial cells. Monitoring Na⁺And HCO₃ ^-The change in secretion may be a sensitive tool to monitor the increase in mucosal barrier function caused by the subject's diet irradiated orally.

HCO under irradiation₃ ^-Secretion is NKCC1 dependent

To determine HCO₃ ^-Whether or not to contribute to anion secretion in the absence of bath Cl^-Under the conditions ofAnd (6) testing. Consider HCO₃ ^-I to help in stimulation of secondary irradiation or forskolin_scAnd (4) rising. The results show that HCO under irradiation₃ ^-Secretion of not bath Cl^-Dependent; thus, under irradiation, HCO₃ ^-Secretion does not involve Cl in the apical membrane^--HCO₃Exchange transporter (AE 1). In the absence of Cl^-In 3Gy irradiated mice (1.0. + -. 0.2. mu. eq.h)^-1.cm^-2vs0.3±0.1μeq.h^-1.cm^-2；P＝ns)I_scAnd forskolin-stimulated (1.7 + -0.2 μ eq.h)^-1.cm^-2For 0.3 +/-0.1 mu eq.h^-1.cm^-2；P＜0.001)I_scLower (fig. 3D). Forskolin stimulated I at 0Gy compared to 3Gy_scHigher (P < 0.001), indicating HCO induced by irradiation₃ ^-Secretion is reduced.

To determine whether NKCC1 mediates HCO under basal and forskolin stimulation conditions₃ ^-Exercise, bumetanide was added to both sides in the absence of Cl^-The bath side of the equilibrated tissue in solution. The results show that bumetanide does not inhibit the basal I_scElevated and forskolin stimulated I_sc(ii) is increased; lack of this inhibition indicates HCO at the substrate-side membrane₃ ^-Absorbed NKCC1 is independent of mechanism.

HCO under irradiation₃ ^-The secretion being luminal Cl^-Independent of

HCO in irradiated mice₃ ^-Direct measurement of secretion showed comparison to non-irradiated mice (0.8. + -. 0.2. mu. eq.h)^-1.cm^-2For 6.7 +/-0.2 mu eq.h^-1.cm^-2) Reduced HCO phase ratio₃ ^-And (4) secreting. By removal of intracavity Cl^-HCO in irradiated mice₃ ^-Secretion was unchanged (table 3). NPPB (0.2. + -. 0.01. mu. eq.h) in irradiated mice^-1.cm^-2) And glibenclamide (0.11. + -. 0.1. mu. eq.h)^{- 1}.cm^-2) Mucosal addition of (instead of DIDS) ended HCO₃ ^-And (4) secreting. Watch with a watch bodyMing HCO₃ ^-Secretion is mediated by anion channels (CFTR channels) rather than by Cl^--HCO₃ ^-Mediated by crossover (fig. 19B).

In contrast, HCO in unirradiated mice₃ ^-The secretion being luminal Cl^-Dependent and Cl^-Is not dependent. The transepithelial electrical measurement result shows that the electricity generation HCO is generated₃ ^-Secretion; however, this does not indicate HCO₃ ^-Secretion is channel-mediated and/or through electrically neutral Cl^--HCO₃ ^-And (4) exchanging.

In the absence of intracavity Cl^-To study Cl in unirradiated mice^--HCO₃ ^-And (4) exchanging. In the absence of inner Cl^-In solution of (2), HCO₃ ^-Lower secretion (4.5 +/-0.1 mu eq.h)^-1.cm^-2And P is less than 0.01). This indicates that basal HCO in unirradiated mice₃ ^-Secretion is in part luminal Cl^-Dependent, in part, on Cl^-Independent (table 3). Addition of 100. mu.M 4, 4-diisothiocyanato-2, 2' -stilbene disulfonic acid (DIDS) (Sigma-Aldrich Co., USA) partially inhibited HCO₃ ^-Secretion (P < 0.001), the inhibition being similar to that with luminal Cl^-Observed in the removal.

Forskolin stimulated luminal Cl^-Independent HCO₃ ^-Secretion of

Addition of forskolin to bath solutions showed a significant increase in basal HCO for 0Gy mice₃ ^-Secretion (P < 0.001) without lumenal Cl^-Change by removal (8.4. + -. 0.4. mu. eq.h)^-1.cm^-2For 8.7 +/-0.4 mu eq.h^-1.cm^-2(ii) a n-6). NPPB eliminates forskolin stimulated HCO₃ ^-Secretion (0.2. + -. 0.01. mu. eq.h)^-1.cm^-2(ii) a n ═ 6); this indicates that anion channels are in cAMP stimulated HCO₃ ^-A role in secretion.

cAMP stimulationHCO of (2)₃ ^-Secretion is NKCC1 independent

To determine cAMP stimulated HCO₃ ^-Whether the apical CFTR channel is required for secretion, glibenclamide is added to the luminal side. Glibenclamide inhibition (0.1 + -0.1 mu eq.h^-1.cm^-2)HCO₃ ^-Secretion (Table 3 and FIG. 19), indicating that cAMP not only inhibits neat HCO₃ ^-Secreted basic Cl^--HCO₃ ^-Exchange components and induce apical anion channel mediated HCO₃ ^-And (4) secreting. Forskolin stimulation of irradiated mice showed involvement with basal HCO₃ ^-Little increase in secretion (0.6. + -. 0.2. mu. eq.h)^-1.cm^-2For 0.78 +/-0.2 mu eq.h^-1.cm^-2). This also indicates the lumen Cl^-Independent HCO₃ ^-Secretion or inhibition of Cl^--HCO₃ ^-And (4) exchanging.

Transepithelial electrical measurements show that HCO₃ ^-The reduction in motion was also NKCC1 independent. Minimal HCO in irradiated mice under both basal and forskolin-stimulated conditions₃ ^-Secretion was not affected by the addition of bumetanide (table 3). Similarly, bumetanide did not alter forskolin-stimulated HCO in unirradiated mice₃ ^-Secretion of HCO indicating cAMP stimulation₃ ^-Secretion was NKCC1 independent (8.4. + -. 0.4. mu. eq.h)^-1.cm^-2For 8.6 +/-0.4 mu eq.h^-1.cm^-2) (Table 3 and FIG. 3B).

HCO₃ ^-The secretion is bath Cl^-Independent of

Required bath Cl^-For substrate side HCO₃ ^-The transport process of absorption is shown in fig. 3B. The results show that bumetanide does not alter cAMP stimulated HCO₃ ^-And (4) secreting. This indicates that, under irradiation, Cl^--HCO₃ ^-The exchange (AE2) transporter is inhibited. Bath Cl^-Can also inhibit HCO related to NKCC1 and AE2₃ ^-Absorption (table 3). Removal of Cl from bath solution^-Without changing HCO₃ ^-Secretion (6.7 +/-0.3 mu eq.h)^-1.cm^-2For 7.1 +/-0.6 mu eq.h^-1.cm^-2)。

HCO₃ ^-The secretion is bath Na⁺Dependent on

Na⁺Coupled substrate-side HCO₃ ^-The incoming transfer process is shown in fig. 3B. FIGS. 3C and 3D show that NKCC1 does not affect HCO₃ ^-And (4) secreting. Addition of 1mM 3-methylsulfonyl-4-piperidinobenzoylguanidine hydrochloride (HOE694) to the bath side by reaction with Cl^--HCO₃ ^-Exchange of coupled NHE1 to eliminate HCO₃ ^-And (4) absorbing. Couinlon, Scholz et al (1993) also describe that 1mM 3-methylsulfonyl-4-piperidinobenzoylguanidine hydrochloride (HOE694) inhibits Na⁺-H⁺Crossover (NHE 1).

HOE694 does not inhibit cAMP-stimulated HCO₃ ^-Secretion (8.4 +/-0.4 mu eq.h)^-1.cm^-2For 7.2 +/-0.9 mu eq.h^{- 1}.cm^-2). In unirradiated mice, bath Na was replaced by N-methyl-D-glucamine (NMDG)⁺HCO with elimination of forskolin stimulation₃ ^-Secretion (8.4 +/-0.4 mu eq.h)^-1cm^-2For 0.3 +/-0.01 mu eq.h^-1cm^-2) Which indicates Na⁺Coupled HCO₃ ^-Cotransport (NBC) (fig. 3E).

TABLE 3 HCO measured in the jejunum of non-irradiated (0Gy) and irradiated (3Gy) mice₃ ^-Secretion of

Note that: values represent mean ± SEMn ═ 6 tissues. P is less than 0.001,

comparison between 0Gy and 3Gy groups.Comparison between existing groups.

In bumetanide experiments in unirradiated mice, tissues were treated with 10mM forskolin.

Abbreviations: ns, no significance between groups; 4, 4-diisothiocyanato-2, 2' -stilbenedisulfonic acid, DIDS

For active HCO at the apical Membrane₃ ^-Secretion, requires its basolateral uptake. Involving HCO at the substrate-side membrane, directly or indirectly₃ ^-Four known switching mechanisms for motion are: 1) na (Na)⁺-K⁺-2Cl^-Co-transport as a possible HCO₃ ^-A transporter protein; 2) cl taken up into cells by NKCC1^-Through the substrate side Cl^--HCO₃ ^-Recycled by exchange (AE2), resulting in a net HCO at the substrate-side membrane₃ ^-Absorption; 3) na (Na)⁺-H⁺The exchange squeezes protons into the intracellular space, resulting in reduced intracellular HCO₃ ^-Concentration, which then stimulates the electrically neutral Cl at the tip^--HCO₃ ^-Exchanging; and 4) Na⁺Coupled HCO₃ ^-And (4) carrying out cooperative transport. These transporters may be electrically neutral or electrogenic, depending on Na per molecule⁺Transported HCO₃ ^-Number of molecules (fig. 3B).

In unirradiated mice, HCO₃ ^-Absorbing Na passing through the side surface of the substrate⁺Coupled cotransporter (NBCe 1-A/B). Apical output through Na⁺-H⁺Exchange bound electrically neutral Cl^--HCO₃ ^-Exchange and through CFTR (electrogenic anion secretion) occurs. Elevation of intracellular cAMP by addition of forskolin leads to increased Cl^-And HCO₃ ^-And simultaneously inhibit the electroneutral Na⁺And Cl^-Absorption (Na)⁺-H⁺Exchange with Cl^--HCO₃ ^-SwitchingCoupling). Cl^-Uptake occurs via NKCC1, HCO₃ ^-Absorption occurs through NBCe 1-A/B; cl^-And HCO₃ ^-The output of both occurs through the CFTR of the tip surface.

According to the present invention, it has been found that irradiation inhibits the electroneutral Na⁺And Cl^-And (4) absorbing. Irradiation also inhibited NBCe1-A/B, which inhibition resulted in HCO at the substrate-side film₃ ^-The absorption decreases and eventually it is exported at the top membrane. Thus, irradiation results in the production of electricity Cl^-Secreted, and electrically neutral and electrogenic HCO₃ ^-Selective inhibition of both secretions (fig. 19B).

Irradiation causes elevated NKCC-1 protein expression and reduced NBCe1-A/B expression in small intestine epithelial tissue. Irradiation also inhibited apical Cl^--HCO₃ ^-Exchange transporter (AE 1). HCO in radiation diarrhea₃ ^-The secretion is Na⁺Dependent, but the lumen Cl^-Independent and NKCC-1 independent. Cl under irradiation^-Transport involves the basal-lateral NKCC-1 transporter, not Cl^--HCO₃ ^-Exchange transporter (AE 1).

As shown in FIG. 19B, irradiation also alters electrolytes (e.g., HCO) in the gastrointestinal tract₃ ^-And Cl^-) And (4) transferring. Irradiated mice show predominantly Cl^-Minimal secretion and HCO₃ ^-And (4) secreting. Presume minimum HCO due to irradiation₃ ^-Secretion is caused by inhibition of HCO₃ ^-Caused by absorption. In contrast, active Cl is present in secretagogue (secretagogue) -induced diarrhea^-And HCO₃ ^-And (4) secreting.

Example 8 irradiation induced reduction of glucose uptake

This example shows that glucose uptake is dose-dependently reduced in IR-induced enteritis. In addition, the presence of unabsorbed glucose in the intestinal lumen mayResulting in osmotic diarrhea and also worsening of IR-related diarrheal conditions. FIG. 7A shows irradiation induced I_scDose-dependently decreased. FIG. 7B shows that irradiation increases K in glucose transport in a dose-dependent manner_m。

SGLT1 is a multifunctional transporter. SGLT1 maintains its function in infectious diarrhea (e.g., cholera). Conservation of SGLT1 function in infectious diarrhea has been used for Na⁺Oral rehydration therapy by absorption.

To investigate the function of SGLT-1 and its effect on glucose absorption after irradiation, the small intestinal mucosa of Swiss mice was obtained on day 6 after exposure with 0, 1, 3, 5 or 7 GyIR. Measurement of short-circuit current (I) for glucose stimulation in the Euler cell_sc) To study SGLT1 transport function. Survival studies were performed on TBI and 15.6-sub-TBI mice at 9 Gy.

Specifically, 8-week-old Balb/C mice from the National Institutes of Health (NIH) were subjected to 137C sub-total (sub-total) irradiation (sub-TBI) (one leg was protected from irradiation) and total-body (total-body) irradiation (TBI).

In animal survival studies, mice were divided into 2 groups: 9GyTBI and 15.6Gy sub-TBI. Control mice were treated with saline; other mice were treated with 5% glucose. Treatment was given on alternate days, 5 days before and up to 10 days after irradiation, using gavage during the experiment.

A multi-channel voltage/current clamp (physiologic instruments, san diego, CA) was used in the ews cell study. Sections of jejunum of mice for placement were bathed in regular modified ringer's solution and treated with 95% O₂And 5% CO₂Inflation for short circuit measurement I_sc. All mice were sacrificed 6 days after irradiation.

To study SGLT-1 kinetics, substrate (glucose) concentrations were started at 0.05mM and ended at 10 mM. Glucose was added at a rate starting at 0.05mM and progressing to 0.1mM, 0.5mM and 1 mM. The results were analyzed using Origin8 software (originlabcorp., Northhampton, MA). I is_scPlotted as Y-axis, glucose concentrationPlotted as the X-axis. The curve is fitted to the Hill's equation (Hill' section).

To prepare a jejunal whole cell lysate, mucosal debris from normal and irradiated mice were lysed in a triacylglycerol hydrolase buffer containing 25mM HEPES, 10% glycerol, 1% TritonX-100, and a protease inhibitor cocktail (10mM iodoacetamide, 1mM phenylmethylsulfonyl fluoride, and 2. mu.g/ml leupeptin, pH 7.4).

To prepare brush border membrane vesicle lysates, mucosal debris from normal and irradiated mice were homogenized in 2mM Tris-HCl (pH7.1)/50mM KCl/1MPMSF solution. The samples were centrifuged at 8000RPM and 13,000RPM using a centrifuge, respectively, and then homogenized using a tuberculin (turberculin) syringe (27G needle) and a TEFLON homogenizer. The samples were then centrifuged at 4,000RPM and then at 15,000 RPM. The samples were resuspended in conventional ringer's solution containing a protease inhibitor cocktail (10mM iodoacetamide, 1mM phenylmethylsulfonyl fluoride and 2. mu.g/ml leupeptin, pH 7.4).

Protein concentrations of SGLT-1 protein were analyzed by Western blotting for both jejunal whole cell lysates and brush border membrane vesicles. Protein equivalent loads from the irradiated samples and the control samples were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein was transferred to a polyvinylidene fluoride (PVDF) membrane, and the SGLT-1 protein was detected using affinity-purified polyclonal antibody.

As shown in fig. 8 to 14, the results demonstrate that: 1) irradiation reduces glucose-stimulated I in a dose-dependent manner_sc(ii) a 2) K for glucose at 0, 1, 3, 5 and 7Gy_mThe values are respectively (mM)0.38 plus or minus 0.04, 0.49 plus or minus 0.06, 1.76 plus or minus 0.16, 1.91 plus or minus 0.3 and 2.32 plus or minus 0.4; 3) at 0, 1, 3, 5 and 7Gy, V of glucose_maxThe values are 387.4 + -16.2, 306.6 + -16.4, 273.2 + -14.9, 212.9 + -9.14 and 188.1 + -9.12 respectively; 4) about 14 days after IR, K_mAnd V_maxThe value returns to the normal level; 5) inhibition of glucose uptake in the first 10 days after irradiation improved survival; 6) western blot analysis of SGLT-1 brush border membranes showed dose of SGLT-1 protein levels with IRAnd increased by increasing.

SGLT-1K_mThe increase in (A) indicates that the affinity of SGLT-1 for glucose is decreased due to irradiation. V_maxThe decrease in (b) indicates a loss of villous epithelial cells due to irradiation as also demonstrated by histopathological examination. As shown in Western blot analysis, an increase in protein levels in IR-treated mouse tissues indicates that the SGLT1 transporter is expressed, but is non-functional.

The results also demonstrate that oral glucose feeding causes malabsorption of glucose and electrolytes, which results in osmotic diarrhea, thus increasing IR-induced GI toxicity. In contrast, inhibition of glucose from oral feeding on the first 14 days after IR prevented or reduced the symptoms of diarrhea and improved overall survival.

Example 9 irradiation causes reduced Glutamine transport

Although glutamine is a non-essential amino acid, it is a major nutrient for intestinal cells and is present in high concentrations in plasma (26%) and skeletal muscle (75%). As the body's demand for glutamine increases, glutamine levels decrease in post-operative patients, trauma patients, or critically ill patients. Thus, glutamine is considered to be important for the proper functioning of the digestive system, the renal system, the immune system, and the nervous system.

This example shows that irradiation causes a dose-dependent reduction in glutamine transport into cells. At IR.gtoreq.7 Gy, glutamine becomes present primarily in the intestinal lumen, causing osmotic diarrhea. Saturation kinetics of the glutamine Transporter show K_mIs dose-dependently increased (fig. 15), indicating a decrease in the affinity of the glutamine transporter for glutamine.

Example 10 irradiation causes dose-dependent increase in lysine transport

Addition of lysine to the luminal side of the small intestine caused I_scAn increase, indicating electrotransport of lysine (figure 16). Tissues from non-IR mice showed K of 1.16. + -. 0.04mM_mWhile 3GyIR tissue has a K of 0.27. + -. 0.01mM_m. Unlike glucose and glutamine, the results show that irradiation increases the affinity of lysine transporters for lysine, and thus, increases lysine uptake.

Example 11 Effect of oral lysine feeding on mouse survival

This example shows that inhibition of non-absorbed nutrients from oral feeding while selective feeding of absorbed nutrients prevents or reduces diarrhea and improves survival after irradiation.

In the first series of experiments, glucose (10mMi/m, 5 days, then every other day) was administered orally to IR-transmice. The results show that glucose administration reduced overall survival (fig. 17B). In contrast, as gastric lavage, lysine (20 mg/mouse/day) was orally administered to IR-passed mice for 5 days, followed by every other day. Mice treated with lysine showed increased survival compared to the control group (fig. 17A). Thus, reducing or limiting oral intake of non-absorbed nutrients (e.g., glucose), as well as elevated oral intake of absorbed nutrients (e.g., lysine), can prolong the survival of irradiated patients.

Example 12 Change in ion Transporter expression levels by irradiation

This example illustrates the change in the level of transporter expression caused by irradiation.

Specifically, on day 6 post-irradiation, tissues were harvested for Western blotting. As shown in fig. 18, Western blot of ileal tissue revealed that irradiation of 1 to 5Gy resulted in elevated NKCC1 protein levels; on the other hand, the increase in NKCC1 expression was decreased in the tissues receiving 7GyIR compared to the tissues receiving 1 to 5GyIR (a).

After irradiation, the NBCe1-a/B protein level decreased significantly, even at doses as low as 1Gy (B). CFTR protein levels were significantly elevated in jejunal tissue after 3Gy irradiation compared to 0Gy jejunal tissue (C). The NBCe1-a/B specific antibody showed increased expression levels in the jejunum (D) compared to the duodenum, ileum and colon in non-irradiated mice. Jejunal tissue has the highest NBCe1-a/B protein levels (D) compared to the duodenum, ileum and colon. Changes in transporter levels correspond to changes in function observed after IR. The expression pattern of transporter protein after irradiation can be used to monitor the effectiveness of an orally irradiated diet, as compared to tissues without IR.

Example 13 variation of nutrient and electrolyte absorption Capacity in mice with Small intestinal mucosal Damage

Similar changes in intestinal absorptive capacity were observed in C57BL/6 mice treated enterally with radiation, chemotherapy, and with enteritis. The radiation model was constructed as described in examples 1 to 12.

In the chemotherapy model, all mice were injected with a single dose of 5-FU or cisplatin. In some mice, a second dose of 5-FU or cisplatin was injected 3 days after the first injection. After each injection, transepithelial Isc (indicator of net anion secretion) was measured using ews' chamber at the time points shown in figure 20. For each measurement a minimum of 32 tissues were examined.

The results show that net anion secretion was significantly elevated on day 3 in mice injected with single doses of cisplatin (FIG. 20B) or 5-FU (FIG. 20A). Also, mice injected with a second dose of chemotherapeutic agent showed significantly higher net increase in anion secretion than mice receiving a single dose.

In the crohn's disease model, mice were injected with anti-CD 3mAb (an acute inflammatory model that mimics the condition of crohn's disease). The net anion secretion (determined based on the paracellular conductance) and paracellular permeability of the small intestine are also significantly increased. Changes in nutrient and electrolyte absorption capacity were also observed.

Disease models with small intestinal mucosal lesions (i.e., radiation models, chemotherapy models, and crohn's disease models) were used to determine changes in the absorptive capacity of nutrients. Specifically, candidate nutrients were administered orally to control mice and mice that received irradiation, chemotherapy, and anti-CD 3mAb, respectively. In addition, compositions comprising various combinations of candidate nutrients are administered orally.

The candidate nutrient is selected from: lysine, histidine, valine, leucine, phenylalanine, cysteine, tyrosine, arginine, isoleucine, threonine, glycine, alanine, methionine, tryptophan, proline, serine, asparagine, glutamine, aspartic acid, glutamic acid, and glucose.

To determine the absorption capacity of each nutrient, bioelectrical measurements were performed using the ewings cell. The measurement comprises the following steps: a) sodium-coupled amino acid current (Isc) and conductance changes; b) changes in saturation kinetics and Isc for each nutrient after administration of each nutrient; c) electrolyte uptake studies using isotope flux studies in the presence and absence of specific candidate nutrients. The results show that the absorption capacity changes in a similar manner for all amino acids and glucose studied in the radiation model, the chemotherapy model and the crohn's disease model. In particular, the results show that oral administration of each of the amino acids selected from the following improves small intestine healing, reduces paracellular conductance (thereby improving small intestine mucosal barrier mechanisms), improves electrolyte absorption and/or improves animal survival: lysine, glycine, threonine, valine, tyrosine, aspartic acid, isoleucine, tryptophan, asparagine, and serine. The results also show that oral administration of glucose and/or glutamine damages the small intestinal mucosal barrier and has an adverse effect on mouse survival in radiation, chemotherapy, and crohn's disease models.

Example 14 improvement of Small bowel function in mice that have received chemotherapy

This example shows that the therapeutic compositions of the present invention improve the healing of the small intestine in mice that have received chemotherapy. Of all the chemotherapeutic drugs studied, 5-FU showed the greatest toxicity to the small intestine. Thus, 5-FU was used to characterize changes in electrolyte and nutrient transport in chemotherapeutic models.

NIHSwiss mice were injected with 5-FU. Intestinal tissue from mice was isolated 5 or 6 days after injection and studied in ewings chamber, exposed to ringer's solution or therapeutic compositions of the invention. The therapeutic composition comprises lysine, glycine, threonine, valine, tyrosine, aspartic acid, isoleucine, tryptophan, asparagine, and serine; water; and a therapeutically acceptable carrier, electrolyte and buffer. The therapeutic composition is weakly basic (ph 7.4). The therapeutic composition does not contain glucose, glutamine or methionine.

The results show that the therapeutic composition significantly improved the small bowel function in mice that had received 5-FU. In particular, the therapeutic compositions significantly reduced the pathological increase in transepithelial Isc (fig. 21A) and transepithelial conductance in the intestine of mice injected with 5-FU.

Example 15 determination of changes in GI function caused by irradiation

The main GI functions include: absorption of nutrients, electrolytes and water, which occurs in well-differentiated and mature villous epithelial cells. 80% of fluid and electrolyte absorption occurs in the small intestine. As shown herein, IR results in selective loss of villi and/or crypts depending on the IR dose, resulting in Na⁺、Cl^-And a reduction in nutrient absorption. This example illustrates the experimental design used to determine the changes in GI function caused by multiple doses of IR over time.

Method of producing a composite material

C57BL/6 mice (8 weeks old, male) from NCI were used. Physical observations, cytology, immunohistochemistry, Western analysis, plasma surrogate markers and functional studies were determined as specific indicators of IR-induced GI toxicity. Mice were randomly grouped and the abdomen irradiated with ShepherdMark-I using a Cs source that delivered IR to the abdomen at a dose rate of 1.84 Gy/min. Mice were subjected to 0, 1, 3, 5, 7 and 9Gy of IR. Changes in glucose and amino acid transport were measured on days 0, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25 and 30, with 10 mice per group. Plasma samples were collected prior to tissue harvest. Ileal and jejunal tissues were harvested for histopathology, Western blot, immunohistochemistry and ewings laboratory studies (for separate evaluations).

A) Determination of electrolyte (Na)⁺、Cl^-And HCO₃ ^-) Change of function of

This example illustrates the experimental design for determining the altered function of transporters associated with post-IR electrolyte absorption. Electrolyte transport functions were then correlated with plasma markers, cytology and physical observations (such as daily activity, body weight, stool formation and fecal occult blood). Cytological examination was performed using crypt assays, H & E staining, BrdU staining, immunohistochemistry and Western blot analysis.

First, Na was checked in the Euler's chamber⁺And Cl^-To assess electrolyte absorption after IR. Mice were sacrificed and examined for basal ion transport changes in both non-IR mice and mice treated with different doses of IR. In the regular epithelium Na⁺And Cl^-The absorption is electrically neutral.

In this example, the isotope (A), (B), (C), (²²Na and³⁶cl) displacement studies to determine the basic Na⁺And Cl^-And (6) moving. Briefly, will²²Na and³⁶cl is added to the mucosa or serosal side. At the end of every 30 minutes, 0.5ml samples were collected from the cold side. The unidirectional flux was calculated using standard formulas and expressed as μmol^-1.cm^-2. Calculating the Net flux (J)_Net) As a groupJ of weave pair_msFlux and J_smThe difference in flux. The experiment was performed under short circuit conditions.

In addition, HCO was measured using pH stabilization technique₃ ^-A change in secretion. As shown herein, IR induces HCO in the jejunum₃ ^-Secretion is reduced. HCO₃ ^-Secretion is critical for acid-base equilibrium and acid neutralization of the upper intestine^72-74. These experiments suggest HCO₃ ^-Possible mechanisms of secretion, and indicate 1) intracavity Cl-dependent HCO in normal and irradiated mice₃ ^-Secretion, and 2) luminal Cl^--independent HCO₃ ^-And (4) secreting. Bicarbonate secretion is expressed as follows:

wherein D2 and D1 are the difference between the total acid added between the two time points, 0.025 denotes the normality of the acid added, 2 is H₂SO₄60 represents the time in minutes to finally represent the secretion per hour. 1.13 represents the surface area of the tissue used in the ewings chamber at time t. HCO studied Using pH stabilization technique₃ ^-Secretion will complement transepithelial Na⁺And Cl^-And (6) measuring the flux.

Ion flux experiments, pH stabilization studies, and transepithelial electrical measurements may illustrate transport processes in non-IR mice and in trans-IR mice.

B) Determination of changes in nutrient absorption function caused by irradiation

Intestinal malabsorption of nutrients affects the nutritional status after IR. As described herein, selective absorption of nutrients occurs after IR. The presence of unabsorbed nutrients in the intestine causes osmotic diarrhea, which further complicates the damage caused by irradiation. This example illustrates the experimental design used to determine the nutrient absorbed from the intestine after IR.

Easily absorbable nutrients can be included in the therapeutic/dietary compositions of the present invention to examine the effect of various IR doses on glucose absorption over time.

Specifically, changes in glucose transport were determined in ewings chamber after IR. The time required for the glucose transporter to restore its normal function (levels without IR) was also investigated. The formulation (ORD) was obtained based on the ability of the mice to tolerate oral glucose. Glucose was subtracted by oral support protocol until glucose transport began to improve.

In addition, changes in amino acid (a.a) transport after IR were examined. Electrogenic amino acid transport is detectable in ews's chamber due to the net charge movement that occurs when an amino acid is transported. These transports were studied in the brush border membrane vesicle study (BBMV) because there was no charge movement associated with electrically neutral amino acids. Both electrogenic and charge neutral amino acids were studied in the BBMV for comparison between different experimental methods.

Specifically, the four major types of amino acid transport systems were studied by testing the absorption of the representative amino acids L-leucine (neutral amino acid), L-proline (imino acid (IMINOacid)), L-glutamic acid (acidic amino acid), and L-cysteine (sulfur-containing amino acid) in Brush Border Membrane Vesicles (BBMV) from non-IR and IR-passed mice.

Changes in electrogenic amino acid transport by IR

Amino acids are broadly classified as neutral, cationic and anionic because their transport properties are based primarily on charge (table 4). Electrogenic amino acid transport can be via B^0/+(neutral and cationic amino acids) or X^- _AGAnd occurs. By the presence or absence of luminal Na⁺Experiments were performed below to determine Na-coupled and Na-independent amino acid transport. In addition, use¹⁴C-labeled amino acids study charge-neutral amino acid transport in BBMV.

TABLE 4 amino acid transport System in Brush border membranes of the Small intestine

Preparation of BBMV to study amino acid transport and Western blot

Isolation of BBMV using magnesium precipitation method⁷⁵. Determination of the Total protein content of BBMV Using the Bradford method⁷⁶. The vesicles were stored in liquid nitrogen or at-80 ℃.

Evaluation of amino acid uptake by BBMV

At 25 ℃ using a mixture of Hopfer et al⁷⁵The described rapid filtration technique (slightly modified) performs amino acid uptake of BBMV. BBMV suspension (5. mu.l) was added to an incubation medium (45. mu.l) containing 1mmol/L of unlabelled amino acid, 25. mu. Ci/ml of radiolabeled substrate L- [ U-¹⁴C]Leucine, L- [ U-¹⁴C]Proline, L- [ U-¹⁴C]Glutamic acid or L-, [ solution of ] A³⁵S]Cysteine, 100mmol/l NaSCN or KSCN, 100mmol/l mannitol, 0.1mmol/l MgSO₄And 10mmol/l HEPES (pH 7.4). In the presence of Na⁺Gradient (using media containing NaSCN) and absence of Na⁺The time course of amino acid uptake was measured under conditions of a gradient (KSCN containing medium). At specific time intervals, the absorption process was terminated by adding 5ml of ice-cold stop solution (containing 150mmol/l KSCN and 10mmol/l Tris-HEPES (pH 7.4)). The suspension was immediately poured onto a pre-wetted microporous filter (millipore filter) which was washed 3 times with 3ml ice-cold stop solution and immersed in 5ml of scintillator Hisafe3 fluid (LKBProducts, Bromma, Sweden). The filters were then counted in a fluid scintillation counter. Nonspecific binding to the filter has been previously measured and subtracted from the total absorption. Results are expressed as picomoles of amino acid absorbed per mg of protein.

C) Determination of changes in paracellular permeability caused by IR

The following technique was used to determine the change in paracellular permeability. i) A diluting potential; ii) TEER; iii) penetration of large non-ionic solutes of different sizes; FITC-conjugated dextran and rhodamine B isothiocyanate-dextran (isothionate-dextran).

post-IR mitigation of dilution potential changes

Dilution potential measurements were used to determine Na using Nernsetz's equation⁺With Cl^-Change in the permeation ratio therebetween. The results of these experiments were compared between the group of non-IR mice and the IR-experienced mice. Results from the paracellular permeability and plasma endotoxin studies were correlated with electrophysiology and survival data.

Perfusing ringer's solution (containing different concentrations of Na) through the mucosa⁺) To induce a dilution potential, mannitol was used to adjust the total osmolarity to maintain an equimolar osmolarity between experiments. The contribution of other ions to the conductance was estimated to be less than 5% and therefore ignored. The transmembrane potential difference was measured using an AgCl-AgCl electrode and a multimeter (VCCMC8, physiologins instruments inc.). The dilution potential is corrected for junction potential (junction potential) variations (typically less than 1 mV). These experiments allowed the following formula to be used to calculate the chlorine and sodium conductance of the paracellular pathway.

Permeability (β) ═ permeabilityPCl/PNa(ii) a T310 (kelvin)

Non-ionic solute osmotic changes through the paracellular space after IR

Paracellular permeability to water-soluble uncharged solutes of various sizes was studied in small intestinal tissue mounted in the ews chamber using FITC-conjugated dextran and rhodamine B isothiocyanate-dextran (Sigma). These studies allow the determination of IR-induced changes in paracellular permeability.

The intercellular barrier formed by tight junctions (tightjunctions) is highly controlled and sensitive to size and ions. Thus, the intercellular barrier represents a semi-permeable diffusion barrier. Experiments were designed to determine the paracellular permeability under basal conditions in normal epithelium and epithelium exposed to radiation to water-soluble uncharged solutes of different sizes in ileal or jejunal tissues housed in ewings' chambers.

FITC-conjugated dextran and rhodamine B isothiocyanate-dextran (Sigma) at a concentration of 3mg/ml dissolved in ringer's solution were added to the mucosal side of the ews chamber and maintained at 37 ℃ for 60 minutes. The solution in the substrate side bath solution was sampled to quantify the fluorescently labeled dextran. FITC-dextran: exc485nm and Em: 544nm, rhodamine B isothiocyanate-dextran: exc520nm and Em590 nm. A standard curve was obtained from the ileum or jejunum tissue of mice mounted in the bathews chamber to examine any change in permeability over time. These values were then compared for tissues from IR-and non-IR mice.

D) Determination of irradiation effects

Tissues from mice sacrificed for ewings chamber and pH stabilization studies were used for H & E staining, BrdU, fecal formation, occult blood, body weight, immunohistochemistry and Western analysis. These results were then compared to changes in function of electrolytes and nutrients and changes in paracellular permeability in non-IR and IR-passed mice.

Pathological analysis by crypt assay, H & E, BrdU staining

a) Crypt assay/Microcolony survival assay

A cell killing model that speculates that clonogenic ('structure-rescue') cells in multicellular structures act according to poisson statistics (poissonnstatistics) is used to fit a target curve to the data. Structures are presumed to remain intact until on average less than 3 cells per structure survive; cell survival was exponential in the dose range analyzed (exponential); and the structure can be regenerated from one or more surviving cells (regrow). Each epithelial center is believed to represent the survival of one or more clonogenic stem cells capable of producing a regenerative crypt.

Mice were sacrificed 3.5 days after IR for crypt microcoloni assays. This interval is located at or near the peak of mitotic recovery in the post-IR crypts. It is used to study the acute effects of IR.

For the biological response to irradiation, D was calculated₀Value sum D₁₀The value is obtained. Studies have shown that although D₀Lack of statistically significant difference between values, but with respect to D₀The variance of (a) strongly depends on the number of mice and the interval per data point. By increasing the number of intervals greater than two and the number of mice greater than four, a reduction in the value of the coefficient of variation (-5%) cannot be obtained. Thus, the study was designed with 3 intervals per mouse and 6 mice per data point.

b) BrdU staining to detect post-IR mitotic activity

Mice were injected with BrdU (30mg/kg body weight) and animals were sacrificed at 12, 24, 48 or 72 hours while tissues were harvested for functional studies. BrdU injections were repeated every 24 hours and BrdU labeling studies were continued beyond 24 hours after their injections. After BrdU labeling, paraffin sections of mouse small intestine were prepared and stained with anti-BrdU antibody (Ab). Cells were scored for the entire crypt and villus unit. At least 60 crypts and corresponding villi were analyzed for each mouse. BrdU-labeled cells were normalized to the total cell number per crypt or villus. The resulting percentages are then plotted against induction time. These studies allow the determination of the rate of transformation of crypt progenitors into the post-mitotic villous compartment, a direct correlation with the rate of cell division in the crypts and the kinetics of migrating crypts⁷⁷。

Variation of body parameters after IR

Body weight, fecal formation and fecal occult blood in mice were studied to detect changes in the nutritional status of the animals after IR. For daily activity and signs of disease, all mice were observed once daily for diarrhea, lack of grooming (grooming), cocked hair (ruffledpain), reduced diet habits, lethargy, etc. and carefully recorded.

The findings from these studies were compared to plasma analysis of surrogate markers, pathology observations, Western blots, immunohistochemistry, and functional studies.

Western blot analysis for determining molecular changes in transport processes involved in electrolyte and nutrient transport

Changes in the activity of the following transporters involved directly or indirectly in electrolyte and nutrient uptake were examined. The transporter protein comprises: CFTR Activity (with Electricity generating Cl)^-Secretion associated), NHE3 activity (with Na)⁺Absorption related), NBCe1-A/B activity in villi (with HCO)₃ ^-Secretion-related), NKCC1 (Na)⁺、K⁺And Cl^-Basal side uptake into cells), SGLT-1 (glucose uptake), B⁰、B^0/+、b^0/+PAT (proton coupled electrotransport system) and X^- _AG(Table 2). These studies were compared to functional data without IR, with IR and with ORD.

Immunohistochemistry for detection of changes in expression patterns of transporter, crypt and villous cell markers

When sacrificed animals were used for functional studies and for the use of various transporters (CFTR, NHE3, NKCC, NBCe1-A/B, SGLT, B)⁰、B^0/+、b^0/+PAT1 and X^- _AG) Frozen sections were prepared for immunostaining with specific antibodies. In addition, the pattern of cell surface marker expression was examined to provide insight into the ratio of crypts to villous cells. These studies allow the determination of altered expression patterns of IR or ORD treated transporters.

E) Identification of surrogate markers for irradiation

While several studies have attempted to identify surrogate markers to determine the radiation dose and time from the start of irradiation to determine the onset of GI toxicity, these studies have been largely unsuccessful. This example illustrates the experimental design that allows identification of surrogate markers to predict the onset of GI toxicity, demonstrating that it can also be used in situations involving multiple organs.

Specifically, plasma was collected when animals were sacrificed for functional assessment (ewings chamber). Mice were sacrificed on days 1, 2, 3, 6, or 9 after exposure to IR doses of 0, 1, 3, 5, 7, or 9 Gy. To identify surrogate markers, intestinal peptides, cytokines and endotoxins were studied.

Plasma assay for endotoxin

Plasma endotoxin levels were measured. Changes in plasma endotoxin levels were correlated with changes in paracellular permeability, plasma gut peptide levels, disease and survival.

Plasma analysis of cytokines

Changes in plasma cytokine levels were examined in IR-and non-IR mice using the LUMINEX multiple bead array technique.

Plasma analysis of intestinal peptide

Gut-specific peptides were studied, including insulin, glucagon, secretin (secretin), cholecystokinin (cholecystokinin), citrulline (citrullin), somatostatin, peptide YY, ghrelin (ghrelin), NPY and GLP-2. All these intestinal peptide kits were purchased from PhoenixPharmaceuticals, Inc (CA, USA). The experiments were performed according to the manufacturer's instructions.

Statistical analysis

Functional differences between normal and IR tissues were compared. Statistical significance was calculated using analysis of variance (ANOVA). The data from these measurements were compared. The correlation coefficient (R) is analyzed to determine the best functional label. Version 9.1 of the SAS System with Window for all statistical analyses2002-. If violation occursWith the distribution assumptions associated with a particular statistical step, appropriate conversion or non-parametric alternatives are used. Receiver Operating Characteristic (ROC) curves were constructed and the area under the ROC curve (AUC) in various functional tests was compared using the non-reference method of Delong et al (1988). The overall class 1error rate (family-wiseType1 error) was controlled to 0.05 using Tukey's method for multiple comparisons. Pearson's correlation coefficient and correlation p-value with 95% confidence interval are reported.

Example 16-development of an ideal oral regimen for the treatment of IR-induced gastrointestinal injury

This example illustrates the experimental design for developing oral therapeutic compositions for treating or ameliorating radiation-induced GI toxicity. It also determines when an Oral Rehydration Diet (ORD) should begin and how long the composition should be administered at least after exposure to multiple doses of IR. The time required to administer ORD depends on K_mThe time required to return to the base level.

Method of producing a composite material

C57BL/6 mice (8 weeks old, male) from NCI were used. To determine the affinity of the transporter, saturation kinetics were calculated by using elevated concentrations of each nutrient. Preliminary studies have shown that some amino acids have increased absorption, while some show reduced absorption, where K after IR_mAnd V_maxA change occurs. Addition of elevated concentrations of amino acids to the ileum or jejunum (evaluated separately) caused I_scAnd (4) rising. Known concentrations of amino acids are given to I_scThe mapping allows the saturation kinetics to be determined. Amino acids selectively absorbed by gastric lavage following IR administration increased survival of mice. Determination of K for Nutrients in 0, 1, 3, 5, 7 or 9Gy in day 0, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25 and 30 IR mice_mAnd V_maxEach group had 10 mice.

A) K for developing essential amino acids and glucose for an ideal Oral Radiation Diet (ORD)_mAnd V_maxIs determined

As described herein, irradiation causes changes in the transport kinetics of nutrients, indicating altered affinity for each transporter. The affinity for the glucose transporter determined using this technique showed a significant drop and required about two weeks to return to basal levels. The presence of unabsorbed glucose and nutrients in the intestinal lumen is known to cause diarrhea. Determining K of nutrients in mice exposed to different doses of IR and tracked for up to 30 days after IR_mAnd V_max. These studies can be used to formulate an ORD based on its absorption pattern over time and radiation dose. Furthermore, IR post shows that absorption enhancing nutrients can be used as an alternative energy source for the system. Thus, the formulation (ORD) can be used in survival studies.

K for glucose transport in post-IR ewings chamber_mAnd V_maxVariations in

Glucose transport was studied. In particular, K of glucose was investigated_mAnd V_max. Increasing concentrations of glucose were added to the luminal side in the ussing laboratory experiment and I was recorded_scIs increased. Glucose was rejected from the oral subtraction protocol until glucose transport began to improve. The formulation is based on the ability of mice to tolerate oral glucose.

K for post-IR amino acid (a.a) transport_mAnd V_maxVariations in

By determining K for each amino acid_mAnd V_maxThe kinetic pattern of amino acids based on IR dose and time after IR was studied. Kinetic indices of the electrogenic amino acids were determined in the ewings chamber set up as described above. Briefly, increasing the concentration of amino acid added to the lumen fluid results in increased I_scIn response, saturation occurred at a specific amino acid concentration. Calculating K from the saturation Curve_mAnd V_max。

BBMV was used to study electrically neutral amino acids. Amino acid uptake by BBMV was carried out in the presence of different concentrations of substrate (0.025mmol/l to 7mmol/l) at a fixed transit time of 3s (19). Each assay was performed in triplicate using BBMV pool (n-12) for each experimental group. Maximum velocity (V)_max) Expressed as picomoles of substrate per milligram of protein in 3 seconds, and transport affinity constant (K)_m) Expressed as millimoles per liter.

Optimizing ORD treatment to reduce GI toxicity and improve survival

To optimize the ORD regimen by selecting the appropriate dose, frequency and interval of administration, the effect of ORD on survival for 7 days, fecal formation, occult blood and body weight were analyzed at lethal dose (15.6Gy ═ 1.2 × LD_50/7Value) before 3 hours after IR, the dose range of IR is defined by K for each amino acid or glucose_mA value is determined. According to the recommended daily amounts of glucose and essential amino acids currently used in adults, from K_mTo calculate the concentration of glucose or amino acids for gastric lavage. Dose conversion from human to mouse is based on K_mFactor(s)⁷⁸. Thus, K_mThere is an inverse correlation with the daily dosage of nutrients. If IR increases K_m(indicating a decrease in affinity for transporters), the daily dose of each nutrient is proportionally decreased. Two additional doses of ORD were formulated, i.e., higher and lower than the calculated dose by a factor of 3. The optimal ORD dose was determined from survival studies.

Gastric lavage was repeated once daily for 7 days. The dose frequency and interval of the ORD gastric lavage were varied based on the results of the survival study. GI toxicity peaks from day 2 to 3 after IR and then gradually recovers by day 7 if ORD is effective. Mice were observed daily to 7 days post IR to monitor their survival.

All mice receiving the regular diet died or were sacrificed (moribund; defined as a combination of 20% weight loss, no longer able to comb, reduced activity, and reduced curiosity) within 7 days after IR. If mice that received ORD treatment were protected from IR-induced lethality, survival experiments were repeated with an additional 10 mice/group at the same treatment to ensure reproducibility of results. Survival data were analyzed by Fisher's exact test (exact).

The sample size for each group of 10 animals ensured a high enough efficacy (power) (> 80%) to detect survival differences of close to 0% for the vehicle group versus 60% or higher for each interference (pairwise comparison at adjusted alpha levels of 0.017 ≈ 0.05/3) to ensure an overall alpha level of 5%. In events where no statistically significant differences were observed or only partial remission was achieved by ORD processing, the protocol of the new cycle described previously was optimized to ensure maximum efficacy of remission against IR-induced lethality. After selecting the optimal dose, it was evaluated whether more frequent (twice a day) ORD gastric lavage was required to achieve greater radiation reduction and more rapid crypt recovery.

Window to determine the effectiveness of DMF and ORD on post-IR treatment

Dose Modification Factor (DMF) is one of the most important parameters for measuring the effectiveness of a radiation moderator, and is defined asWherein T is ORD treatment group, C is control group on regular diet⁵¹. To determine the efficacy of ORD treatment in reducing IR-induced lethality, groups averaging 10C 57BL/6 mice (10 to 20 mice/group varied according to IR dose) were treated with vehicle or ORD using the optimal protocol defined by previous experiments. Vehicle-treated mice were exposed to 11Gy to 13GyIR, using increments of 0.5Gy to 1 Gy. Survival of these mice was recorded at an observation period of 7 days after IR. Mice were euthanized at the end of the observation period or when they became necropsied.

The small intestine and plasma were collected after euthanasia. Blood samples were used for intestinal peptide analysis, while small intestine tissue samples were used to study IR-induced intestinal injury. LD_50/7Values are a good indicator of IR-induced GI toxicity.

Vehicle treated mice based on our pre-laboratory observationsMouse LD_50/7Close to 13 Gy. The ORD treated groups were exposed to IR ranging from 14.5 to 16.5GyIR (increments of 0.5 to 1 Gy) as observed and examined by the methods described above for vehicle treated mice. If a large number of mice survived in the ORD treated group (even after exposure to 16.5 Gy), the mice were given higher IR doses in subsequent studies. Calculating the LD from the survival curves of ORD-treated animals_50/7The value, then the DMF of the ORD is calculated. ORD-treated mouse LD_50/7DMF was greater than 1.2.

To determine how long to give ORD treatment after IR, group 5 animals were given ORD at 0, 1, 3, 5, 7, 9, 12 and 24 hours post-IR, followed by scheduled ORD treatment, observed for 7 days, concurrently with positive control (3 hours post-IR treatment) and negative control (saline vehicle). The survival of the animals was compared according to the survival at the 7 day time point.

In this model, several logistic-regression models (outcome variables, death/survival at 7 days) and multiple time trends of eight groups of applied ORDs after IR were considered. Both linear models (most likely to reduce survival because of the increased delay in treatment) and non-linear models (exponential decrease in survival) were considered.

Comparison of post-IR 3 hour administration to vehicle was performed in a pairwise fashion using Fisher's exact test. Paired comparisons (3 hours after IR, ORD and vehicle for different time groups) were present to maintain individual assay alpha levels at 0.005(≈ 0.05/10).

a) Survival rate

One of the main indicators for the treatment effect of an ORD is to determine survival. Two recordings were made each day and survival curves were generated.

b) Daily activity or signs of disease

All mice were observed once daily for signs of illness such as diarrhea, lack of grooming, cocked hair, reduced diet habit, lethargy, etc. and carefully recorded.

c) Body weight, fecal formation and occult blood

To determine whether or not the ORD can reverse some of the effects of IR-induced GI toxicity, the colon will be removed at the time of sacrifice of those animals for the functional studies described herein, mapped against the stool and analyzed for stool occult blood. These studies allow for determining whether demulcents are able to maintain the integrity of the GI mucosa and its function as seen by the naked eye.

d) Immunohistochemistry

Sections from H & E staining of the jejunum or ileum were used to analyze inflammatory cell infiltration in the lamina propria (laminapropria). The frequency of distribution of lymphoid follicles was carefully determined.

The optimal dose, start time and ORD regimen for acute GI toxicity were determined sequentially. Mice were treated with different dose formulations of ORD after IR exposure. The optimal dose was determined in a logistic regression model by determining survival (yes vs no) over 7 days as the response variable and dose level as the explanatory variable. Due to the uncertainty of the dose response curve, several plausible dose response models were proposed. After the dose response model is determined, the Minimum Effective Dose (MED) is calculated. The start time and optimal duration of treatment were answered by equivalent tests using the variance in the ANOVA model and estimated mean response.

Example 17 determination of functional improvement of GI function

In this example, electrophysiological experiments were performed to determine how the ORD helped restore the IR damaged intestinal mucosa to absorb electrolytes and nutrients. Functional changes were correlated with plasma surrogate markers, cytology and physical observations (such as daily activity, body weight and stool formation). Fecal occult blood, cytology such as crypt assays, H & E staining, BrdU staining, immunohistochemistry and Western blot analysis. These studies allow to determine the protective effect of the ORD on GI function at the molecular, cellular and functional level.

Method of producing a composite material

C57BL/6 mice (8 weeks old, male) from NCI were used. Functional studies, physical observations, cytology, immunohistochemistry, Western analysis were performed, and plasma surrogate markers were used as specific indicators of IR-induced GI toxicity. Mice were randomly grouped and the abdomen irradiated with ShepherdMark-I using a Cs source delivering IR at a dose rate of 1.84 Gy/min. Mice were irradiated with 1, 3, 5, 7, and 9Gy, and then given ORD. Mice were treated with ORD. Mice were sacrificed on day 6 and tissues used for functional studies, histopathology, Western blotting, and immunohistochemistry.

A) Correlation of the role of ORD with functional improvement in electrolyte and nutrient absorption

The treatment effect was evaluated using a series of indicators: 1) mice were weighed daily and observed closely for any signs of disease; 2) blood samples and physical parameters were analyzed when animals were sacrificed for functional studies (electrolyte and nutrient uptake), crypt assay, immunohistochemistry and western blot analysis. Blood samples were used to measure plasma endotoxin (an indicator of intestinal barrier dysfunction), cytokines, intestinal peptides (insulin, glucagon, secretin, cholecystokinin, citrulline, somatostatin, peptide YY, ghrelin, NPY and GLP2), citrulline, glucose and insulin.

Na in Yousi laboratory study⁺And Cl^-Determination of transepithelial flux of

To study the functional improvement of the ORD, jejunal and ileal slices (for separate evaluation) from mice were placed in ewings chamber and experiments were performed as described in example 15. Comparison of Na in groups of mice treated with IR, IR and ORD without IR⁺And Cl^-And (4) absorbing.

Determination of pfHCO Using pH stabilization techniques₃ ^-Secretion of

The experiment was performed as described in example 15. ORD-treated HCO₃ ^-Recovery of secretion indicates improved function. Comparison of HCO in groups of mice treated with IR, IR and ORD without IR₃ ^-And (4) secreting.

Determination of nutrient absorption in ewings chamber and vesicle study

The uptake of glucose, electrogenic amino acids and electrically neutral amino acids was determined as described in example 15. The results of these studies were compared in groups of mice treated with no IR, IR and ORD.

Determination of the modified paracellular permeability change after IR

Decreased paracellular permeability upon treatment with ORD indicates improved epithelial integrity. These changes would indicate a concomitant improvement in plasma endotoxin levels.

ORD effects were correlated with crypt assays, H & E staining, BrdU, stool formation, occult blood, body weight, immunohistochemistry and Western analysis

The study was similar to that described in example 15, and the results of the groups of mice treated without IR, with IR and with ORD were compared.

Histopathological analysis to determine anatomical improvement

Samples were processed for H & E staining and pathology analysis, including crypt determination, BrdU staining, as shown in example 15. Briefly, tissues were fixed in formalin, processed in paraffin blocks, and stained with H & E.

Immunohistochemistry to detect changes in expression profiles (expressionpatterns) of transporter, crypt and villus cell markers

The method adopts the method of the treatment of various transporters (NHE3, NBCe1-A/B, SGLT, B)^0/+、b^0/+、X^- _AG) And cell surface markers (Lgr5, EphB2, and EphB3), and the harvested tissue was used for immunostaining. The process is similar to that described in example 15. These studies will help determine the extent of villus and crypt cell formation after treatment with the ORD.

Western blot analysis to study molecular changes in transport processes involved in electrolyte and nutrient transport

The process is similar to that described in example 15. CFTR activity (with electrogenic Cl) will be examined^-Secretion associated), NHE3 activity (with Na)⁺Absorption related), NBCe1-A/B activity in villi (with HCO)₃ ^-Secretion associated), SGLT-1, B^0/+、b^0/+Or X^- _AG。

Plasma analysis with surrogate markers correlated with the effects of ORD

Preliminary studies have shown changes in intestinal peptide in mice after IR. Changes in the levels of surrogate markers relative to basal levels were examined and the results would indicate systemic improvement upon treatment with ORD.

Statistical analysis

The mean and standard deviation of the raw data are calculated using a graphical technique such as bar graph. The primary method used to compare the two groups (treatment vs vehicle) employed a mixed-action model (linear or non-linear) based on longitudinal data.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The terms "a," "an," and "the" are used in the context of describing the invention to mean one or more than one unless otherwise indicated herein or clearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise indicated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a particular factor or measurement can be understood to also provide a corresponding approximate measurement, modified by "about" where appropriate).

The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any element as essential to the practice of the invention unless explicitly stated.

The description herein of any aspect or embodiment of the invention using terms such as "comprising," "having," "including," or "containing" to refer to an element is intended to provide support for a similar aspect or embodiment of the invention that "consists of," "consists essentially of," or "comprises" the particular element, unless the context clearly dictates otherwise, or clearly contradicts by context, (e.g., a composition described herein comprising the particular element should be understood to also describe a composition consisting of that element, unless the context clearly dictates otherwise, or clearly contradicts by context).

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

The following corresponds to the original claims in the parent application, which are now incorporated as part of the specification:

1. a sterile therapeutic composition for improving small intestine health, in particular in the case of villous atrophy, wherein the composition is formulated for enteral administration and comprises lysine, glycine, threonine, valine, and tyrosine, as free amino acids; and water; and wherein the total osmolality of the composition is from 230mosm to 280 mosm.

2. The therapeutic composition of item 1, wherein the composition does not comprise one or more ingredients selected from the group consisting of glucose, glutamine, and methionine, or, if such ingredients are present, glucose is present at a concentration of less than 1g/l, glutamine is present at a concentration of less than 300mg/l, and methionine is present at a concentration of less than 300 mg/l.

3. The therapeutic composition of item 2, further comprising aspartic acid, isoleucine, tryptophan, asparagine, and/or serine.

4. The therapeutic composition of claim 1, wherein the composition does not comprise glucose, glutamine, or methionine.

5. The composition of item 1, wherein the composition further comprises electrolytes, vitamins, minerals, and/or flavoring agents.

6. A method for treating a subject with villous atrophy, wherein the method comprises administering to the subject the composition of item 1 by enteral administration.

7. The method of item 6, wherein the subject is a human.

8. The method of item 6, wherein the total number of small intestinal epithelial cells in the villus region of the subject is reduced by at least 10% compared to normal.

9. The method of item 6, wherein the height of villi in the small intestine of the subject is lost at least 10% compared to normal.

10. The method of item 6, wherein the composition does not comprise one or more ingredients selected from the group consisting of glucose, glutamine, and methionine, or, if one or more of these ingredients are present, glucose is present at a concentration of less than 1g/l, glutamine is present at a concentration of less than 300mg/l, and methionine is present at a concentration of less than 300 mg/l.

11. The method of item 6, wherein the composition further comprises aspartic acid, isoleucine, tryptophan, asparagine, and/or serine.

12. The method of item 6, wherein the composition does not comprise one or more ingredients selected from glucose, glutamine, or methionine.

13. The method of item 5, wherein the villous atrophy is caused by disease, radiation, chemotherapy, proton therapy, abdominal surgery, and/or cytotoxic agents.

14. The method of item 13, wherein the composition is administered for a period of 1 to 14 days after the subject receives radiation, chemotherapy, proton therapy, or a cytotoxic agent.

15. The method of item 13, for treating radiation enteritis.

16. The method according to item 13, for treating small intestine injury caused by chemotherapy or a cytotoxic agent selected from: cisplatin, 5-fluorouracil (5-FU), hydroxyurea, etoposide, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine, methotrexate, steroids, or combinations thereof.

17. The method of item 13, for treating Inflammatory Bowel Disease (IBD), ulcerative colitis, duodenal ulcers, or crohn's disease.

18. A method for preparing a sterile therapeutic composition for promoting intestinal health in a subject with villous atrophy, wherein the method comprises combining one or more ingredients selected from the group consisting of: free amino acids, dipeptides, monosaccharides, disaccharides, or combinations thereof,

wherein it has been determined that each component maintains at least 50% of its absorptive capacity in the small intestine of a subject with villous atrophy as compared with normal, and

wherein the composition does not comprise glucose at a concentration above 1g/l or glutamine at a concentration above 300 mg/l.

19. A composition produced by the method of item 18.

20. The composition of item 19, comprising water and having an osmolality of from 230mosm to 280 mosm.

21. The composition of item 19, which is in the form of a powder.

22. A kit comprising the composition of item 1 or a powder that when combined with water forms the composition of item 1, wherein the kit further comprises instructions for administering the composition to a patient having or expected to have villous atrophy.

Reference to the literature

1.Wolfe，B.M.，etal.Experiencewithhomeparenteralnutrition，AmJSurg146，7-14(1983).

2.Beer，W.H.，Fan，A.&Halsted，C.H.Clinicalandnutritionalimplicationsofradiationenteritis.AmJClinNutr41，85-91(1985).

3.Donaldson，S.S.Nutritionalconsequencesofradiotherapy.CancerRes37，2407-2413(1977).

4.Theis，V.S.，Sripadam，R.，Ramani，V.&Lal，S.ChronicRadiationEnteritis.ClinOncol(RCollRadiol)(2009).

5.Gunnlaugsson，A.，etal.Dose-volumerelationshipsbetweenenteritisandirradiatedbowelvolumesduring5-fluorouracilandoxaliplatinbasedchemoradiotherapyinlocallyadvancedrectalcancer.ActaOncol46，937-944(2007).

6.Dickerson，J.W.Nutritioninthecancerpatient：areview.JRSocMed77，309-315(1984).

7.Bounous，G.，etal.Dietaryprotectionduringradiationtherapy.Strahlentherapie149，476-483(1975).

8.Alpers，D.H.Glutamine：dothedatasupportthecauseforglutaminesupplementationinhumans？Gastroenterology130，S106-116(2006).

9.Hauer-Jensen，M.，Wang，J.，Boerma，M.，Fu，Q.&Denham，J.W.Radiationdamagetothegastrointestinaltract：mechanisms，diagnosis，andmanagement.CurrOpinSupportPalliatCare1，23-29(2007).

10.Tankel，H.I.，Clark，D.H.&Lee，F.D.Radiationenteritiswithmalabsorption.Gut6，560-569(1965).

11.Yeoh，E.K.，etal.Gastrointestinalfunctioninchronicradiationenteritis--effectsofloperamide-N-oxide.Gut34，476-482(1993).

12.Gavazzi，C.，Bhoori，S.，Lovullo，S.，Cozzi，G.&Mariani，L.Roleofhomeparenteralnutritioninchronicradiationenteritis.AmJGastroenterol101，374-379(2006).

13.Traber，P.G.，Yu，L.，Wu，G.D.&Judge，T.A.Sucrase-isomaltasegeneexpressionalongcrypt-villousaxisofhumansmallintestineisregulatedatlevelofmRNAabundance.AmJPhysiol262，G123-130(1992).

14.Minhas，B.S.&Field，M.Localizationofbicarbonatetransportalongthecrypt-villousaxisinrabbitileum.Gastroenterology106，1562-1567.(1994).

15.Welsh，M.J.，Smith，P.L.，Fromm，M.&Frizzell，R.A.Cryptsarethesiteofintestinalfluidandelectrolytesecretion.Science218，1219-1221.(1982).

16.Rijke，R.P.，vanderMeer-Fieggen，W.&Galjaard，H.Effectofvillouslengthoncellproliferationandmigrationinsmallintestinalepithelium.CellTissueKinet7，577-586(1974).

17.Wright，N.A.&Irwin，M.Thekineticsofvillouscellpopulationsinthemousesmallintestine.I.Normalvilli：thesteadystaterequirement.CellTissueKinet15，595-609(1982).

18.Roberts，S.A.，Hendry，J.H.&Potten，C.S.Intestinalcryptclonogens：anewinterpretationofradiationsurvivalcurveshapeandclonogeniccellnumber.CellProlif36，215-231(2003).

19.Roberts，S.A.&Potten，C.S.Clonogencontentofintestinalcrypts：itsdeductionusingamicrocolonyassayonwholemountpreparationsanditsdependenceonradiationdose.IntJRadiatBiol65，477-481(1994).

20.Potten，C.S.，Owen，G.&Roberts，S.A.Thetemporalandspatialchangesincellproliferationwithintheirradiatedcryptsofthemurinesmallintestine.IntJRadiatBiol57，185-199(1990).

21.MacNaughton，W.K.Reviewarticle：newinsightsintothepathogenesisofradiation-inducedintestinaldysfunction.AlimentPharmacolTher14，523-528(2000).

22.Rodier，J.F.Radiationenteropathy--incidence，aetioiogy，riskfactors，pathologyandsymptoms.Tumori81，122-125(1995).

23.PiadelaMaza，M.，etal.Acutenutritionalandintestinalchangesafterpelvicradiation.JAmCollNutr20，637-642(2001).

24.Leiper，K.&Morris，A.I.Treatmentofradiationproctitis.ClinOncol(RCollRadiol)19，724-729(2007).

25.Denton，A.S.，Andreyev，H.J.，Forbes，A.&Maher，E.J.Systematicreviewfornon-surgicalinterventionsforthemanagementoflateradiationproctitis.BrJCancer87，134-143(2002).

26.Andreyev，J.Gastrointestinalcomplicationsofpelvicradiotherapy：aretheyofanyimportance？Gut54，1051-1054(2005).

27.DeCosse，J.J.，etal.Thenaturalhistoryandmanagementofradiationinducedinjuryofthegastrointestinaltract.AnnSurg170，369-384(1969).

28.Libotte，F.，etal.Survivalofpatientswithradiationenteritisofthesmallandthelargeintestine.ActaChirBelg95，190-194(1995).

29.Galland，R.B.&Spencer，J.Thenaturalhistoryofclinicallyestablishedradiationenteritis.Lancet1，1257-1258(1985).

30.Classen，J.，etal.Radiation-inducedgastrointestinaltoxicity.Pathophysiology，approachestotreatmentandprophylaxis.StrahlentherOnkol174Suppl3，82-84(1998).

31.Donaldson，S.S.，etal.Radiationenteritisinchildren.Aretrospectivereview，clinicopathologiccorrelation，anddietarymanagement.Cancer35，1167-1178(1975).

32.Voitk，A.J.，Brown，R.A.，McArdle，A.H.，Hinchey，E.J.&Gurd，F.N.Clinicalusesofanelementaldiet：preliminarystudies.CanMedAssocJ107，123-129(1972).

33.Klimberg，V.S.，etal.Prophylacticglutamineprotectstheintestinalmucosafromradiationinjury.Cancer66，62-68(1990).

34.Klimberg，V.S.，etal.Oralglutamineaccelerateshealingofthesmallintestineandimprovesoutcomeafterwholeabdominalradiation.ArchSurg125，1040-1045(1990).

35.Jensen，J.C.，etal.Preventionofchronicradiationenteropathybydietaryglutamine.AnnSurgOncol1，157-163(1994).

36.Kozelsky，T.F.，etal.PhaseIIIdouble-blindstudyofglutamineversusplaceboforthepreventionofacutediarrheainpatientsreceivingpelvicradiationtherapy.JClinOncol21，1669-1674(2003).

37.Silvain，C.，etal.Long-termoutcomeofsevereradiationenteritistreatedbytotalparenteralnutrition.DigDisSci37，1065-1071(1992).

38.Ekelund，M.，Kristensson，E.&Ekblad，E.Totalparenteralnutritioncausescircumferentialintestinalatrophy，remodelingoftheintestinalwall，andredistributionofeosinophilsintheratgastrointestinaltract.DigDisSci52，1833-1839(2007).

39.Jackson，W.D.&Grand，R.J.Thehumanintestinalresponsetoenteralnutrients：areview.JAmCollNutr10，500-509(1991).

40.Burrin，D.G.，etal.Minimalenteralnutrientrequirementsforintestinalgrowthinneonatalpiglets：howmuchisenough？AmJClinNutr71，1603-1610(2000).

41.Drucker，D.J.，etal.Biologicpropertiesandtherapeuticpotentialofglucagon-likepeptide-2.JPENJParenterEnteralNutr23，S98-100(1999).

42.Niinikoskd，H.，etal.OnsetofsmallintestinalatrophyisassociatedwithreducedintestinalbloodflowinTPN-fedneonatalpiglets.JNutr134，1467-1474(2004).

43.Matheson，P.J.，Wilson，M.A.&Garrison，R.N.Regulationofintestinalbloodflow.JSurgRes93，182-196(2000).

44.Nowicki，P.T.，Stonestreet，B.S.，Hansen，N.B.，Yao，A.C.&Oh，W.Gastrointestinalbloodflowandoxygenconsumptioninawakenewbornpiglets：effectoffeeding.AmJPhysiol245，G697-702(1983).

45.vanGoudoever，J.B.，etal.Secretionoftrophicgutpeptidesisnotdifferentinbolus-andcontinuouslyfedpiglets.JNutr131，729-732(2001).

46.Knickelbein，R.，Aronson，P.S.，Schron，C.M.，Seifter，J.&Dobbins，J.W.Sodiumaudchloridetransportacrossrabbitilealbrushborder.II.EvidenceforCl-HCO3exchangeandmechanismofcoupling.TheAmericanJournalofPhysiology249，G236-245(1985).

47.Field，M.，Fromm，D.&McColl，I.Iontransportinrabbitilealmucosa.I.NaandClfluxesandshort-circuitcurrent.AmJPhysiol220，1388-1396(1971).

48.Sellin，J.H.&DeSoignie，R.Rabbitproximalcolon：adistincttransportepithelium.AmJPhysiol246，G603-610(1984).

49.Turnberg，L.A.，Bieberdorf，F.A.，Morawski，S.G.&Fordtran，J.S.Interrelationshipsofchloride，bicarbonate，sodium，andhydrogentransportinthehumanileum.JClinInvest49，557-567(1970).

50.Bach，S.P.，Renehan，A.G.&Potten，C.S.Stemcells：theintestinalstemcellasaparadigm.Carcinogenesis21，469-476(2000).

51.Kaur，P.&Potten，C.S.Cellmigrationvelocitiesinthecryptsofthesmallintestineaftercytotoxicinsultarenotdependentonmitoticactivity.CellTissueKinet19，601-610(1986).

52.Qiu，J.M.，Roberts，S.A.&Potten，C.S.Cellmigrationinthesmallandlargebowelshowsastrongcircadianrhythm.EpithelialCellBiol3，137-148(1994).

53.Al-Dewachi，H.S.，Wright，N.A.，Appleton，D.R.&Watson，A.J.Theeffectofasingleinjectionofhydroxyureaoncellpopulationkineticsinthesmallbowelmucosaoftherat.CellTissueKinet10，203-213(1977).

54.Hendry，J.H.，etal.Theresponseofmurineintestinalcryptstoshort-rangepromethium-147betairradiation：deductionsconcerningclonogeniccellnunbersandpositions.RadiatRes118，364-374(1989).

55.Okine，E.K.，Glimm，D.R.，Thompson，J.R.&Kennelly，J.J.Influenceofstageoflactationonglucoseandglutaminemetabolisminisolatedenterocytesfromdairycattle.Metabolism44，325-331(1995).

56.Alpers，D.H.Isglutamineauniquefuelforsmallintestinalcells？CurrOpinGastroenterol16，155(2000).

57.Wu，G.Intestinalmucosalaminoacidcatabolism.JNutr128，1249-1252(1998).

58.Tome，D.&Bos，C.Lysinerequirementthroughthehumanlifecycle.JNutr137，1642S-1645S(2007).

59.Vayro，S.，Lo，B.&Silverman，M.FunctionalstudiesoftherabbitintestinalNa+/glucosecarrier(SGLT1)expressedinCOS-7cells：evaluationofthemutantA166CindicatesthisregionisimportantforNa+-activationofthecarrier.BiochemJ332(Pt1)，119-125(1998).

60.Loo，D.D.，Zeuthen，T.，Chandy，G.&Wright，E.M.CotransportofwaterbytheNa+/glucosecotransporter.ProcNatlAcadSciUSA93，13367-13370(1996).

61.Benson，A.B.，3rd，etal.Recommendedguidelinesforthetreatmentofcancertreatment-induceddiarrhea.JClinOncol22，2918-2926(2004).

62.Mehta，D.I.，Horvath，K.，Chanasongcram，S.，Hill，I.D.&Panigrahi，P.Epidermalgrowthfactorup-regulatessodium-glucosecotransportinenterocytemodelsinthepresenceofcholeratoxin.JPENJParenterEnteralNutr21，185-191(1997).

63.Thomson，A.B.，Cheeseman，C.I.&Walker，K.Lateeffectsofabdominalradiationonintestinaluptakeofnutrients.RadiatRes107，344-353(1986).

64.Porteous，J.W.Intestinalmetabolism.EnvironHealthPerspect33，25-35(1979).

65.Balda，M.S.&Matter，K.ThetightjunctionproteinZO-1andaninteractingtranscriptionfactorregulateErbB-2expression.EmboJ19，2024-2033(2000).

66.Stefani，E.&Cereijido，M.Electricalpropertiesofculturedepithelioidcells(MDCK).JMembrBiol73，177-184(1983).

67.Gonzalez-Mariscal，L.，ChavezdeRamirez，B.，Lazaro，A.&Cereijido，M.Establishmentoftightjunctionsbetweencellsfromdifferentanimalspeciesanddifferentsealingcapacities.JMembrBiol107，43-56(1989).

68.Souba，W.W.，Scott，T.E.&Wilmore，D.W.Intestinalconsumptionofintravenouslyadministeredfuels.JPENJParenterEnteralNutr9，18-22(1985).

69.CardonaPera，D.[Administrationofglutamineanditsdipeptidesinparenteralnutrition.Whichpatientsarecandidates？].NutrHosp13，8-20(1998).

70.Joiner，W.J.，etal.ActiveK+secretionthroughmultipleKCa-typechannelsandregulationbyIKCachannelsinratproximalcolon.AmJPhysiolGastrointestLiverPhysiol285，G185-196(2003).

71.Vidyasagar，S.&Ramakrishna，B.S.Effectsofbutyrateonactivesodiumandchloridetransportinratandrabbitdistalcolon.JPhysiol(Lond)539，163-173(2002).

72.Vidyasagar，S.，Barmeyer，C.，Geibel，J.，Binder，H.J.&Rajendran，V.M.RoleofShort-ChainFattyAcidsinColonicHCO3Secretion.AmJPhysiolGastrointestLiverPhysiol288，G1217-1226(2005).

73.Vidyasagar，S.，Rajendran，V.M.&Binder，H.J.ThreedistinctmechanismsofHCO3-secretioninratdistalcolon.AmJPhysiolCellPhysiol287，C612-621(2004).

74.Zhang，H.，Ameen，N.，Melvin，J.E.&Vidyasagar，S.Acuteinflammationaltersbicarbonatetransportinmouseileum.JPhysiol581，1221-1233(2007).

75.Hopfer，U.，Nelson，K.，Perrotto，J.&Isselbacher，K.J.Glucosetransportinisolatedbrushbordermembranefromratsmallintestine.JBiolChem248，25-32(1973).

76.Bradford，M.M.Arapidandsensitivemethodforthequantitationofmicrogramquantitiesofproteinutilizingtheprincipleofprotein-dyebinding.AnalBiochem72，248-254(1976).

77.Heath，J.P.Epithelialcellmigrationintheintestine.CellBiolInt20，139-146(1996).

78.Reagan-Shaw，S.，Nihal，M.&Ahmad，N.Dosetranslationfromanimaltohumanstudiesrevisited.FASEBJ22，659-661(2008).

Claims (16)

1. A therapeutic composition for improving small intestine health, in particular in the case of villous atrophy, wherein the composition is formulated for enteral administration and comprises at least two amino acids selected from the group consisting of lysine, glycine, threonine, valine, and tyrosine, as free amino acids; and water; and wherein the composition does not comprise glucose, glutamine and methionine, or, if these ingredients are present, glucose is present at a concentration of less than 1g/l, glutamine is present at a concentration of less than 300mg/l and methionine is present at a concentration of less than 100 mg/l.

2. The composition of claim 1, wherein the composition has a total osmolarity of from 230mosm to 280 mosm.

3. The composition of claim 1 or 2, further comprising aspartic acid, isoleucine, tryptophan, asparagine, and/or serine.

4. The composition of claims 1-3, wherein the composition does not comprise glucose, glutamine or methionine.

5. The composition according to any one of claims 1 to 4, wherein the composition further comprises electrolytes, vitamins, minerals and/or flavoring agents.

6. The composition of any one of claims 1 to 5, which is sterile.

7. The composition of any preceding claim, for use in treating a subject with villous atrophy.

8. The composition for use according to claim 7, comprising administering the composition to the subject, preferably a human, via enteral administration.

9. The composition for use according to claim 7 or 8, wherein the total number of small intestinal epithelial cells in the villus region of the subject is reduced by at least 10% compared to normal.

10. The composition for use according to any one of claims 7-9, wherein the height of villi in the small intestine of the subject is lost by at least 10% compared to normal.

11. The composition for use according to any one of claims 7-10, wherein the villous atrophy is caused by disease, radiation, chemotherapy, proton therapy, abdominal surgery, and/or cytotoxic agents.

12. The composition for use according to any one of claims 7-11, wherein the composition is administered for a period of 1 to 14 days after the subject receives radiation, chemotherapy, proton therapy, or a cytotoxic agent.

13. The composition for use according to any one of claims 7-11, for use in the treatment of radiation enteritis.

14. The composition for use according to any one of claims 7-11, for treating small intestine injury caused by chemotherapy or a cytotoxic agent selected from the group consisting of: cisplatin, 5-fluorouracil (5-FU), hydroxyurea, etoposide, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine, methotrexate, steroids, or combinations thereof.

15. The composition for use according to any one of claims 7-11, for use in the treatment of Inflammatory Bowel Disease (IBD), ulcerative colitis, duodenal ulcers or crohn's disease.

16. A kit comprising the composition of any one of claims 1 to 6 or a powder that when combined with water forms the composition of any one of claims 1 to 6, wherein the kit further comprises instructions for administering the composition to a patient who has or is expected to have villous atrophy.