Treatments to cure dysregulation in GI tissue

Abstract

The present disclosure relates to the treatment of dysregulated regeneration and / or maturation of the digestive tract (also referred to herein as the GI tract), particularly in infants, such as preterm infants (also referred to herein as newborns), small-for-gestational-age infants, and / or infants born after preeclampsia, by administering a therapeutically effective amount of IGF-I, particularly as a complex with an IGFBP, e.g., IGFBP-3. Treatment is also useful in other patient populations, including adult populations, with GI tract conditions / diseases / disorders and / or infections.

Description

[Technical Field]

[0001] The present disclosure relates to the treatment of dysregulated regeneration and / or maturation of the digestive tract (also referred to herein as the GI tract), particularly in infants, such as preterm infants (also referred to herein as newborns), small-for-gestational-age infants, and / or infants born after preeclampsia. Treatment is also useful in other patient populations, including adult populations with GI tract conditions / diseases / disorders and / or infections. [Background technology]

[0002] Approximately 20,000 protein-coding genes are expressed in human cells, and 75% of these genes are expressed in at least one of the various parts of the digestive system. Over 600 of these genes are more specifically expressed in one or more parts of the GI tract, and the corresponding proteins have functions related to food digestion and nutrient intake.

[0003] The GI tract is an extremely complex arrangement that can be divided into four concentric layers: the mucosa, the submucosa, the muscularis and the adventitia or serosa.

[0004] The mucosa consists of: Epithelium - the innermost layer that is primarily responsible for the processes of digestion, absorption, and secretion; it defines the interior space (lumen) of the tube. Lamina propria - connective tissue with a cellular composition, and Muscularis mucosae - smooth muscle involved in GI motility and peristalsis.

[0005] Embedded in the mucosa are functional elements such as nerves, lymphatic tissue, ducts from the glands, and glandular mucosa.

[0006] The submucosa is a dense, irregular layer of connective tissue containing glands, large blood vessels, lymphatics, and nerves that branch out into the mucosa and muscularis.

[0007] The muscle layer includes an inner circular layer and an outer longitudinal layer.

[0008] The adventitia and serosa comprise several layers of connective tissue.

[0009] The structure of the GI tract is crucial to the health and function of the digestive system. Simply put, the functions of the GI tract are to: ·Keeping the contents of the duct, including pathogens, contained and separated from the rest of the body; and Digesting food, and ·Delivering nutrients to the body, and - Eliminate waste products.

[0010] When the GI tract becomes inflamed and diseased, this containment structure can become leaky. Cells within the GI tissue begin to break down, resulting in trauma, damage, and "holes" in various layers of the structure. If the situation becomes severe enough, the vasculature of the GI tract can become leaky, allowing pathogens to breach the GI tract and become systemic, causing, for example, sepsis. This process can be life-threatening.

[0011] There are multiple diseases and illnesses that can lead to these symptoms, e.g., bacterial infections / autoimmune diseases / premature birth. Interestingly, some individuals are better able to cope with and recover from these problems than others who are more susceptible to this pathology.

[0012] Studies of individuals with these conditions reveal that some markers, such as inflammatory markers, are elevated and others, such as IGF-1, are decreased. In this condition, there may be a negative feedback loop that perpetuates and / or worsens the condition or disease.

[0013] However, without being bound by theory, it is believed that some individuals may have dysregulation of GI tract regeneration before disease / disorder and pathology become apparent, or may have dysregulation of GI tract regeneration caused by disease.

[0014] Research suggests that celiac disease patients have a predisposing condition to the damaging effects of gliadin, such as constitutive changes in intestinal cell proliferation. It has been suggested that Paneth cell dysfunction leads to an imbalanced gut microbiome, which in turn may predispose to the development of Crohn's disease. Abnormal Paneth cells with reduced expression or secretion of defensins HD-5 and HD-6 (in humans) and antimicrobial peptides are associated with inflammatory bowel disease. Therefore, when a "challenge" such as a bacterial infection occurs in these individuals, the body is unable to defend itself and heal efficiently.

[0015] The above applies to preterm infants, who are born before their GI tract is fully operational. Preterm infants have immature mucosal barriers and may also have abnormal gut microbiota. Therefore, the ability to heal and / or mature rapidly is a matter of life and death in this patient population. Necrotizing enterocolitis (NEC) is a life-threatening disease in preterm infants, commonly resulting in severe sepsis, intestinal necrosis, and intestinal death. Specific treatments for treating or preventing NEC are currently unavailable because its pathogenesis is not fully understood. While NEC appears to be a multifactorial disease involving the involvement of an immature immune response, inadequate breastfeeding, and gut microbiota disruption, the present inventors suggest a role for impaired development of the neonatal intestinal system.

[0016] Surprisingly, the inventors have demonstrated that the underlying mechanisms involved in dysregulation of GI regeneration can be addressed to support bodily function and minimize severe GI damage even in the most frail patient populations, such as preterm infants. Summary of the Invention

[0017] This disclosure is summarized in the following sections: 1. A method of treating or preventing dysregulated GI tract regeneration (e.g., in infants, such as in preterm infants (also referred to herein as newborns) and / or infants born small for gestational age or following preeclampsia) by administering a therapeutic amount of a composition comprising IGF-1 and an IGF binding protein (such as IGFBP-3), particularly as a complex. In one embodiment, the patient, particularly a human, has dysregulated GI tract regeneration and / or is at risk for dysregulated GI tract regeneration.

[0018] 1A. A composition comprising IGF-1 and an IGF binding protein (such as IGFBP-3), particularly as a complex, for use in the treatment or prevention of dysregulated intestinal regeneration, particularly in the patient populations disclosed herein.

[0019] 1B. A composition comprising IGF-1 and an IGF binding protein (such as IGFBP-3), particularly as a complex, for use in the manufacture of a medicament for the treatment or prevention of dysregulated intestinal regeneration, particularly in patient populations disclosed herein.

[0020] 1C. Methods of treatment or prevention by stimulating GI cell maturation (e.g., to increase and / or maintain tight junctions and / or) in infants, such as preterm infants (also referred to herein as newborns) and / or small-for-gestational-age infants, by administering a therapeutic amount of a composition comprising IGF-1 and an IGF binding protein (such as IGFBP-3), particularly as a complex.

[0021] 2. The method or composition of any of the preceding claims, wherein the composition is administered parenterally.

[0022] 3. The method or composition of paragraph 2, wherein the parenteral administration is by infusion, e.g., continuous infusion (e.g., to infants, premature and / or small-for-gestational-age infants and / or infants born after preeclampsia), e.g., continuous infusion for at least 1 week (e.g., 2-6 weeks, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks).

[0023] 4. The method or composition of paragraph 2 or 3, wherein the parenteral administration is intraperitoneal and / or subcutaneous administration, for example, at least once daily, for example, once, twice, or three times daily. In one embodiment, a treatment is provided for infants, for example, preterm infants, infants born low for gestational age, and / or infants born after preeclampsia, wherein the treatment is administered for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after birth, particularly for 1 to 7 days after birth. In one embodiment, a treatment is provided for infants according to the present disclosure, for example, preterm infants, particularly treated by subcutaneous administration, for example, subcutaneous bolus administration, for at least 1 week, for example, 2 to 6 weeks, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. In one embodiment, the bolus injection is administered once daily. Treatment may begin, for example, within the first 24 hours after birth, with a continuous infusion for a period beginning in the first 12, 6, 5, 4, 3, 2 or 1 hour, which may be accompanied and / or followed by intraperitoneal and / or subcutaneous administration, i.e., subsequently, when the infusion is terminated, administration may be continued solely by intraperitoneal and / or subcutaneous administration for a period of time.

[0024] 5. The method or composition of any of the preceding paragraphs, wherein treatment reduces inflammation.

[0025] 6. The method or composition of paragraph 5, wherein systemic inflammation is reduced, for example, IL-6 and / or CXCL1 is reduced.

[0026] 7. The method or composition of paragraph 5 or 6, wherein the inflammation is in the GI tract, such as the digestive tract and intestine, e.g., selected from the digestive tract, intestine, and / or colon, particularly the intestine. In one embodiment, the inflammation is not systemic.

[0027] 8. The method or composition of any of the preceding paragraphs, wherein one or more cytokines selected from IL-4, IL-6, IL-10, and combinations of two or three thereof are reduced by treatment, for example, by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more, compared to untreated patients with enteritis (such as necrotizing enterocolitis).

[0028] 9. A method or composition according to any of the preceding paragraphs, wherein inflammation-induced destruction of IGFBP-3 in tissues is minimized and / or destruction (including removal) of IGF-1 is minimized.

[0029] 10. A method or composition according to any of the preceding paragraphs, wherein a positive feedback loop for IGF-1 production is supported, stabilized, maintained, initiated, and / or increased.

[0030] 11. A method or composition according to any of the preceding paragraphs, wherein vascular permeability is reduced, e.g., compared to an untreated patient with necrotizing enterocolitis, in particular vascular leakage into the intestinal tissue is minimized and / or leakage from the GI tract into the vasculature is minimized. In one embodiment, microvascular integrity is preserved, stabilized, and / or improved by treatment.

[0031] 12. A method or composition according to any of the preceding paragraphs, wherein tight junctions are promoted, preserved, supported, stabilized, maintained, initiated, and / or increased by the treatment, e.g., the proportion of tight junctions is preserved or increased.

[0032] 13. A method or composition according to any of the preceding paragraphs, wherein the treatment supports the maintenance of epithelial barrier function and / or epithelial integrity, including, for example, preventing basement membrane rupture, and / or basal stromal cells are not exposed to luminal contents.

[0033] 14. A method or composition according to any of the preceding paragraphs, wherein VEGF and VEGFR2 protein expression is preserved, supported, maintained, initiated, and / or increased, particularly at healthy levels, particularly in GI tract tissue, e.g., the digestive tract.

[0034] 15. A method or composition according to any of the preceding claims, wherein angiogenesis in the GI tract, e.g., the digestive tract and / or intestine, particularly the intestine, is maintained, supported, initiated, and / or increased by the treatment, e.g., the treatment provides increased microvascular density.

[0035] 16. A method or composition according to any of the preceding paragraphs, wherein endothelial cell proliferation is preserved, supported, maintained, initiated, and / or increased by the treatment, e.g., villous endothelial cell proliferation is preserved, supported, maintained, initiated, and / or increased by the treatment.

[0036] 17. A method or composition according to any of the preceding paragraphs, wherein epithelial cell migration is preserved, supported, maintained, initiated, and / or increased by the treatment.

[0037] 18. A method or composition according to any of the preceding paragraphs, wherein epithelial cell apoptosis, eg, intestinal cell apoptosis, eg, apoptosis induced by oxidative stress, is minimized (modulated) by treatment.

[0038] 19. A method or composition according to any of the preceding paragraphs, wherein the treatment promotes intestinal cell proliferation, eg, compared to an untreated patient.

[0039] 20. A method or composition according to any of the preceding claims, wherein enterocyte function, e.g., a function selected from ion uptake, water uptake, sugar uptake, peptide uptake, amino acid uptake, lipid uptake, vitamin B12 uptake, immunoglobulin secretion, and combinations of two or more thereof, e.g., all functions, is preserved, supported, maintained, initiated, and / or increased.

[0040] 21. A method or composition according to any of the preceding paragraphs, wherein Paneth cell function, such as Wnt signaling and Notch signaling and / or secretion of defensins HD-5 and HD-6, is preserved, supported, maintained, initiated, and / or increased.

[0041] 22. A method or composition according to any of the preceding paragraphs, wherein Paneth cell dysfunction is minimized by treatment, for example compared to untreated patients.

[0042] 23. A method or composition according to any of the preceding paragraphs, wherein Paneth cell proliferation is increased by treatment, eg, compared to an untreated patient.

[0043] 24. A method or composition according to any of the preceding paragraphs, wherein the treatment reduces the incidence of severe damage to the GI tract, e.g., severe intestinal damage, e.g., by promoting and / or modulating the healing process.

[0044] 25. The method or composition of paragraph 24, wherein severe damage to the villi, such as destruction of the villi and / or malformation of the villi, is minimized.

[0045] 26. A method or composition according to any preceding claim for use in the treatment of colitis (including necrotizing enterocolitis), C. difficile infection, Shigella infection, toxic megacolon, and the like.

[0046] 27. The method or composition according to paragraph 22, wherein the significant incidence of severe necrotizing enterocolitis, for example a score of 2 or higher (e.g., 3, 4, 5, and 6), is reduced compared to untreated patients with necrotizing enterocolitis.

[0047] 28. A method or composition according to any of the preceding paragraphs, wherein the incidence of sepsis is reduced in treated patients.

[0048] 29. A method or composition according to any of the preceding paragraphs, wherein the treatment has a paracrine component, which may, for example, stimulate angiogenesis (such as neovascularization) and / or stimulate the production of IGF-1.

[0049] 30. A method or composition according to any of the preceding paragraphs, wherein the treatment has an endocrine component.

[0050] 31. A method or composition according to any preceding claim, wherein the treatment has an autocrine component.

[0051] 32. A method or composition according to any of the preceding claims, wherein macrophages in the GI tract, e.g., the digestive tract and / or intestine (especially the intestine), are activated, particularly to secrete IGF-1, particularly wherein the macrophages have a "healthy" morphology in contrast to the morphology adopted in NEC.

[0052] 33. A method or composition according to any of the preceding paragraphs, wherein the level of macrophages is higher in treated patients than in untreated patients.

[0053] 34. A method or composition according to any of the preceding paragraphs, wherein patients receiving treatment have a lower risk of death than corresponding patients without treatment.

[0054] 35. The method or composition of any of the preceding paragraphs, wherein the complex is provided at a ratio of 0.5 to 1.5 IGF-1 to 1 IGFBP protein (such as IGFBP-3), such as a 1:1 ratio.

[0055] 36. The method or composition of any of the preceding claims, wherein the dose for infants, such as preterm infants, infants born low for gestational age and / or after preeclampsia, is a 200-500 μg / Kg / 24 hour complex, e.g., 350-500 μg / Kg / 24 hours, 400 μg / Kg / 24 hours, particularly by continuous infusion.

[0056] 37. The method or composition according to any of the preceding paragraphs, wherein 55 to 110 μg / Kg / 24 hours of IGF-1 is administered.

[0057] 38. Serum IGF-1 levels for infants, such as preterm infants, range from 28 to 109 ng / mL, with averages of 28 ng / mL, 29 ng / mL, 30 ng / mL, 31 ng / mL, 32 ng / mL, 33 ng / mL, 34 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 46 ng / mL, 47 ng / mL, 48 ng / mL, 49 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 66 ng / mL, 67 ng / mL, 68 ng / mL, 69 ng / mL, 70 ng / mL, 71 ng / mL, 72 ng / mL, 73 ng / mL, 74 ng / mL, 75 ng / mL, 76 ng / mL, 77 ng / mL, 78 ng / mL, 79 ng / mL, 80 ng / mL, 81 ng / mL, 82 ng / mL, 83 ng / mL, 84 ng / mL, 85 ng / mL, 86 ng / mL, 87 ng / mL, 88 ng / mL, 89 ng / mL, 90 ng / mL, 91 ng / mL, 92 ng / mL, 93 ng / mL, 94 ng / mL, 95 ng / mL, 96 ng / mL, 97 ng / mL, 98 ng / mL, 99 ng / mL, 100 ng / mL, 101 ng / mL, 102 ng / mL, 103 ng / mL, 104 ng / mL, 105 ng / mL, 10. The method or composition of any of the preceding claims, wherein the serum albumin is maintained at 100ng / mL, 65ng / mL, 70ng / mL, 75ng / mL, 80ng / mL, 85ng / mL, 90ng / mL, 95ng / mL, 100ng / mL, 101ng / mL, 102ng / mL, 103ng / mL, 104ng / mL, 105ng / mL, 106ng / mL, 107ng / mL, 108ng / mL or 109ng / mL.

[0058] 39. The method or composition of any of the preceding claims, wherein the patient is a human, e.g., an infant, particularly a premature infant (also referred to herein as a newborn), a small-for-gestational-age infant, and an infant born after preeclampsia, particularly a premature infant.

[0059] 40. A method or composition according to any preceding paragraph, wherein the infant has a birth weight of 2.2 pounds or less (1 kg or less).

[0060] 41. A method or composition according to any of the preceding paragraphs, wherein the preterm infant is between 23 and 37 weeks of gestation, for example between 23 and 34 weeks, or between 23 and 30 weeks, or between 24 and 29 weeks, at the time treatment is initiated.

[0061] 42. The method or composition of any preceding clause, wherein treatment of an infant (such as a preterm infant, an infant born low for gestational age and / or an infant born after preeclampsia) according to the present disclosure is initiated within 24 hours of birth, e.g., within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours and within 24 hours of birth, particularly within 1 hour, 2 hours or 3 hours of birth.

[0062] 43. A method or composition according to the present disclosure for use in treating celiac disease (or a predisposition to celiac disease), including inflammatory bowel disease and / or non-responsive celiac disease.

[0063] 44. The method or composition of any of the preceding paragraphs, wherein administration is designed to increase tissue-specific levels of IGF-1 and / or IGFBP-3.

[0064] 45. The method or composition of any preceding paragraph, wherein tissue-specific levels of at least IGF-1 and / or IGFBP-3 are increased.

[0065] 46. ​​The method or composition of any of the preceding paragraphs, wherein serum levels of IGF-1 are increased by treatment.

[0066] In one embodiment, destruction of IGFBP-3, eg, proteolysis by IL-6, is minimized.

[0067] In one embodiment, the binding protein is IGFBP-3.

[0068] In one embodiment, the binding protein is IGFBP-5.

[0069] In one embodiment, the complex is a mixture of IGF-1 / IGFBP-3 and 5.

[0070] In one embodiment, the present disclosure relates to increasing tissue-specific levels of IGF-1 and / or IGFBP-3. Without wishing to be bound by theory, such halting or reversing the negative feedback loop in which inflammation destroys IGFBPs (e.g., IGFBP-3) would in turn promote rapid clearance of IGF-1.

[0071] In one embodiment, the present disclosure is not directed to increasing serum levels of free IGF-1.

[0072] In one embodiment, treatment maintains expression of the intestinal IGF-1 receptor, which helps protect against, for example, intestinal damage.

[0073] In one embodiment, the levels of VEGF and / or VEGFR, particularly protein expression levels, are stabilized or increased, particularly locally in GI tissue.

[0074] In one embodiment, the treatment provides stabilized and / or improved oxygen delivery to the intestinal tissue.

[0075] In one embodiment, the treatment provides stabilized and / or improved nutrient delivery / absorption.

[0076] In one embodiment, there is stabilized blood flow to the GI tract.

[0077] In one embodiment, treatment provides stabilized and / or improved removal of waste products from local cells in the GI tract, such as enterocytes, including removal of waste products via the circulatory system.

[0078] In one embodiment, treatment provides stabilized and / or improved function of local cells in the GI tract, such as enterocytes.

[0079] In one embodiment, treatment provides stabilized and / or improved motility of the GI tract and / or its substructures, e.g., microvilli and / or villi. In one embodiment, the arrangement of the microvilli and / or villi is more "normal" in treated patients, e.g., less flattened and / or less flaccid. In one embodiment, local GI cell, e.g., enterocyte, tone is normalized.

[0080] In one embodiment, treatment of human patients is provided, including infants (i.e., under 1 year of age, including premature infants, as defined herein), children (e.g., 1-12 years of age), adolescents (e.g., 12-17 years of age), and adults (18 years of age or older). In one embodiment, the patient is female, e.g., in the age range of 15-60 years of age, e.g., 15-40 years of age or 40-60 years of age.

[0081] In one embodiment, patients, such as infants / young children, receiving treatment according to the present disclosure have a lower mortality rate (eg, all-cause) than patients not receiving treatment.

[0082] In one embodiment, infants, such as preterm infants, low birth weight infants, and infants born after preeclampsia, particularly preterm infants. Treatment in this patient population can result in further assimilation / differentiation / maturation of GI cells and structures, such as enterocytes, villi, and / or microvilli.

[0083] In one embodiment, the premature infant is between 23 and 34 weeks of gestation (e.g., 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 weeks) at the time treatment is initiated.

[0084] In one embodiment, patients, such as infants or children, receiving treatment have less severe diseases and conditions, such as Crohn's disease (present in children but not in infants), colitis, necrotizing enterocolitis, toxic megacolon, and particularly severe necrotizing enterocolitis is reduced in infants, such as preterm infants, low birth weight infants, and infants born after preeclampsia, particularly preterm infants.

[0085] In one embodiment, the premature infant is treated by infusion, eg continuous infusion, particularly for at least 1 week, such as 2-6 weeks, such as 2, 3, 4, 5 or 6 weeks.

[0086] In one embodiment, the treatment of the present disclosure acts on endothelial cells, including reducing membrane permeability, increasing and / or stabilizing angiogenesis, increasing and / or stabilizing blood vessel formation, stabilizing and / or increasing epithelial cell migration, and / or inhibiting epithelial cell apoptosis.

[0087] In one embodiment, epithelial cell migration, e.g., intestinal epithelial cell migration, is increased by the treatment, e.g., it is reduced by NEC. The increase may be in comparison to patients who did not receive the treatment.

[0088] In one embodiment, treatment according to the present disclosure reduces intestinal vascular permeability, e.g., reduces an NEC-induced increase in intestinal vascular permeability, which increase can be in comparison to patients who did not receive the treatment.

[0089] In one embodiment, microvessel density is increased in the GI tract, particularly compared to patients with NEC who have not received treatment (such as the types of patients disclosed herein).

[0090] In one embodiment, patients treated according to the present disclosure exhibit better, improved, or faster healing than untreated patients.

[0091] In one embodiment, treatment according to the present disclosure is administered to a patient following surgery on the GI tract.

[0092] In one embodiment, treatment according to the present disclosure is administered to patients with severe gastric and / or intestinal ulcers, eg, to improve prognosis.

[0093] In one embodiment, patients treated in accordance with the present disclosure are better able to cope with the onslaught of disease, disorders, trauma and infections of the GI tract.

[0094] In one embodiment, the patient undergoing treatment is protected from severe GI tract damage.

[0095] In one embodiment, the IGF-1 / IGFBP (such as IGFBP-3) complex is administered locally to the gastrointestinal tract, optionally in combination with other routes of administration disclosed herein.

[0096] In one embodiment, the basement membrane is denser or thicker after treatment compared to an untreated control.

[0097] In one embodiment, VEGF inhibitors and / or VEGFR2 inhibitors are contraindicated in patients according to the present disclosure.

[0098] In one embodiment, treatment according to the present disclosure is not directed to increasing VEGF levels systemically and / or locally.

[0099] In embodiments, the treatment reduces intestinal vascular permeability.

[0100] In one embodiment, treatment improves / increases intestinal epithelial cell migration (eg, towards "normal" levels).

[0101] In one embodiment, the reduction in the level of bound IGF-1 is minimized and / or the increase in bound IGF-1 is promoted (particularly IGF-1 / IGFBP-3 binding).

[0102] In one embodiment, proteolysis of IGFBP-3, eg, IL-6 mediated proteolysis, is minimized.

[0103] In one embodiment, the increased clearance of free IGF-1 is minimized and / or the half-life of free IGF-1 is optimized.

[0104] Treatment according to the present disclosure has one or more (e.g., all) of the following effects: increased VEGF and VEGFR2 protein expression, reduced vascular permeability (e.g., intestinal vascular permeability), maintained intestinal endothelial cell proliferation, promoted enterocyte proliferation and migration while reducing enterocyte apoptosis, and attenuated systemic and small intestinal tissue inflammation.

[0105] In one embodiment, apoptosis of intestinal cells, such as enterocytes, is minimized (eg, normalized—particularly reduced to normal levels).

[0106] In one embodiment, serum levels of CXCL1 are stabilized, minimized, and / or reduced by treatment.

[0107] In one embodiment, serum levels of IL-6 are stabilized, minimized, and / or reduced by treatment.

[0108] In one embodiment, intestinal levels of IL-4, IL-6 and IL-10 and combinations thereof are stabilized, minimized and / or reduced by treatment.

[0109] In one embodiment, treatment protects the intestine against injury (including severe intestinal injury) and / or promotes healing of intestinal injury.

[0110] In one embodiment, the newborn does not develop ALS.

[0111] Patients treated according to the present disclosure may have improved survival rates and reduced incidence of severe intestinal damage.

[0112] Detailed Disclosure As used herein, a preterm infant (or newborn) refers to an infant born before 40 weeks of gestation, e.g., within 37 weeks of gestation, such as between 22 and 37 weeks (gestational age), particularly within 23, 24, 25, 26, 27, 28, 29, 30, 31, 33, 34, 35, or 36 weeks. Currently, newborns born at 22 weeks of gestation are borderline survivable, and most may not respond to resuscitation. There have been cases of newborns born at 22 weeks of gestation surviving.

[0113] Generally, the premature infant is a human.

[0114] The lowest weight infants (not necessarily the most premature) may benefit most from the therapy of the present disclosure. Some newborns do not grow adequately in utero. Even if such newborns are "older," they are still small for their gestational age. Treatment according to the present disclosure is particularly useful for these infants, such as those in the bottom quartile of birth weight.

[0115] As used herein, dysregulation of GI tract regeneration refers to a decreased ability of the GI tract, such as the digestive tract (stomach) and / or intestine, to develop, produce, repair, and / or disproportionately increase cell destruction in the above locations. Dysregulation of GI tract regeneration includes decreased proliferation of epithelial cells (e.g., enterocytes located in the microvilli), decreased migration of epithelial cells (e.g., enterocytes), increased apoptosis of cells in GI tissue, increased permeability (e.g., increased vascular permeability, e.g., intestinal vascular permeability), loss or decrease in the proportion of tight junctions, decreased levels of VEGF / VEGFR, loss or decrease in macrophages in GI tissue to secrete IGF-1, increased inflammatory cytokines in GI tissue (e.g., cytokines selected from IL-4, IL-6, IL-10, and combinations of two or three thereof).

[0116] As used herein, severe intestinal damage refers to damage to the GI tract caused by inflammation (including chronic inflammation), including, for example, one or more of ulcers, malabsorption, necrosis, ischemia, bleeding, pain, persistent diarrhea, weight loss, fatigue, bloating, disruption of the intestinal barrier, leaky membranes, and pathologies that make the patient more susceptible to bacterial infections, sepsis, and combinations thereof.

[0117] Gastrointestinal tract and GI tract are used interchangeably herein and include the digestive tract (stomach), intestines and colon, particularly the digestive tract and intestines, particularly the intestines.

[0118] The tissue levels (e.g., intestinal tissue levels) of IGF-1 / IGFBP (e.g., IGFBP-3) profiles in tissues and serum appear to be different, for example, serum levels of IGFBP-3 may be higher than levels in organ tissues such as the intestine, which may be related to different functions.

[0119] IGF-1 and / or IGF-1 / IGFBP and / or IGFBP-3 have endocrine, paracrine and autocrine functions in the body.

[0120] Endocrine activity arises from components within the circulatory system.

[0121] Paracrine activity results from components secreted from another cell.

[0122] Autocrine activity results from components secreted by the cell itself.

[0123] Increasing the levels of one or more of IGF-1 and IGFBP, such as IGFBP-3, in a tissue, particularly intestinal tissue, can be done by one or more of the following methods: Increasing the levels of one or more IGF-1 and IGFBPs, such as IGFBP-3, by intravenous infusion (e.g., continuous infusion) to achieve transport or migration of one or more of IGF-1 and IGFBPs, such as IGFBP-3, into tissues, which may require a threshold level of the complex to be achieved in the blood to facilitate this. This mechanism is an endocrine mechanism. Increasing the level of the complex directly in epithelial tissues by subcutaneous injection, which may in particular stimulate paracrine and / or autocrine mechanisms. Increasing the levels of the complex directly in the peritoneal cavity, for example by IP injection, may stimulate, inter alia, paracrine and / or autocrine mechanisms.

[0124] A combination of the above delivery strategies may be used.

[0125] "IGFBP" or "IGF-binding protein" refers to a protein or polypeptide from the insulin-like growth factor-binding protein family that normally associates with, binds to, or complexes with IGF-1, whether circulating (i.e., in serum or tissue). Such binding proteins do not include receptors. This definition includes IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, Mac25 (IGFBP-7), and prostacyclin production-stimulating factor (PSF) or endothelial cell-specific molecule (ESM-1), as well as other proteins with high homology to IGFBPs. Mac25 is described, for example, in Swisshelm et al., Proc. Natl. Acad. Sci. USA, 92: 4472-4476 (1995) and Oh et al., J. Biol. Chem., 271: 30322-30325 (1996). PSF is described in Yamauchi et al., Biochemical Journal, 303: 591-598 (1994). ESM-1 is described in Lassalle et al., J. Biol. Chem., 271: 20458-20464 (1996).Other identified IGFBPs are described, for example, in European Patent No. 375,438, issued June 27, 1990; European Patent No. 369,943, issued May 23, 1990; International Publication No. WO 89 / 09268, published October 5, 1989; Wood et al., Molecular Endocrinology, 2: 1176-1185 (1988); Brinkman et al., The EMBO J., 7: 2417-2423 (1988); Lee et al., Mol. Endocrinol., 2: 404-411 (1988); Brewer et al., BBRC, 152: 1289-1297 (1988); European Patent No. 294,021, issued December 7, 1988; Baxter et al. See, e.g., WO 89 / 08667, published September 21, 1989; WO 89 / 09792, published October 19, 1989; and Binkert et al., EMBO J., 8: 2497-2502 (1989).

[0126] "IGFBP-3" refers to insulin-like growth factor binding protein 3. IGFBP-3 is a member of the insulin-like growth factor binding protein family. IGFBP-3 can be derived from any species, including bovine, ovine, porcine, and human, in native sequence or in mutant forms, including, but not limited to, naturally occurring allelic variants, particularly human. IGFBP-3 can be derived from natural, synthetic, or recombinant sources, so long as it binds IGF-I at the appropriate site. IGFBP-3 can be produced by recombinant techniques, as described in WO 95 / 04076.

[0127] A therapeutic composition, as used herein, is defined as comprising IGF-1 or an analog thereof in combination with its binding protein, e.g., IGFBP-3 or an analog thereof. In some embodiments, IGF-1 is produced by recombinant technology. In some embodiments, IGFBP-3 is produced by recombinant technology. In some embodiments, IGF-1 and IGFBP-3 are complexed prior to administration to a subject. In some embodiments, IGF-1 and IGFBP-3 are complexed in equimolar amounts.

[0128] Therapeutic compositions may also contain other substances, such as carriers such as water, minerals, proteins, and other excipients known to those skilled in the art.

[0129] In the context of this specification, "comprising" should be interpreted as "including." Embodiments of the invention comprising certain features / elements are also intended to cover alternative embodiments "consisting of" or "consisting essentially of" the associated elements / features.

[0130] Where technically appropriate, embodiments of the invention may be combined.

[0131] Technical references, such as patents and applications, are incorporated herein by reference.

[0132] Any embodiment specifically and explicitly described herein may form the basis of a disclaimer either alone or in combination with one or more additional embodiments.

[0133] The subject headings herein are used to divide the document into sections and are not intended to be used to interpret the meaning of the disclosure provided herein.

[0134] The Background Art section contains technical information related to the present invention and may be used as a basis for modification.

[0135] Specific values ​​may be taken from the examples in combination with the general disclosure of this description.

[0136] The concepts disclosed in the examples are not closely related to the parameters of the specific examples and the general concepts that apply to the entire presently described invention, and these concepts in the examples may be separated and used as a basis for amending the claims.

[0137] This application claims priority to U.S. Patent Application No. 63 / 484,954, filed February 14, 2023, and UK Patent Application No. 2319443.4, filed December 18, 2023, both of which are incorporated herein by reference and may be used as a basis for any amendment herein.

[0138] The invention is further described, by way of illustration only, in the following examples. [Brief explanation of the drawings]

[0139] [Figure 1] IGF-1-binding IGF-binding protein 3 (IGFBP3) is reduced in the serum of NEC pups. Pups less than 24 hours old underwent a 24-hour NEC protocol or were left with their dams to nurse. 1–3 ml of serum from NEC pups (n=7) or dam-fostered controls (n=7) was subjected to Western blot analysis (probed with antibodies against IGF-1) under non-reducing conditions: (A) a representative blot is shown here; (B) the relative IGF-1 / BP3 and IGF-1 / other BP band densities of NEC pups were normalized to the mean band density of dam-fostered pups, demonstrating that IGF-1 / BP3, but not IGF-1 / other BPs, is reduced in NEC. Data are the combined values ​​of three experiments. P values ​​were calculated using a two-tailed Student's t-test. ***p<0.001. [Figure 2]Serum concentrations of "human" IGF-1 in pups injected with rhIGF-1 / BP3 (2 mg / kg or 10 mg / kg, i.p.) or vehicle control approximately 8 hours after birth. Serum was collected 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours after injection of rhIGF-1 / BP3. *p≦0.05, **p≦0.01. n=4-5 pups / group for 30 minutes, 1 hour, 2 hours, and 8 hours; n=3 pups / group for 4 hours and 24 hours; n=2 pups / group for the 24 hour vehicle control group. [Figure 3] rhIGF-1 / BP3 maintains intestinal VEGF / VEGFR2 expression, endothelial cell proliferation, and vascular permeability. One-day-old neonatal mice were subjected to an experimental NEC model and injected with rhIGF-1 / BP3 or vehicle control as described. Dam-fostered littermates served as baseline controls. Intestinal tissues were harvested 24 hours after the initiation of experimental NEC: (A) VEGF and VEGFR2 proteins were assessed by Western blot; a representative blot is shown; (B-C) quantified VEGF and VEGFR2, respectively, and band density measurements from A are shown; n = 6–8 per group; (D) Tissue sections were stained with antibodies against BrdU (red) and CD31 (green); a representative image is shown; scale bar = 100 mm; (E) Bar graphs represent the average number of proliferating endothelial cells (BrdU+CD31+ cells, arrows in D) per 20x field (at least 3 fields / sample) for each sample; n = 5 per group. (F) One-day-old neonatal mice were subjected to the experimental NEC model and injected with rhIGF-1 / BP3 (n = 9) or vehicle control (n = 9) as described. Dam-fostered littermates were used as baseline controls (n = 8). Twenty-four hours after initiating NEC, 10 μl of Evans blue solution was intravenously injected, and EBA leaked into the small intestinal tissue was quantified 1 hour later. P values ​​were calculated using one-way anova followed by the Tukey-Kramer multiple comparison test (B, C, E, and F). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 4]IGF-1 / IBP3 maintained epithelial cell proliferation and migration while reducing enterocyte apoptosis during experimental NEC. One-day-old neonates were subjected to the experimental NEC protocol or left alone with their dams (DF). Two hours before the initiation of NEC, half of the pups in each group were injected with rhIGF-1 / BP3 (6 mg / kg / d, i.p.). (A-D) At the initiation of NEC, all pups in the DF and NEC groups were injected i.p. with 50 mg / kg BrdU. At 24 or 48 hours after the initiation of NEC, intestines were harvested, and sections were stained with BrdU antibody and DAPI. Representative images are shown here; scale bar = 100 mm (A and B). (C) The number of proliferating enterocytes per villi was counted in 24-hour NEC and DF controls with or without rhIGF-1 / BP3 treatment. (D) The distance of intestinal cell migration was measured using Photoshop. For the dam-fostering group, n = 4 pups / group (3 litters combined), and for the NEC group, n = 4–8 pups / group from 4 litters combined. (E–F) Intestinal tissues were harvested 48 h after initiating NEC and stained with anti-cleaved caspase 3 antibody. The number of apoptotic cells per 20x field was counted. (E) Representative images are shown; scale bar = 25 mm. (F) Number of apoptotic cells per 20x field. n = 4–6 / group. P values ​​were calculated using one-way anova followed by the Tukey-Kramer multiple comparison test. *p<0.05, **p<0.01, ***p<0.001. [Figure 5]IGF-1 / IBP3 reduces systemic and local proinflammatory cytokine production. One-day-old neonatal mice were subjected to the experimental NEC model and injected with rhIGF-1 / BP3 (n = 8 pups / 8 samples) or vehicle control (n = 8 pups / 8 samples) as described. Untreated dam-fostered littermates and LPS-treated dam-fostered littermates were used as baseline and positive controls (n = 6 pups pooled in two samples for both the DF and LPS groups). 24 hours after initiating NEC, intestinal tissue lysates (A–C) and serum (D–E) were prepared, and cytokines were measured by ELISA. P values ​​were calculated using one-way anova followed by the Tukey-Kramer multiple comparison test (A–F). p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. [Figure 6] rhIGF-1 / BP3 improves survival and reduces tissue damage in experimental NEC. One-day-old neonates were subjected to experimental NEC and treated twice daily with rhIGF-1 / BP3 or vehicle control as described (see Methods section): (A) 60-hour survival curves are shown, and P values ​​were calculated using the log-rank (Mantel-Cox) test. n = 55 and 50 for the control and treatment groups, respectively; (B) Histological scores of intestinal damage (left graph) and the percentage of severe NEC (score ≥ 2, right graph) are shown, and P values ​​were calculated using the chi-square test. n = 54 and 44 for the NEC group and the NEC-rhIGF-1 / BP3-treated group, respectively. *p < 0.05, **p < 0.01. [Figure 7] IGF-1 / IBP3 maintained epithelial cell proliferation, which was attenuated by inhibiting VEGFR2 signaling. Intestinal cell proliferation was assessed in tissue sections from 36-hour NEC pups treated with rhIGF-1 / BP3, vehicle control, or rhIGF-1 / BP3 and Ki8751 and compared with dam-fostered (DF) controls, n=8-11 pups per group. ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0140] Example reagent Recombinant human IGF-1 / BP3 (rhIGF-1 / BP3, SHP607 DS, Lot#PR20151178-ENG) was provided by Takeda Pharmaceuticals USA (Deerfield, IL). Anti-CD31 (ab28364, for immunofluorescence staining) and rat anti-BrdU (catalog no. ab-6326) antibodies were purchased from Abcam (Cambridge, UK). Anti-VEGF-A (sc-7269) antibodies for Western blotting were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-IGF-1 antibodies (NBP2-16929) for Western blotting were purchased from Novus Biologicals (Littleton, CO). Goat anti-rabbit (A11034) and goat anti-rat (A11007) antibodies were obtained from Fisher Scientific (Waltham, MA). Anti-VEGFR2 (2479) antibody was purchased from Cell Signaling Technology (Danvers, MA). Evans blue, albumin, and formamide were purchased from Sigma (St. Louis, MO).

[0141] Animal strains and rearing strategies C57BL / 6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice ranging in age from 8 weeks to 6 months were routinely housed. Breeders were fed breeding chow and housed at a maximum ratio of 1 male to 3 females. An overnight rearing strategy was used to produce experimental pups. Pregnant dams were separated from their breeding cages and housed individually 3 days before expected delivery. Newborn pups with pigmented spots on the scrotum were identified as males. Pups were distributed to experimental groups based on sex and weight to limit bias. All procedures, animal breeding, maintenance, and procedures performed were approved by the Animal Care and Use Committee and in accordance with the regulations of the Northwestern University Center for Comparative Medicine.

[0142] Animal experiments Pups less than 24 hours old were either left with their dams to be bred or (1) exposed to a standardized adult mouse commensal bacterial preparation (10 8 (2) initial oral gavage with 200 mL of Esbilac infant formula (200 mL / kg) and LPS (5 mg / kg) every 3 hours; -1 day -1 and (3) a murine NEC model involving brief hypoxia (100% N2 for 60 seconds) followed immediately by cold stress (4°C for 10 minutes) twice daily. This well-established and widely used protocol was found to cause intestinal damage ranging from epithelial injury to transmural necrosis resembling human NEC, typically developing after 36 hours.

[0143] rhIGF-1 / BP3 or vehicle control was injected twice daily i.p. into pups undergoing the NEC protocol or into dam-fostered littermates. The first dose was administered 2 hours before the initiation of NEC, with daily doses of 3 mg / kg / d or 6 mg / kg / d. Pups were carefully observed and euthanized if they showed signs of distress. Whole intestinal tissue was collected. Hematoxylin and eosin-stained tissue sections were evaluated and scored by two investigators blinded to the group assignments described above. Severe NEC was defined as a histological score of ≥2. Pups found dead without direct observation were excluded from histological analysis. Alternatively, small intestinal tissue was collected 6–48 hours after the initiation of NEC for the studies described below.

[0144] Western blot Small intestinal tissue lysates were obtained by homogenizing tissue in Celllytic™ MT Cell Lysis Reagent (Sigma) containing protease inhibitors. Protein concentrations of tissue lysates were measured using the Bradford method. 20–50 μg of non-reduced protein was separated on a 4–15% precast gradient SDS-PAGE gel. For serum IGF-1 detection, native and non-reduced proteins were used for Western blot analysis. Proteins were transferred to nitrocellulose membranes and then blocked with 5% milk / Tween 20 for 60 minutes. Primary antibodies were added and incubated overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Target proteins were detected using standard Pierce enhanced chemiluminescence techniques. β-actin was probed on the same membrane as an internal control.

[0145] Endothelial cell proliferation test Pups were intraperitoneally injected with 50 mg / kg BrdU 4 hours before euthanasia and tissue collection. 5 mm-thick tissue sections were prepared from formalin-fixed, paraffin-embedded intestines. Sections were boiled in EDTA antigen retrieval buffer, pH 8.5, for 20 minutes, followed by deparaffinization and sequential hydration. After blocking with 10% normal goat serum for 1 hour at room temperature, sections were incubated with anti-CD31 (1:50) and anti-BrdU antibodies (1:200) overnight at 4°C. Fluorescently conjugated secondary antibodies were applied to the sections and incubated for 1 hour at room temperature. Images were taken with a Keyence BZ-800 microscope after adding mounting medium containing DAPI. Proliferating endothelial cells staining positive for both CD31 and BrdU were counted in at least three fields per sample.

[0146] Testing intestinal epithelial cell proliferation and migration NEC pups and DF littermates were injected i.p. with 50 mg / kg BrdU (BD Biosciences) at the onset of NEC. At 24 or 48 h after NEC initiation, pups were euthanized by decapitation, and their entire small intestines were fixed in 10% buffered formalin and processed for paraffin embedding and sectioning. Antigen retrieval of deparaffinized tissue sections (5 μm) was performed for 20 minutes as described above. Sections were then stained with rat anti-BrdU antibody (1:200), and anti-BrdU antibody was detected using Alexa Fluor 594-conjugated goat antibody against rat IgG (1:1000). Nuclei were counterstained with DAPI. Slides were examined under a Keyence BZ-X800 fluorescence microscope using appropriate filters. The distance of epithelial cell migration was assessed by measuring the gap between the base of the villi and the highest labeled cell within the villus shaft. At least 42 villi were evaluated per sample, and the average migration distance was used for subsequent analysis. Only intact villi were selected to examine how enterocytes migrate along the villus axis. Proliferation of intestinal epithelial cells was measured from tissue harvested 24 hours after NEC initiation. To do this, BrdU + The number of epithelial cells was counted per villi, with at least 9 villi counted per sample.

[0147] Apoptosis analysis Five-micrometer-thick sections from formalin-fixed, paraffin-embedded intestinal tissue were subjected to antigen retrieval as described above. Slides were blocked with 10% normal goat serum for 1 hour at room temperature and then incubated with anti-cleaved caspase 3 antibody (cell signaling #9664, 1:500) overnight at 4°C. Fluorescently conjugated goat anti-rabbit Ab was applied to the sections and incubated for 1 hour at room temperature. Images were taken with a Keyence BZ-800 microscope after adding mounting medium containing DAPI. Cleaved caspase-positive cells were evaluated in at least three fields per sample (20x magnification).

[0148] Intestinal vascular permeability assay Pups were anesthetized by ice hypothermia and intravenously injected with 10 μl of Evans Blue Albumin (EBA) solution (0.5% Evans Blue / 40 mg / ml BSA). One hour later, intestinal vascular permeability was measured as previously published. Saline (500 μl) was injected intracardially to remove stain accumulated in the circulation, and small intestinal tissue was harvested immediately after perfusion. The pancreas and surrounding connective tissue were carefully removed under a dissecting microscope, and small intestinal tissue lysates were prepared as described above. The EBA stain diffused into the intestinal tissue was extracted with formamide and quantified spectrophotometrically. Results were compared to serial dilutions of EBA at known concentrations and normalized to tissue weight.

[0149] Enzyme-linked immunosorbent assay For determination of serum rhIGF-1 / BP3 concentrations, pups were injected i.p. with 2 mg / kg or 10 mg / kg rhIGF-1 / BP3 approximately 8 hours after birth, and pups were euthanized 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, or 24 hours later. Blood was collected after decapitation, and serum was obtained by centrifugation. ELISA was performed using the Mediagnost (E20) IGFBP Block Human Insulin-Like Growth Factor ELISA according to the manufacturer's instructions (Mediagnost; Reutlingen, Germany).

[0150] For cytokine analysis, two to three blood samples were pooled for analysis as needed. Intestinal tissue lysates were prepared by homogenizing tissue in Celllytic™ MT Cell Lysis Reagent (Sigma) containing protease inhibitors. Proinflammatory cytokines, including IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, KC / GRO, IL-10, IL-12p70, and TNF-α, from serum and intestinal tissue lysates were detected using the V-PLEX Inflammatory Panel 1 Mouse Kit from Meso Scale Discovery according to the manufacturer's instructions. A MESO QuickPlex SQ120 instrument was used to read electrochemiluminescence signals. Cytokine concentrations were calculated using known standards. Data analysis was performed using DISCOVERY WORKBENCH assay analysis software, and intestinal results were normalized to tissue weight.

[0151] Statistical analysis. The statistical software GraphPad Prism (version 8.1.2) was used for statistical analysis. Animal survival data were analyzed by the log-rank or Gehan-Breslow-Wilcoxon test. Nonparametric chi-square (χ 2 ) test was used to compare the incidence of severe NEC (grade ≥ 2) between two groups. For other data, Student's two-tailed t-test was used for comparison between two groups, and One-way Anova was used for comparison of three or more groups. In the ANOVA test, correction for multiple comparisons was applied for pairwise comparisons after performing the ANOVA. Data were normalized when normalization tests using the Shapiro-Wilk test failed. Results are expressed as mean ± standard error of the mean (SEM). Differences were considered statistically significant when P ≤ 0.05.

[0152] result The IGF-1 / IBP3 / ALS complex is decreased in serum before the onset of experimental NEC. R: Both intestinal tissue and serum IGF-1 levels are reduced prior to the onset of experimental NEC. Given that IGF-1 is highly regulated in serum by its multiple binding proteins, serum IGF-1 bound to binding proteins was assessed during NEC development by Western blot using non-denaturing conditions. When pups were subjected to the 24-hour experimental NEC protocol, the IGF-1 ternary complex (IGF-1 / IBP3 / ALS) was significantly reduced in serum, whereas the levels of other complexes were not significantly different (Figure 1A-B).

[0153] rhIGF-1 / BP3 maintains intestinal VEGF / VEGFR2 expression and endothelial cell proliferation To determine the dose of rhIGF-1 / BP3 that improves plasma IGF-1 levels in neonates, dams and nursing pups were injected i.p. with 2 mg / kg or 10 mg / kg rhIGF-1 / BP3 (or vehicle control) approximately 8 hours after birth. Pups were euthanized, and serum was collected 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, or 24 hours after rhIGF-1 / BP3 injection for detection of human IGF-1 by ELISA. Injection of 10 mg / kg, but not 2 mg / kg, rhIGF-1 / BP3 significantly increased serum IGF-1 concentrations in neonatal serum within 30 minutes, with a peak (165.7 ± 49.7 ng / ml) reached at 2 hours (Figure 2), and the effect was not significant after 8 hours. Based on literature data for both humans and mice, a physiological range of 30-110 ng / ml was targeted, and therefore a dose of 6 mg / kg / day was used in subsequent experiments.

[0154] In the intestine, VEGF and VEGFR2 protein were downregulated, and endothelial cell proliferation and intestinal microvascular density were reduced in NEC-induced pups compared with dam-fed controls, which were ameliorated by exogenous IGF-1. To determine whether rhIGF-1 / BP3 maintains intestinal microvascular development during NEC, VEGF and VEGFR2 expression and endothelial cell proliferation were assessed in the intestines of neonatal mice injected with rhIGF-1 / BP3 or vehicle control. In pups subjected to the 24-hour NEC model, treatment with rhIGF-1 / BP3 (6 mg / kg / day) maintained intestinal VEGF and VEGFR2 expression (Figures 3A-C). Furthermore, administration of exogenous rhIGF-1BP3 at the same dose ameliorated the NEC-induced decrease in villous endothelial cell proliferation, but not the vehicle control (Figures 3D-E).

[0155] Exogenous rhIGF-1 / BP3 ameliorates NEC-induced increased intestinal vascular permeability Next, we sought to determine whether NEC affected intestinal vascular permeability and whether permeability was improved by administration of rhIGF-1 / BP3. To do this, rhIGF-1 / BP3 (6 mg / kg / day) or vehicle control was administered to pups subjected to the NEC model. Vehicle-treated dam-fostered pups served as controls. 24 h later, intestinal vascular permeability was assessed by quantifying EBA leakage into intestinal tissue after iv injection. Mean EBA concentrations were found to be significantly higher in the intestinal tissue of pups subjected to the NEC protocol compared with dam-fostered controls, suggesting higher intestinal vascular permeability. Furthermore, rhIGF-1 / BP3 significantly improved NEC-induced intestinal EBA leakage (Figure 3F).

[0156] Exogenous rhIGF-1 / BP3 promotes epithelial cell proliferation and migration during experimental NEC To determine the effects of rhIGF-1 / BP3 on epithelial cell proliferation and migration, pups less than 24 hours old were subjected to the NEC protocol or dam nursing for 24 or 48 hours. Starting 2 hours before the onset of NEC, subgroups of pups were injected i.p. with 6 mg / kg / day of rhIGF-1 / BP3 or vehicle. All pups were injected i.p. with 50 mg / kg BrdU at the onset of NEC to label proliferating cells, and intestinal tissue was harvested at 24 or 48 hours to assess epithelial cell proliferation and migration. We found that at 24 hours, epithelial cell proliferation was significantly reduced in the intestinal tissue of NEC pups (Figures 4A and 4C), and that rhIGF-1 / BP3 treatment reversed the NEC-induced epithelial cell proliferation defect (Figures 4A and 4C). Furthermore, at 48 hours, intestinal epithelial cell migration was attenuated in NEC pups, and rhIGF-1 / BP3 treatment increased intestinal cell migration in both NEC and DF pups (Figures 4A, 4B, 4D).

[0157] IGF-1 / BP3 protects animals against NEC-induced intestinal epithelial cell apoptosis To determine whether IGF-1 / BP3 protects against intestinal cell apoptosis during experimental NEC, 24-hour-old pups were subjected to NEC and treated with IGF-1 / BP3 (6 mg / kg / day, i.p.) or vehicle. At 48 hours after the initiation of NEC, intestinal tissue was Cleaved caspase 3 We stained for IGF-1 / BP3. We observed a significantly higher number of apoptotic cells (mainly epithelial cells) in NEC tissue compared to DF controls. Furthermore, the number of apoptotic enterocytes was reduced when exogenous IGF-1 / BP3 was administered (Figure 4E-F).

[0158] IGF-1 / IBP3 reduces systemic and local proinflammatory cytokine production To determine whether rhIGF-1 / BP3 has an effect on systemic inflammation during NEC development, pups exposed to the NEC protocol for 24 hours were treated with either rhIGF-1 / BP3 or vehicle control and compared with dam-fostered controls treated with or without LPS (1 mg / kg, i.p., for 6 hours—negative and positive controls, respectively). Pup serum and intestinal tissue lysates were assessed for inflammatory cytokines by ELISA. Intestinal IL-4, IL-6, and IL-10 (Figures 5A-C) and serum IL-6 and CXCL1 (Figures 5D-E) were significantly increased during NEC and significantly reduced by administration of exogenous rh / IGF-1 / BP3.

[0159] rhIGF-1 / BP3 improves survival and reduces tissue damage in experimental NEC To determine whether administration of rhGF-1 / BP3 protects the intestine against injury in a neonatal NEC model, pups were injected i.p. with rhIGF-1 / BP3 or vehicle control twice daily (6 mg / kg / day), with the first dose administered 2 hours before the onset of NEC. When treated with rhGF-1 / BP3, NEC pups had significantly improved survival compared with vehicle-treated NEC controls (median survival of 64 hours in the rhIGF-1 / BP3-treated group compared with 52 hours in the vehicle-treated group) (Fig. 6A). Furthermore, the incidence of severe histological intestinal damage (grade 2) was significantly lower in rhGF-1 / BP3-treated pups compared with those treated with vehicle control (13 / 44 (30%) vs. 27 / 54 (50%) -χ 2 = 4.20, p 0.05 - Figure 6B).

[0160] Consideration Exogenous rhIGF-1 / BP3 is undergoing a phase IIb multicenter clinical trial to determine its efficacy in diseases affecting preterm infants, such as ROP and BPD (ClinicalTrials.gov registries NCT03253263 and NCT03253263). However, the effects of exogenous rhIGF-1 / BP3 on the neonatal intestine remain unknown.

[0161] In pups subjected to experimental NEC, serum IGFBP3-bound IGF-1 was reduced, and exogenous administration of rhIGF-1 / BP-3 (6 mg / kg / d) maintained VEGF and VEGFR2 protein expression, reduced vascular permeability, and maintained intestinal endothelial cell proliferation. Furthermore, rhIGF-1 / BP-3 promoted enterocyte proliferation and migration while reducing enterocyte apoptosis and attenuated systemic and intestinal tissue inflammation. Finally, we demonstrated that rhIGF-1 / BP-3 improved survival and reduced the incidence of severe intestinal damage in experimental NEC. Thus, supplemental rhIGF-1 / BP-3 (6 mg / kg / d) protects neonatal mice against experimental NEC through multiple mechanisms.

[0162] Because IGF-1 is bound by several IGFBPs and is decreased during experimental NEC, we sought to determine which form of IGF-1 is decreased during NEC development.

[0163] Western blot analysis of mouse serum, as well as adult human blood, revealed that the majority of serum IGF-1 was present in a protein complex of 160-170 kDa in size, likely corresponding to IGF-1 trapped in a ternary complex consisting of IGF-1, one molecule each of IGFBP-3 or IGFBP-5, and an acid-labile subunit. A small amount of serum IGF-1 was present in a complex of approximately 74 kDa in size, presumably bound to other IGFBPs, and a small amount of IGF-1 existed as a free form of <13 kDa in size.

[0164] Interestingly, we found that only IGFBP3 / ALS-bound IGF-1 was reduced, while the other two forms remained unchanged 24 h after NEC onset. The decrease in bound IGF-1 levels may be related to IL-6-mediated proteolysis of IGFBP-3 with increased clearance of free IGF-1.

[0165] Herein, we show that rhIGF-1 / BP3 (6 mg / kg / d) promotes intestinal VEGF / VEGFR2 expression and endothelial cell proliferation, both of which are significantly reduced during NEC.

[0166] Decreased intestinal cell migration and proliferation in neonatal rats and mice in multiple pathologies. Here, we found that epithelial cell proliferation and migration, which are substantially reduced before tissue injury, were significantly improved in pups treated with rhIGF-1 / BP3.

[0167] Epithelial cell proliferation, differentiation, and migration are required for the maintenance of an intact epithelial layer. Apoptosis of intestinal epithelial cells precedes experimental NEC. Here, we found that exogenous rhIGF-1 / BP3 attenuated intestinal cell apoptosis.

[0168] Our present study showed that administration of rhIGF-1 / BP3 significantly reduced the production of several inflammatory cytokines, including IL-6, both locally and systemically during experimental NEC.

[0169] Together, our studies suggest that in neonatal mice, supplementation with rhIGF-1 / BP3 at a dose of 6 mg / kg / day may protect against severe intestinal injury through multiple mechanisms: preserving intestinal microvascular development and integrity, promoting enterocyte proliferation and migration while reducing enterocyte apoptosis, and reducing local and systemic inflammation.

[0170] References 1. Neu J, Walker WA. Necrotizing enterocolitis. N Engl J Med 2011;364:255-64. 2. Bowker RM, Yan X, Managlia E, et al. Dimethyloxalylglycine preserves the intestinal microvasculature and protects against intestinal injury in a neonatal mouse NEC model: role of VEGF signaling. Pediatr Res 2018;83:545-553. 3. Yan X, Managlia E, Liu SX, et al. Lack of VEGFR2 signaling causes maldevelopment of the intestinal microvasculature and facilitates necrotizing enterocolitis in neonatal mice. Am J Physiol Gastrointest Liver Physiol 2016;310:G716-25. 4. Yan X, Managlia E, Zhao YY, et al. Macrophage-derived IGF-1 protects the neonatal intestine against necrotizing enterocolitis by promoting microvascular development. Commun Biol 2022;5:320. 5. Hellstrom A, Engstrom E, Hard AL, et al. Postnatal serum insulin-like growth factor I deficiency is associated with retinopathy of prematurity and other complications of premature birth. Pediatrics 2003;112:1016-20. 6. Holgersen K, Gao X, Narayanan R, et al. Supplemental Insulin-Like Growth Factor-1 and Necrotizing Enterocolitis in Preterm Pigs. Front Pediatr 2020;8:602047. 7. Yakar S, Wu Y, Setser J, et al. The role of circulating IGF-I: lessons from human and animal models. Endocrine 2002;19:239-48. 8. Rajaram S, Baylink DJ, Mohan S. Insulin-like growth factor-binding proteins in serum and other biological fluids: regulation and functions. Endocr Rev 1997;18:801-31. 9. Boisclair YR, Rhoads RP, Ueki I, et al. The acid-labile subunit (ALS) of the 150 kDa IGF-binding protein complex: an important but forgotten component of the circulating IGF system. J Endocrinol 2001;170:63-70. 10. Jeschke MG, Bolder U, Chung DH, et al. Gut mucosal homeostasis and cellular mediators after severe thermal trauma and the effect of insulin-like growth factor-I in combination with insulin-like growth factor binding protein-3. Endocrinology 2007;148:354-62. 11. Baregamian N, Song J, Jeschke MG, et al. IGF-1 protects intestinal epithelial cells from oxidative stress-induced apoptosis. J Surg Res 2006;136:31-7. 12. Hunninghake GW, Doerschug KC, Nymon AB, et al. Insulin-like growth factor-1 levels contribute to the development of bacterial translocation in sepsis. Am J Respir Crit Care Med 2010;182:517-25. 13. Ozen S, Akisu M, Baka M, et al. Insulin-like growth factor attenuates apoptosis and mucosal damage in hypoxia / reoxygenation-induced intestinal injury. Biol Neonate 2005;87:91-6. 14. Wolterink-Donselaar IG, Meerding JM, Fernandes C. A method for gender determination in newborn dark pigmented mice. Lab Anim (NY) 2009;38:35-8. 15. Tian R, Liu SX, Williams C, et al. Characterization of a necrotizing enterocolitis model in newborn mice. Int.J.Clin.Exp.Med. 2010;3:293-302. 16. Bu HF, Zuo XL, Wang X, et al. Milk fat globule-EGF factor 8 / lactadherin plays a crucial role in maintenance and repair of murine intestinal epithelium. J Clin Invest 2007;117:3673-83. 17. Yan X, Managlia E, Tan XD, et al. Prenatal inflammation impairs intestinal microvascular development through a TNF-dependent mechanism and predisposes newborn mice to necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 2019;317:G57-g66. 18. Diaz-Gomez NM, Domenech E, Barroso F. Breast-feeding and growth factors in preterm newborn infants. J Pediatr Gastroenterol Nutr 1997;24:322-7. 19. Hellstrom A, Ley D, Hansen-Pupp I, et al. Role of Insulinlike Growth Factor 1 in Fetal Development and in the Early Postnatal Life of Premature Infants. Am J Perinatol 2016;33:1067-71. 20. Andersen AD, Sangild PT, Munch SL, et al. Delayed growth, motor function and learning in preterm pigs during early postnatal life. Am J Physiol Regul Integr Comp Physiol 2016;310:R481-92. 21. Cakir B, Hellstrom W, Tomita Y, et al. IGF1, serum glucose, and retinopathy of prematurity in extremely preterm infants. JCI Insight 2020;5. 22. De Benedetti F, Meazza C, Oliveri M, et al. Effect of IL-6 on IGF binding protein-3: a study in IL-6 transgenic mice and in patients with systemic juvenile idiopathic arthritis. Endocrinology 2001;142:4818-26. 23. Bowker RM, Yan X, De Plaen IG. Intestinal microcirculation and necrotizing enterocolitis: The vascular endothelial growth factor system. Semin Fetal Neonatal Med 2018;23:411-415. 24. Slomiany MG, Rosenzweig SA. Hypoxia-inducible factor-1-dependent and -independent regulation of insulin-like growth factor-1-stimulated vascular endothelial growth factor secretion. J Pharmacol Exp Ther 2006;318:666-75. 25. Feng J, Besner GE. Heparin-binding epidermal growth factor-like growth factor promotes enterocyte migration and proliferation in neonatal rats with necrotizing enterocolitis. J.Pediatr.Surg. 2007;42:214-220. 26. Cetin S, Ford HR, Sysko LR, et al. Endotoxin inhibits intestinal epithelial restitution through activation of Rho-GTPase and increased focal adhesions. J.Biol.Chem. 2004;279:24592-24600. 27. Leaphart CL, Cavallo J, Gribar SC, et al. A critical role for TLR4 in the pathogenesis of necrotizing enterocolitis by modulating intestinal injury and repair. J Immunol 2007;179:4808-20. 28. Fholenhag K, Arrhenius-Nyberg V, Sjogren I, et al. Effects of insulin-like growth factor I (IGF-I) on the small intestine: a comparison between oral and subcutaneous administration in the weaned rat. Growth Factors 1997;14:81-8. 29. Alexander AN, Carey HV. Oral IGF-I enhances nutrient and electrolyte absorption in neonatal piglet intestine. Am J Physiol 1999;277:G619-25. 30. Houle VM, Schroeder EA, Odle J, et al. Small intestinal disaccharidase activity and ileal villus height are increased in piglets consuming formula containing recombinant human insulin-like growth factor-I. Pediatr Res 1997;42:78-86. 31. Jilling T, Lu J, Jackson M, et al. Intestinal epithelial apoptosis initiates gross bowel necrosis in an experimental rat model of neonatal necrotizing enterocolitis. Pediatr.Res. 2004;55:622-629. 32. Zhang W, Frankel WL, Adamson WT, et al. Insulin-like growth factor-I improves mucosal structure and function in transplanted rat small intestine. Transplantation 1995;59:755-61. 33. Baregamian N, Rychahou PG, Hawkins HK, et al. Phosphatidylinositol 3-kinase pathway regulates hypoxia-inducible factor-1 to protect from intestinal injury during necrotizing enterocolitis. Surgery 2007;142:295-302. 34. Tian F, Liu GR, Li N, et al. Insulin-like growth factor I reduces the occurrence of necrotizing enterocolitis by reducing inflammatory response and protecting intestinal mucosal barrier in neonatal rats' model. Eur Rev Med Pharmacol Sci 2017;21:4711-4719. 35. Ge RT, Mo LH, Wu R, et al. Insulin-like growth factor-1 endues monocytes with immune suppressive ability to inhibit inflammation in the intestine. Sci Rep 2015;5:7735. 36. Puzik A, Rupp J, Troger B, et al. Insulin-like growth factor-I regulates the neonatal immune response in infection and maturation by suppression of IFN-γ. Cytokine 2012;60:369-76. 37. Klevebro S, Hellgren G, Hansen-Pupp I, et al. Elevated levels of IL-6 and IGFBP-1 predict low serum IGF-1 levels during continuous infusion of rhIGF-1 / rhIGFBP-3 in extremely preterm infants. Growth Horm IGF Res 2020;50:1-8. 38. Sheng X, Sun X, Li F, et al. Linear growth failure induced by systemic inflammation inhibiting IGF-1 / IGFBP axis in rats with asymptomatic colitis. BMC Gastroenterol 2019;19:96. 39. Hellgren G, Lofqvist C, Hansen-Pupp I, et al. Increased postnatal concentrations of pro-inflammatory cytokines are associated with reduced IGF-I levels and retinopathy of prematurity. Growth Horm IGF Res 2018;39:19-24.

Claims

1. A method of treating or preventing dysregulation of GI tract regeneration, for example in infants, such as preterm infants (also referred to herein as newborns) and / or infants, such as small-for-gestational-age infants or infants born after preeclampsia, by administering a therapeutic amount of a composition comprising IGF-1 and an IGF binding protein (such as IGFBP-3), particularly as a complex. (Claim 1A) 1. A composition comprising IGF-1 and an IGF binding protein (such as IGFBP-3), particularly as a complex, for use in the treatment or prevention of dysregulation of intestinal regeneration in infants, for example preterm infants (also referred to herein as newborns) and / or infants such as small-for-gestational age infants or infants born after preeclampsia. (Claim 1B) 1. A composition comprising IGF-1 and an IGF binding protein (such as IGFBP-3), particularly as a complex, for use in the manufacture of a medicament for the treatment or prevention of dysregulated intestinal regeneration, for example in infants such as preterm infants (also referred to herein as newborns) and / or small-for-gestational-age infants or infants born after preeclampsia. (Claim 1C) A method of treatment or prevention by stimulating GI cell maturation (e.g., to increase and / or maintain tight junctions and / or) in infants, such as preterm infants (also referred to herein as newborns) and / or small-for-gestational-age infants, by administering a therapeutic amount of a composition comprising IGF-1 and an IGF binding protein (such as IGFBP-3), particularly as a complex.

2. 10. The method or composition of any one of claims 1, 1A, 1B and 1C, wherein the treatment reduces inflammation, e.g., inflammation in the GI tract, particularly inflammation produced by cytokines selected from IL-4, IL-6, IL-10 and combinations of two or three thereof.

3. 3. The method or composition of any one of claims 1 to 2, wherein inflammation-initiated destruction of IGFBP-3 in tissues is minimized and / or destruction (including removal) of IGF-1 is minimized.

4. 4. The method or composition of any one of claims 1 to 3, wherein a positive feedback loop for IGF-1 production is supported, stabilized, maintained, initiated and / or increased.

5. 5. The method or composition of any one of claims 1 to 4, wherein vascular permeability is reduced, e.g., compared to untreated patients with necrotizing enterocolitis, in particular vascular leakage into the intestinal tissue is minimized and / or leakage from the GI tract into the vasculature is minimized.

6. 6. The method or composition of any one of claims 1 to 5, wherein tight junctions are promoted, preserved, supported, stabilized, maintained, initiated and / or increased by the treatment, e.g., the proportion of tight junctions is preserved or increased.

7. 7. The method or composition of any one of claims 1 to 6, wherein the treatment supports the maintenance of epithelial barrier function and / or epithelial integrity, including, for example, preventing basement membrane rupture, and / or preventing exposure of basal stromal cells to luminal contents.

8. 8. The method or composition of any one of claims 1 to 7, wherein tight junctions are promoted, preserved, supported, stabilized, maintained, initiated and / or increased by the treatment, e.g., the proportion of tight junctions is preserved or increased.

9. 7. The method or composition of any one of claims 1 to 6, wherein the treatment supports the maintenance of epithelial barrier function and / or epithelial integrity, including, for example, preventing basement membrane rupture, and / or preventing exposure of basal stromal cells to luminal contents.

10. 10. The method or composition of any one of claims 1 to 9, wherein epithelial cell migration is preserved, supported, maintained, initiated and / or increased by the treatment.

11. 11. The method or composition of any one of claims 1 to 10, wherein epithelial cell apoptosis, e.g., intestinal cell apoptosis, e.g., apoptosis induced by oxidative stress, is minimized (modulated) by treatment.

12. 12. The method or composition of any one of claims 1 to 11, wherein the treatment promotes intestinal cell proliferation, e.g., compared to an untreated patient.

13. 13. The method or composition of any one of claims 1 to 12, wherein the function of the intestinal cells, such as a function selected from ion uptake, water uptake, sugar uptake, peptide uptake, amino acid uptake, lipid uptake, vitamin B12 uptake, immunoglobulin secretion, and combinations of two or more thereof, such as all functions, is preserved, supported, maintained, initiated and / or increased.

14. 14. The method or composition of any one of claims 1 to 13, wherein Paneth cell function, such as Wnt signaling and Notch signaling and / or secretion of defensins HD-5 and HD-6, is preserved, supported, maintained, initiated and / or increased.

15. 15. The method or composition of any one of claims 1 to 14, wherein Paneth cell dysfunction is minimized by treatment, e.g., compared to untreated patients.

16. 16. The method or composition of any one of claims 1 to 15, wherein the treatment reduces the incidence of severe damage to the GI tract, such as severe intestinal damage, in particular reducing the significant incidence of severe necrotizing enterocolitis, for example by promoting and / or modulating the healing process.

17. 17. The method or composition of claim 16, wherein severe damage to the villi, such as destruction of the villi and / or malformation of the villi, is minimized.

18. 18. The method or composition of any one of claims 1 to 17 for use in the treatment of colitis (including necrotizing enterocolitis), C. difficile infection, Shigella infection, or toxic megacolon.

19. 17. The method or composition of claim 16, wherein the significant incidence of severe necrotizing enterocolitis, e.g., a score of 2 or higher (such as 3, 4, 5, and 6), is reduced compared to untreated patients with necrotizing enterocolitis.

20. 20. The method or composition of any one of claims 1 to 19, wherein the incidence of sepsis is reduced in treated patients.

21. 21. The method or composition of any one of claims 1 to 20, wherein the dose for infants, such as preterm infants, infants low for gestational age and / or infants born after preeclampsia, is a complex of 200-500 μg / Kg / 24 hours, such as 350-500 μg / Kg / 24 hours, 400 μg / Kg / 24 hours, especially by continuous infusion.

Images