Parenteral nutrition comprising glucogenic amino acids and galactose

Abstract

The present invention relates to parenteral nutrition (PN). In particular, the present invention relates to PN composition comprising glucogenic amino acids and galactose, method for producing said PN composition, and the PN composition for use in the treatment or prevention of infection, such as preventing or delaying sepsis in a subject by optimizing the immune system of said subject.

Description

[0001] 85583PC01

[0002] 1

[0003] Parenteral Nutrition Comprising Glucogenic Amino Acids

[0004] Technical field of the invention

[0005] The present invention relates to parenteral nutrition (PN). In particular, the present invention relates to a PN composition comprising glucogenic amino acids and galactose, method for producing said PN composition, and the PN composition for use in the treatment or prevention of infection, such as preventing or delaying sepsis in a subject by optimizing the immune system of said subject.

[0006] Background of the invention

[0007] Infection and poor glycemic control are major concerns in very preterm infants, especially when they receive glucose-rich parenteral nutrition in early life due to gut immaturity. Hyperglycemia during infection can exaggerate glycolysis-induced inflammation and lead to life-threatening sepsis, while glucose restriction decreases sepsis but induces severe hypoglycemia. It is unclear how nutritional intake and composition affect glucose dysregulation and sepsis.

[0008] Newborn infants, particularly those born very preterm (< 32 weeks of gestation), are extremely susceptible to infections that can lead to life-threatening sepsis with severe tissue damage and organ dysfunction. The high risk of infection in preterm infants has conventionally been attributed to their immature immune systems with a limited capacity to respond to exogenous stimuli. However, the existing evidence suggests that neonatal sepsis develops due to dysregulated host infection responses causing tissue damage.

[0009] Neonates have limited energy reserves and high energy demands for growth. As a result, newborns are programmed to prioritize their energy for vital organ functions rather than for inflammatory responses to infection, leading them to tolerate invading pathogens. However, when pathogen growth exceeds a certain threshold, the glycolysis-mediated resistance mechanism can occur in an excessive manner, causing sepsis with collateral tissue damage and organ dysfunctions.

[0010] Nourishing newborn preterm infants is challenging, as their immature gut may not tolerate high amounts of enteral nutrition. Therefore, they often receive glucose-rich parenteral nutrition (PN) shortly after birth to ensure adequate nutrition and85583PC01

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[0012] to avoid hypoglycemia. However, due to their poor glucose homeostatic control (insulin resistance), this practice often leads to hyperglycemia, which may amplify glycolysis-induced inflammation upon pathogen invasion, increasing sepsis risk. Commercial PN compositions comprises a high level of glucose and various amino acids.

[0013] US 2021 / 169115 Al discloses parenteral neonatal nutrition formulations, especially for preterm or very preterm neonates, that reduce the

[0014] incidence of infections and necrotising enterocolitis and enhance

[0015] immune response. The PN formulations comprises GAAs in combination with glucose.

[0016] Abstract 403 of JOURNAL OF PAEDIATRICS AND CHILD HEALTH, BLACKWELL SCIENTIFIC PUBL, MELBOURNE, AU, vol. 59, 24 September 2023 discloses glucose restriction combined with supplementation of GAAs maintains normoglycaemia and reduces the risk of sepsis. There is no explicit disclosure of the concentration of the GAAs in the parenteral formulation.

[0017] Baek Ole et al. (Altered hepatic metabolism mediates sepsis preventive effects of reduced glucose supply in infected preterm newborns"),

[0018] Section for Comparative Pediatrics and Nutrition, Department of Veterinary and Animal Sciences, University of Copenhagen, 1 July 2024) discloses metabolic challenges in preterm infants and their relation to the development of neonatal sepsis. Parental nutrition with a reduced glucose content is recommended for preventing sepsis in preterm animals, and that glucogenic amino acids may contribute to the maintenance of glucose homeostasis under reduced glucose supply. This document does not disclose a PN comprising GAAs in a specified amount.

[0019] Hence, an improved glucose homeostatic control and balanced defence strategy would be advantageous, and in particular an improved parenteral nutrition composition with less glucose would be advantageous.85583PC01

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[0021] Summary of the invention

[0022] In preterm neonates, hypoglycemia is among the most common metabolic disturbances shortly after birth, which is partly derived from their low levels of fat and glycogen stores. On the other hand, they are often insulin resistant, which causes high risks of hyperglycemia upon nourishment with glucose-rich PN.

[0023] Neonatal hyperglycemia is associated with many adverse outcomes, including increased risk of sepsis, adverse neurological outcomes, and reduced long-term growth. Mechanistically, hyperglycemia during neonatal infection can trigger excessive glycolysis-induced resistance response, risking the development of lethal sepsis. Hence, newborn health maintenance upon infection requires a delicate balance of host glucose homeostasis to control defence strategies with appropriate levels of inflammatory responses.

[0024] The inventors have anticipated that improved glucose homeostatic control and balanced defence strategies can be lifesaving during neonatal infection. Using a neonatal sepsis model in preterm piglets infected with Staphylococcus epidermidis, which is the most common pathogen causing neonatal sepsis in preterm infants and administering a PN composition comprising a high amount of glucogenic amino acids (GAAs) and galactose, and they have shown a dampened hepatic glycolysis and inflammation. It was found that adding further glucogenic amino acids (GAAs) and galactose to a commercial PN composition or replacing a major fraction of monosaccharides with glucogenic amino acids and galactose simultaneously enhanced disease resistance and tolerance, in turn maximizing infection survival. Thus, the PN composition administered to a subject comprises a reduced amount of glucose while adding galactose and an additional amount of glucogenic amino acids (GAAs) on top of the amino acids already present in a (commercial) PN composition.

[0025] GAAs are converted into glucose through a process called gluconeogenesis.

[0026] Galactose is converted to glucose via the Leloir pathway, comprising the latter stage of a two-part process that converts p-D-galactose to UDP-glucose.

[0027] Hence, glucose is slowly generated from GAAs and galactose via different metabolic pathways allowing for a better control and balance of the glucose homeostasis, thus preventing sudden glucose spikes and hyperglycaemia, as well as preventing excessive glycolysis-induced inflammatory responses.85583PC01

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[0029] Thus, an object of the present invention relates to the provision of a parenteral nutrition (PN) composition comprising glucogenic amino acids and galactose, for optimizing immune function and thereby preventing, treating, or delaying sepsis in a subject, such as a preterm infant.

[0030] In particular, it is an object of the present invention to provide a PN composition comprising glucogenic amino acids and galactose, and optionally less or no glucose, which solves the above-mentioned problems of the prior art with hyperglycemia and glycolysis-induced inflammation due to conventional glucose-rich parenteral nutrition. The PN composition according to the invention is a stock PN composition, which can be added to a second PN composition, such as a commercial PN composition, resulting in a final combination PN composition, which is administered to a subject.

[0031] Thus, one aspect of the invention relates to a parenteral nutrition (PN) composition comprising:

[0032] a) one or more glucogenic amino acids (GAAs) in an amount of at least 0.5% by weight of the PN composition, and

[0033] b) galactose in an amount of at least 1% by weight of the PN composition.

[0034] Another aspect of the invention relates to a combination parenteral nutrition (PN) composition comprising:

[0035] a) a first PN composition, wherein the first PN composition is the PN composition according to the invention, and

[0036] b) a second PN composition.

[0037] Another aspect of the present invention relates to a method for producing a PN composition according to the invention comprising the steps of:

[0038] a) providing one or more glucogenic amino acids (GAAs) in an amount of at least 0.5% by weight of the PN composition;

[0039] b) providing galactose in an amount of at least 1% by weight of the PN composition;

[0040] c) optionally, providing one or more additional components;

[0041] d) mixing the one or more GAAs, galactose, and the optional one or more additional components; and85583PC01

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[0043] e) obtaining the PN composition.

[0044] A further aspect of the invention relates to a method for producing a combination PN composition according to the invention comprising the steps of:

[0045] 1) providing a first PN composition, wherein the first PN composition is the PN composition according to the invention;

[0046] 2) providing a second PN composition;

[0047] 3) mixing the first PN composition of step a) and the second PN composition of step b); and

[0048] 4) obtaining the combination PN composition.

[0049] Yet another aspect of the present invention is to provide a parenteral nutrition (PN) composition or a combination PN composition according to the invention for use in the treatment and / or prevention of infection or sepsis in a subject.

[0050] Still another aspect of the present invention is to provide use of the PN composition according to the invention for mixing or combining with a second PN composition.

[0051] An aspect of the present invention relates to a kit of parts comprising

[0052] • the PN composition according to the invention,

[0053] • a second PN composition, and

[0054] • optionally, instructions for use.

[0055] Brief description of the figures

[0056] Figure 1 shows that combined glucose restriction and glucogenic amino acids supply prevent lethal sepsis and organ dysfunctions. (A) Experimental design. Forty-four cesarean-delivered preterm piglets were randomly assigned to be exclusively nourished with parenteral nutrition containing either a combination of 1.4 % glucose (2 g / kg / d) and four glucogenic amino acids (aspartate, glutamate, asparagine, valine; each 0.5 g / kg / d) (rGLU-GAAs) or 10% glucose (sGLU; 14.4 g / kg / d). Piglets were infused with S. epidermidis (SE) or control saline (CON). This setup allowed for the division of piglets into four groups: CON-sGLU (n = 6), CON-rGLU-GAAs (n = 5), SE-sGLU (n = 16), and SE-rGLU-GAAs (n = 17). (B)85583PC01

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[0058] Survival curves of the animals throughout the experiment, displayed as the time until humane endpoints or scheduled euthanasia. A log-rank test was used to compare survival between the SE-rGLU-GAAs and SE-sGLU groups. (C-I) Blood gas parameters ((C) blood glucose, (D) pH, (E) oxygen saturation, (F) base excess, (G) hemoglobin, (H) pCC , and (I) lactate) at 3, 6, 12, and 15 h postinoculation or at euthanasia. Data at each time point were analyzed using a linear mixed-effects model, incorporating group, gender, and birth weight as fixed factors and litter as a random factor. *P-value < 0.05, **P-value < 0.01, and ***P-value < 0.001, compared between SE-rGLU-GAAs and SE-sGLU groups at the same time point. Another linear mixed-effects model was employed to probe further disparities spanning the entire experimental duration, incorporating group, time, their interaction, gender, and birth weight as fixed factors, with litter and pig ID as random factors. Ptreat(SE), Ptime(SE), and Pint(SE) denote probability values for group effect (SE-rGLU-GAAs and SE-sGLU) over time, time effects, and the interaction effects between time and group in the linear mixed effects interaction model, respectively. Uninfected animals (CON) served as a reference group and were not compared directly with infected animals (SE). Statistical significance was defined as P-value < 0.05. All data were presented as box and whisker plots showing the range from minimum to maximum values.

[0059] Figure 2 shows that combined glucose restriction and glucogenic amino acid supply enhances both, host disease resistance and tolerance. (A) The density of S. epidermidis in the blood of infected piglets, determined by (CFU) assays. (B-I) Blood immune parameters (blood total leukocytes, lymphocytes, neutrophils, monocytes, platelets, plasma TNF-o, plasma IL-6, and plasma IL-10) at 3, 6, 12, and 15 h post-bacterial inoculation or at euthanasia. Statistics: Data at each time point were analyzed using a linear mixed-effects model, incorporating group, gender, and birth weight as fixed factors and litter as a random factor. *P-value < 0.05, **P-value < 0.01, and ***P-value < 0.001, compared between SE-rGLU-GAAs and SE-sGLU groups at the same time point. Another linear mixed-effects model was employed to probe further disparities spanning the entire experimental duration, incorporating group, time, their interaction, gender, and birth weight as fixed factors, with litter and pig ID as random factors. Ptreat(SE), Ptime(SE), and Pint(SE) denote probability values for group effect (SE-rGLU-GAAs and SE-sGLU) over time, time effects, and the interaction effects between time and group in the85583PC01

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[0061] linear mixed effects interaction model, respectively. Uninfected animals (CON) served as a reference group and were not compared directly with infected animals (SE). Statistical significance was defined as P-value < 0.05. All data were presented as box and whisker plots showing the range from minimum to maximum values.,

[0062] Figure 3 shows that hepatic transcriptomics and metabolomics revealed sepsis preventive effects of rGLU-GAAs supply associated with suppression of glycolysis and inflammation and elevation of gluconeogenesis. (A) GSEA was performed between the SE-rGLU-GAAs and SE-sGLU groups using the Sus scrofa (pig) KEGG knowledgebases. Significant pathways in specific categories such as energy metabolism, carbohydrate metabolism, amino acid metabolism, lipid metabolism, signal transduction, and immune system have been selected for visualization. The size and tone of the dots indicate the gene ratio and FDR values, respectively. Figure 3A has been divided into three sections (top, middle, and bottom). Any indication in any of the sections apply in all sections. (B) shows the effects of rGLU-GAAs supply on hepatic metabolism. GSEA was performed between the CON-rGLU-GAAs and CON-sGLU groups using the Sus scrofa (pig) KEGG knowledgebase. Significant pathways in specific categories such as energy metabolism, carbohydrate metabolism, amino acid metabolism, lipid metabolism, signal transduction, and immune system have been chosen for presentation. The size and tone of the dots indicate the gene ratio and FDR values, respectively. Figure 3B has been divided into two sections (top and bottom). Any indication in any of the sections apply in both sections,

[0063] Figure 4 shows MDAs-based pathways results between SE-rGLU-GAAs and SE-sGLU groups. The tone of a circle indicates the level of enrichment significance (y-axis), with darker grey for high and lighter grey for low, and the size of a circle is proportional to the pathway's impact value (x-axis). The table presents the significant pathways,

[0064] Figure 5 shows MDAs-based pathway results between SE-rGLU-GAAs and SE-sGLU groups. The tone of a circle indicates the level of enrichment significance (y-axis), with darker grey for high and lighter grey for low, and the size of a circle is proportional to the pathway's impact value (x-axis). The table presents the85583PC01

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[0066] significant pathways, highlighting their role between SE-rGLU-GAAs and SE-sGLU groups,

[0067] Figure 6 shows that glucogenic amino acid supplementation is sufficient to modulate clinical parameters and induce metabolic rewiring during sepsis. (A) Schematic overview of the experimental design. Thirteen preterm piglets delivered by cesarean section were randomly assigned to groups exclusively nourished with parenteral nutrition containing restricted glucose levels (1.4 %, 2 mg / kg / day), without (rGLU, n = 7) vs. with supplementation of four glucogenic amino acids (aspartate, glutamate, asparagine, and valine, each 0.5 g / kg / day, rGLU-GAAs, n=6). All piglets were infused with S. epidermidis (SE). (B) Survival curves of the animals throughout the experiment, displayed as the time until humane endpoints or scheduled euthanasia. (C-F) Blood gas parameters ((C) blood glucose, (D) pH, (E) pCO2, and (F) lactate) at 3, 6, 12, and 15 h post-inoculation or at euthanasia.

[0068] (G) Blood urea nitrogen (BUN) at 15 h post-inoculation or at euthanasia. (H) The blood S. epidermidis density was determined by colony-forming units (CFU) assays. (I-L) Blood immune parameters (total leukocytes, plasma TNF-o, IL-6, and IL-10) at 3, 6, 12, and 15 h post-inoculation or at euthanasia.

[0069] Statistics: (C-L) Data at each time point were analyzed using a linear model, incorporating group, gender, and birth weight as fixed factors. *P-value < 0.05, **P-value < 0.01, and ***P-value < 0.001, compared between SE-rGLU-GAAs and SE-rGLU groups at the same time point. (C-F & H-L) A linear mixed-effects model was employed to probe further disparities spanning the entire experimental duration, incorporating group, time, and their interaction as fixed factors, with pig ID as random factors. Ptreat, Ptime, and Pint denote probability values for group effect (SE-rGLU-GAAs vs. SE-rGLU) over time, time effects, and the interaction effects between time and group in the linear mixed effects interaction model, respectively. Uninfected animals (CON) served as a reference and were not included in the statistics. Statistical significance was defined as P-value < 0.05. All data were presented as box and whisker plots showing the range from minimum to maximum values, and

[0070] Figure 7 shows that GAA supply during glucose restriction enhances gluconeogenesis and reduces inflammation during neonatal infection. GSEA was performed between the SE-rGLU-GAAs and SE-rGLU groups using the Sus scrofa85583PC01

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[0072] (pig) KEGG knowledgebase. Significant pathways in specific categories such as energy metabolism, carbohydrate metabolism, amino acid metabolism, lipid metabolism, signal transduction, and immune system have been chosen for presentation. The size and tone of the dots indicate the gene ratio and FDR values, respectively. Figure 7 has been divided into three sections (top, middle, and bottom). Any indication, such as gene ratio, in any of the three sections apply in all sections.

[0073] Figure 8 shows that supply of galactose instead of glucose maintains normoglycemia, improves host disease tolerance, and reduces the risk of sepsis.

[0074] (A) Schematic overview of the experimental design. Forty-two preterm piglets delivered by cesarean section were randomly assigned to groups exclusively nourished with parenteral nutrition containing either a standard glucose solution (sGLU; 10% glucose, 14.4 g / kg / d) or an equivalent concentration of galactose (sGAL, 10% galactose, 14.4 g / kg / d). Piglets then received either live S. epidermidis (SE, 109 CFU / kg, n = 32) or saline control (CON, n = 10). This setup allowed for the division of piglets into four groups: CON-sGLU (n = 6), CON-sGAL (n = 4), SE-sGLU (n = 16), and SE-sGAL (n = 16). (B) Survival curves of the animals throughout the experiment, displayed as the time until 15 h postinoculation or humane euthanasia based on predefined humane endpoints. A logrank test was used to compare survival between the SE-sGAL and SE-sGLU groups. (C-G) Blood gas and immune parameters ((C) blood pH, (D) blood pCC , (E) lactate, (F) glucose, (G) blood neutrophils, (H) total leukocytes, (I) monocytes, (J) hemoglobin, (K) plasma IL-10) at 3, 6, 12, and 15 h postinoculation or at euthanasia. CON-sGLU: Light grey, CON-sGAL: Diagonal stripes, SE-sGLU: White dotted, and SE-sGAL: Dark grey.

[0075] Data at each time point were analyzed using a linear mixed-effects model, incorporating group, gender, and birth weight as fixed factors and litter as a random factor. *P-value < 0.05, **P-value < 0.01, and ***P-value < 0.001, compared between SE-sGAL and SE-sGLU groups at the same time point Another linear mixed-effects model was employed to probe further disparities spanning the entire experimental duration, incorporating group, time, their interaction, gender, and birth weight as fixed factors, with litter and pig ID as random factors.

[0076] Ptreat(SE), Ptime(SE), and Pint(SE) denote probability values for group effect (SE-sGAL and SE-sGLU) over time, time effects, and the interaction effects85583PC01

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[0078] between time and group in the linear mixed effects interaction model, respectively. Uninfected animals (CON) served as a reference group and were not compared directly with infected animals (SE). Statistical significance was defined as P-value < 0.05. All data were presented as box and whisker plots showing the range from minimum to maximum values. (L) Density of S. epidermidis in jugular venous blood samples from infected animals at 3 and 6 h or heart puncture at euthanasia. (M-N) Blood and immune parameters (plasma TNF-o and plasma IL-6) were collected at 3, 6, 12, and 15 h post-inoculation or at euthanasia.

[0079] Figure 9 shows that galactose supply reduces inflammation and enhances multiple hepatic metabolic pathways. (A-B) GSEA was performed between the infected groups (A) and uninfected groups (B), respectively. The Sus scrofa (pig) KEGG knowledgebase was utilized for pathway enrichment analysis. Significant pathways in specific categories such as energy metabolism, carbohydrate metabolism, amino acid metabolism, lipid metabolism, signal transduction, and immune system have been chosen for presentation. The size and tone of the dots indicate the gene ratio and FDR values, respectively. (A) has been divided into three sections (top, middle, and bottom), any indications in any of the sections apply to all three sections.

[0080] Figure 10 shows clinical parameters, blood metabolites, and survival in preterm piglets receiving glucose-based or galactose + glucogenic amino acid (GAA) parenteral nutrition. (A) Time from birth to achievement of early developmental milestones (first stand, first walk, first meconium, and eye opening). (B) Body weight measured at birth, on days 2-4, and at euthanasia. (C) Arterial blood glucose concentrations measured on postnatal days 1 and 2 (09:00 and 21:00, Dl-21, D2-09 and D2-21) and at 0, 3, 6, 12, and 24 hours after Staphylococcus epidermidis inoculation as well as at euthanasia (INF-0H, INF-3H, INF-6H, INF-12H, INF-24H and INF-EUT,). (D) Arterial blood pH measured at corresponding time points. (E) Arterial blood lactate concentrations measured before and after inoculation. (F) Kaplan-Meier survival curves showing the proportion of animals alive following S. epidermidis inoculation. Data are shown as individual values with box-and-whisker plots or mean ± SD, as indicated. Grey shaded areas denote the pre-infection period. Statistical significance is indicated as *: P < 0.05, **: P < 0.01, *: P < 0.001.85583PC01

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[0082] Figure 11 shows blood bacterial burden, bacterial clearance, cytokine responses, and thrombocyte counts following Staphylococcus epidermidis infection in preterm piglets receiving glucose-based or galactose + glucogenic amino acid (GAA) parenteral nutrition. (A) Blood bacterial burden expressed as colony-forming units (CFU) per milliliter measured at 3, 6, and 24 hours after S. epidermidis inoculation and at euthanasia (INF-3H, INF-6H, INF-24H, and INF-EUT). (B) Bacterial clearance expressed as the percentage of bacteria cleared from the blood between 3 and 6 hours, 3 and 24 hours, and 3 and 48 hours after inoculation. (C) Plasma interleukin-10 concentrations measured at 3, 6, 12, and 24 hours after inoculation and at euthanasia (INF-3H, INF-6H, INF-12H, INF-24H, and INF-EUT). (D) Plasma interleukin-6 concentrations measured at corresponding time points. (E) Plasma tumor necrosis factor-o concentrations measured at corresponding time points. (F) Blood thrombocyte counts measured at 0, 6, and 24 hours after inoculation and at euthanasia (INF-OH, INF-6H, INF-24H, and INF-EUT). Data are shown as individual values with bar or box-and-whisker plots. Statistical significance is indicated as *: P < 0.05, **: P < 0.01, *: P < 0.001

[0083] The present invention will now be described in more detail in the following.

[0084] Detailed description of the invention

[0085] Definitions

[0086] Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

[0087] Parenteral nutrition (PN)

[0088] Parenteral nutrition is a method of delivering nutrients directly into the bloodstream, bypassing the digestive system. This is typically done through an intravenous (IV) catheter. It is often used for individuals who cannot use their digestive systems due to certain medical conditions. Parenteral nutrition solutions contain a mix of essential nutrients, including carbohydrates, proteins, fats, vitamins, and minerals.

[0089] The two main types of parenteral nutrition are:85583PC01

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[0091] Partial Parenteral Nutrition (PPN), which supplements other forms of feeding when additional nutrients are needed.

[0092] - Total Parenteral Nutrition (TPN), which provides all nutritional needs intravenously when the digestive system is completely bypassed Glucogenic amino acids

[0093] Glucogenic amino acids are amino acids that can be converted into glucose through a process called gluconeogenesis. This process is known for maintaining blood glucose levels. When glucogenic amino acids are broken down, they are converted into intermediates that enter the gluconeogenesis pathway, ultimately producing glucose.

[0094] Glucose

[0095] Glucose is a simple sugar, or monosaccharide, with the molecular formula CeHizOe. It is a crucial energy source for the body and is often referred to as blood sugar when found in the bloodstream.

[0096] Maintaining balanced glucose levels is essential for overall health. Imbalances can lead to conditions such as hypoglycemia (low blood sugar) or hyperglycemia (high blood sugar).

[0097] Galactose

[0098] Galactose is a type of simple sugar, or monosaccharide, that is an epimer structure to glucose. It is often found in combination with glucose to form lactose, the sugar present in milk. The molecular formula of galactose is C6H12O6.

[0099] The main pathway of galactose metabolism is the Leloir pathway. The Leloir pathway comprises of the latter stage of a two-part process that converts p-D-galactose to UDP-glucose. The initial stage is the conversion of p-D-galactose to o-D-galactose by the enzyme, mutarotase (GALM). The Leloir pathway then carries out the conversion of o-D-galactose to UDP-glucose via three principal enzymes: Galactokinase (GALK) phosphorylates o-D-galactose to galactose-1-phosphate, or Gal-l-P; Galactose-l-phosphate uridyltransferase (GALT) transfers a UMP group from UDP-glucose to Gal-l-P to form UDP-galactose; and finally, UDP galactose-4'-epimerase (GALE) interconverts UDP-galactose and UDP-glucose, thereby completing the pathway.85583PC01

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[0101] Total carbohydrate

[0102] In here, "Total carbohydrate" is to be understood as being the sum of all carbohydrates in, including all different types of carbohydrate. For example, a total carbohydrate in PN is the amount sum of all different types of carbohydrates in the PN.

[0103] Preterm infant

[0104] A preterm infant is a premature newborn baby born before 37 weeks of gestation. The gestational age categories are:

[0105] • Extremely preterm: Born less than 28 weeks of gestation.

[0106] • Very preterm: Born 28 to less than 32 weeks of gestation.

[0107] • Moderate to late preterm: Born 32 to 37 weeks of gestation.

[0108] Preterm infants often face various health challenges, including breathing difficulties, feeding problems, and higher risks of infections.

[0109] "Preterm infants" and "premature infants" may be used interchangeably.

[0110] "Infant", "newborn", "neonate", and "baby" may be used interchangeably.

[0111] Adult person

[0112] An adult person is to be understood as a person that physically is fully grown and developed, thus having reached biological maturity. Biological maturity refers to the point at which the body has completed its major growth and developmental processes, including skeletal, muscular, hormonal, and reproductive systems. Biological maturity is typically reached at the age of between 18 and midtwenties, such as 25.

[0113] Elderly person

[0114] An "elderly person" refers to a person in an advanced stage of life, often beyond middle age. An elderly person may be a person aged 65 or older.

[0115] An elderly person may have variety of health issues such heart disease, osteoporosis, cancers, etc.

[0116] In addition, the immune system of elderly people tends to become less effective, a process known as immunosenescence. Their immune system becomes slower to respond, which increases the risk of infections. Further, there are fewer white blood cells capable of responding to new antigens, making it harder to fight off new infections.85583PC01

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[0118] Vaccines may not work as well or provide long-lasting protection.

[0119] Chronic inflammation becomes more common, which is linked to many age-related diseases.

[0120] The risk of autoimmune disorders increases, as the immune system may mistakenly attack healthy body tissues, leading to autoimmune disorders.

[0121] In an embodiment, an elderly person has a less effective immune system compared to a young or middle-aged adult.

[0122] Suspected infection

[0123] A suspected infection refers to a situation where a healthcare provider believes that a subject may have an infection based on their symptoms, physical examination, and / or initial test results. The suspicion can be validated via diagnostic tests to confirm the presence and type of infection, such as blood tests, urine tests, or imaging studies.

[0124] Infections in preterm infants are difficult to identify, since preterm infants do not immediately show symptoms of infection. In particular, preterm infants are extremely susceptible to infections that can lead to life-threatening sepsis. Thus treatment of infections in preterm infants are usually initiated before they are definitely proven by blood culture or other biomarkers.

[0125] Symptoms of infection

[0126] Infections can present with a variety of symptoms depending on the type of pathogen (bacteria, virus, fungus, or parasite) and the part of the body affected. General symptoms can be fever, chills, sweats, fatigue, headache, muscle and joint aches, nausea and / or vomiting.

[0127] In infants, such as preterm infants, can include temperature instability, poor feeding, lethargy, apnea, bradycardia and respiratory distress.

[0128] Infections are typically validated via tests, such as laboratory tests.

[0129] Low immunity

[0130] A "low immunity" is to be understood as an immunodeficiency, which is a condition where the body's immune system is not functioning as effectively as it should compared to a corresponding normal healthy subject. This can make a person more susceptible to infections and illnesses.85583PC01

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[0132] Low immunity is often seen in newborns and premature infants because at birth, an infant's immune system is not fully developed. It takes a few months for the immune system to mature and become more effective at fighting off infections. Further, newborns have not been exposed to many pathogens, and so their immune system has not had the chance to build up defences against a wide range of infections. In addition, newborns have immune cells that are more focused on regulating and tolerating new antigens rather than aggressively fighting them off. This helps them adapt to the new environment outside the womb but can make them more susceptible to infections.

[0133] Immune function

[0134] "Immune function" refers to the specific activities and processes carried out by the immune system to defend the body. The immune function includes:

[0135] - Identifying and destroying pathogens: Recognizing foreign invaders and eliminating them.

[0136] Memory formation: Remembering past infections to respond more effectively in the future.

[0137] Healing and repair: Assisting in the recovery of damaged tissues.

[0138] Regulation: Balancing immune responses to avoid overactivity, which can cause autoimmune diseases or underactivity, which can lead to infections.

[0139] Thus, immune function describes the actions and processes that the immune system performs to maintain health.

[0140] In here "immune function" and "immune system" may be used interchangeably. The terms "immune function" and "immune system" are closely related but refer to different aspects of the body's defence mechanisms.

[0141] Immune system

[0142] The "immune system" is the entire network of organs, tissues, cells, and molecules that work together to protect the body from harmful invaders like bacteria, viruses, fungi, and parasites. Thus, the immune system is the structural framework.85583PC01

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[0144] Optimizing immune system and / or response

[0145] The immune system is optimized or enhanced by improving the efficiency, effectiveness, or performance of the immune system of a subject. For example, the host defence can be optimized by controlling glycolysis-induced inflammation. This will increase disease resistance and tolerance, and in turn maximize infection survival.

[0146]

[0147] A healthy subject is a subject having an effective and well-functioning immune system, which is able to protect the body from infections and diseases. The immune system can distinguish between the body's own cells and foreign pathogens such as bacteria, viruses, and fungi. It responds quickly to eliminate these pathogens. Thus, a healthy subject has a healthy immune system, which is a well-coordinated defence mechanism that protects the body from a wide range of potential threats.

[0148] >

[0149] Sepsis is a medical condition that occurs when the body's response to an infection causes widespread inflammation, leading to tissue damage, organ failure, and potentially death. Bacterial, viral, or fungal infections can lead to sepsis.

[0150]

[0151] Delaying progression of sepsis is to delay the clinical deterioration during infection, thus delaying sepsis progression increases the window of time between initial infection and sepsis endpoint in order for medical specialists, such as doctors and nurses, to administer other adjunct therapies (e.g., antibiotics or inotropes) for treatment and prevention of septic shock.

[0152] Delaying sepsis progression will not in itself prevent sepsis or eliminate pathogens causing sepsis. Delaying sepsis is a mere postponement of sepsis, which will in any event occur at a later time point.

[0153]

[0154] By preventing sepsis, sepsis will not occur in an infected subject. With respect to the present invention, the pathogens leading to sepsis are not eliminated by the PN composition according to the invention, however the PN composition of the invention prevents lethal sepsis via enhanced host resistance and tolerance and rewired immune-metabolic response. Thus, the PN composition of the invention provides nutrition for the subject and thereby optimizes the immune system and85583PC01

[0155] 17

[0156] immune response of the infected subject for naturally fighting off and eliminating the infection.

[0157]

[0158] Prophylactic treatment is to be understood as to a medical intervention taken to prevent a disease or health condition before it occurs, rather than to cure or manage an existing illness. This reduces the risk of developing a disease or prevent recurrence. Prophylactic treatment is applied before symptoms appear or before exposure to risk factors.

[0159] Parenteral nutrition (PN) composition

[0160] The parenteral nutrition composition according to the present invention is suitable for optimizing the immune system and immune function in a subject, such as the immune function of a preterm human infant or an elderly human. Thus, the present invention relates to a "stock" PN composition comprising a high amount of GAAs and galactose, which may be used for mixing with a second PN composition, such as a commercial PN composition. The PN composition comprises a higher amount or concentration of glucogenic amino acids, which improves glucose homeostasis and disease tolerance to decrease sepsis severity, since the presence of glucogenic amino acids or a higher amount of glucogenic amino acids in the PN dampens hepatic glycolysis and inflammation. The stock PN composition further comprises galactose, which also dampens hepatic glycolysis and inflammation by improving glucose homeostasis and disease tolerance.

[0161] The stock PN composition of the invention comprises or consists of GAAs and galactose, wherein said stock PN composition can be mixed with a second (commercial) PN composition comprising various amino acids, which may be normally present in commercial PN compositions. The combination of the stock PN composition and a commercial PN composition results in one or more of the GAAs and galactose being supplemented. This results in a combination PN composition having an even higher total amount of GAAs and galactose in the combination PN composition of the invention compared to a conventional commercial PN composition, which can be administered to a subject in need thereof.

[0162] By adding the stock PN composition comprising or consisting of GAAs and galactose to a commercial PN composition or by replacing a major fraction of monosaccharides, such as glucose, in a commercial PN composition with85583PC01

[0163] 18

[0164] glucogenic amino acids and / or galactose simultaneously enhances disease resistance and tolerance, in turn maximizing infection survival. Alternatively, the stock PN composition may be co-administered, such as administered separately, with the second (commercial) PN composition. If the stock PN composition is administered separately from the second (commercial) PN composition, the stock PN composition should preferably be diluted prior to administration.

[0165] Thus, the final PN composition administered to a subject comprises a reduced or no amount of glucose while adding an additional amount of glucogenic amino acids on top of the amino acids already present in the PN composition as well as galactose.

[0166] A stock PN composition

[0167] An aspect of the invention relates to a (stock) parenteral nutrition (PN) composition comprising

[0168] a) one or more glucogenic amino acids (GAAs) in an amount of at least 0.5% by weight of the PN composition, and

[0169] b) galactose in an amount of at least 1% by weight of the PN composition. Hence, the present invention relates to a stock PN composition, which can be mixed with a second PN composition, such as a commercial PN composition, to obtain a final combined PN composition. The final combined PN composition will have an increased concentration of GAAs and galactose compared to a conventional commercial PN composition.

[0170] As can be seen in the Examples, the PN composition of the present invention comprises a higher amount of GAAs and galactose, such as via a supplementation of GAAs and galactose in a commercial PN composition. The presence of a higher amount GAAs and galactose effectively limits glycolysis, enhanced gluconeogenesis to prevent severe hypoglycemia, and completely prevents sepsis. In addition, data shows that substitution of PN glucose with galactose alone delays, but does not prevent, clinical deterioration during experimental infection, while substitution of glucose with GAA completely prevented clinical deterioration but leads to hypoglycemia. It is therefore expected that the combined supplementation of galactose and GAA creates a synergistic metabolic environment in which immune and non-immune tissues are supplied with energy substrates that favor oxidative phosphorylation over aerobic glycolysis.85583PC01

[0171] 19

[0172] An aspect of the invention relates to a (stock) parenteral nutrition (PN) composition consisting of

[0173] a) one or more glucogenic amino acids (GAAs) in an amount of at least 0.5% by weight of the PN composition,

[0174] b) galactose in an amount of at least 1% by weight of the PN composition, and

[0175] c) optionally glucose.

[0176] An aspect of the invention relates to a parenteral nutrition (PN) composition comprising one or more glucogenic amino acids (GAAs) in an amount of at least 2% by weight, such as at least 3% by weight, of the PN composition.

[0177] Another aspect of the invention relates to a parenteral nutrition (PN) composition comprising

[0178] a) one or more glucogenic amino acids (GAAs) in an amount of at least 0.5% by weight of the PN composition, and

[0179] b) galactose in an amount of at least 1% by weight of the PN composition, wherein the one or more GAAs are selected from a group consisting of valine, asparagine, aspartate, glutamate, or a combination thereof.

[0180] These glucogenic amino acids valine, asparagine, aspartate, glutamate, or a combination thereof can easily enter the tricarboxylic acid (TCA) cycle, whereas other amino acids require one or more metabolic steps prior to entry. Thus, the combination of GAAs valine, asparagine, aspartate, and / or glutamate enter the TCA cycle in various places, ensuring efficient gluconeogenesis. Hence said four types of GAAs maximize the possibilities to enhance gluconeogenesis, relative to using less than four different GAAs, such as only one type of GAA.

[0181] In an embodiment, the one or more GAAs are in an amount of 2% to 10% by weight, such as 2% to 9% by weight, such as 2% to 8% by weight, preferably 2.5% to 6% by weight, such as 3% to 6% by weight, such as 3% to 5% by weight, such as 0.5% to 4% by weight, such as 1% to 4% by weight, preferably 1% to 3% by weight, more preferably 0.5% to 2% by weight, most preferably 1% to 2% by weight of the PN composition.

[0182] For example, the (stock) PN composition may comprise one or more GAAs in an amount of 1.6% by weight of the PN composition.85583PC01

[0183] 20

[0184] In an embodiment, the one or more GAAs are selected from a group consisting of alanine, arginine, aspartate, asparagine, cysteine, glycine, glutamate, glutamine histidine, hydroxyproline, proline, methionine, serine, threonine, valine, or a combination thereof, preferably selected from a group consisting of methionine, valine, asparagine, aspartate, glutamate, or a combination thereof, more preferably selected from a group consisting of valine, asparagine, aspartate, glutamate, or a combination thereof.

[0185] A PN composition comprising multiple GAAs, such as four GAAs will result in four amino acids entering the TCA cycles at different places, as they are converted to four metabolites within the TCA cycle. This makes the process of gluconeogenesis more efficient compared to a PN composition comprising for example only one GAA.

[0186] In an embodiment, the one or more GAAs are present in an amount of 66% to 100% by weight, such as 67% to 100% by weight, such as 68% to 100% by weight, such as 80% to 100% by weight, preferably 100% by weight relative to the total amount of amino acids of the (stock) PN composition.

[0187] The PN composition of the invention comprises various amino acids as listed above, however one or more specific GAAs are present in a higher amount compared to the remaining amino acids, such as remaining ketogenic amino acids and GAAs. Thereby the total amount of GAAs will be higher in the PN composition of the invention. Said PN composition comprising a higher amount of GAAs have shown a dampened hepatic glycolysis and inflammation. Preferably, the stock PN composition comprises 100% by weight of GAAs relative to the total amount of amino acids in the (stock) PN composition.

[0188] The fraction of GAAs relative to a total amount of amino acids in a commercial PN composition is around 65%, hence the fraction of GAAs relative to a total amount of amino acids in the stock PN composition of the present invention is much higher and above 65%.

[0189] In an embodiment, valine, asparagine, aspartate, and glutamate are in a ratio of 1: 1:1:1.

[0190] The combination of GAAs valine, asparagine, aspartate, and / or glutamate enter the TCA cycle in various places, ensuring efficient gluconeogenesis. Hence said85583PC01

[0191] 21

[0192] four GAAs maximize the possibilities to enhance gluconeogenesis, relative to using only less than four different GAAs.

[0193] In an embodiment, the amount of valine, asparagine, aspartate, and / or glutamate is 0.5% to 5% by weight, such as 0.5% to 4% by weight, such as 1% to 4% by weight, such as 1.5% to 3% by weight, such as 2% to 2.5% by weight, such as 0.5% to 4% by weight, preferably 1% to 3% by weight, more preferably 0.5% to 2% by weight, most preferably 1% to 2% by weight of the PN composition.

[0194] As seen in the Examples, valine, asparagine, aspartate, and / or glutamate are supplemented and thereby resulting in a higher amount of one or more of valine, asparagine, aspartate, and / or glutamate and thereby a higher amount of total GAAs in the final PN composition.

[0195] The combination of valine, asparagine, aspartate, and / or glutamate enter the TCA cycle in various places, ensuring efficient gluconeogenesis. Hence said four GAAs maximize the possibilities to enhance gluconeogenesis, relative to using only less than four different GAAs.

[0196] In an embodiment, valine is present in an amount of 0.25% to 2% by weight, such as 0.25% to 1% by weight, preferably 0.5% by weight of the PN composition.

[0197] In an embodiment, asparagine is present in an amount of 0.25% to 2% by weight, such as 0.25% to 1% by weight, preferably 0.5% by weight of the PN composition.

[0198] In an embodiment, aspartate is present in an amount of 0.25% to 2% by weight, such as 0.25% to 1% by weight, preferably 0.5% by weight of the PN composition.

[0199] In an embodiment, glutamate is present in an amount of 0.25% to 2% by weight, such as 0.25% to 1% by weight, preferably 0.5% by weight of the PN composition.

[0200] In an embodiment, the one or more GAAs are a natural, artificial, or modified amino acid analogue or substitute.85583PC01

[0201] 22

[0202] In an embodiment, the PN composition comprises two or more, such as three or more, preferably four or more GAAs.

[0203] Multiple GAAs in the PN composition, such as a PN composition comprising four GAAs will result in four amino acids entering the TCA cycles at different places, as they are converted to four metabolites within the TCA cycle. Hence, a PN composition comprising a combination of GAAs makes the process of gluconeogenesis more efficient compared to a PN composition comprising only one GAA.

[0204] In an embodiment, the galactose is in an amount of 5% to 50% by weight, such as 10% to 50% by weight, such as 10% to 45% by weight, such as 10% to 30% by weight, preferably 10% to 25% by weight, more preferably 10% to 22% by weight, most preferably 10% to 20% by weight of the PN composition.

[0205] For example, the stock PN composition comprises galactose in an amount of 20% by weight of the stock PN composition.

[0206] In a preferred embodiment, the (stock) PN composition is a solution, such as a liquid solution.

[0207] In an embodiment, the (stock) PN composition further comprises glucose, wherein the glucose in an amount of 0% to 50% by weight, such as 0% to 40% by weight, such as 0% to 30% by weight, such as 0% to 25% by weight, such as 0% to 20% by weight, preferably 0% to 10% by weight, more preferably 0% to 5% by weight, most preferably 0% by weight of the (stock) PN composition.

[0208] A combination PN composition

[0209] As described above, the combination PN composition of the present invention comprises a higher amount of GAAs and galactose, such as via a supplementation of GAAs and galactose from the stock PN composition in a commercial PN composition. The presence of a higher amount GAAs and galactose in a PN composition administered to a subject effectively limits glycolysis, improved glucose homeostasis, enhanced gluconeogenesis to prevent severe hypoglycemia, and completely prevents sepsis.85583PC01

[0210] 23

[0211] Another aspect of the invention relates to combination parenteral nutrition (PN) composition comprising:

[0212] a) a first PN composition, wherein the first PN composition is the PN composition according to the invention, and

[0213] b) a second PN composition.

[0214] The second PN composition may in principle be any suitable commercial PN composition.

[0215] Hence, in an embodiment, the second PN composition is a commercial PN composition.

[0216] In an embodiment, the combination PN composition comprises one or more GAAs in an amount of at least 0.2% by weight, and / or galactose in an amount of at least 5% of the combination PN composition.

[0217] In an embodiment, the combination PN composition comprises one or more GAAs in an amount of 0.2% to 2% by weight, such as 0.5% to 1.5% by weight, preferably 0.5% to 1% by weight, more preferably 0.7% to 1% by weight of the combination PN composition.

[0218] In an embodiment, the combination PN composition comprises galactose in an amount of 5% to 20% by weight, preferably 5% to 15% by weight, more preferably 5% to 10% by weight of the combination PN composition.

[0219] Thus, in an embodiment, the one or more GAAs in the second PN composition have been supplemented with one or more additional GAAs from the first PN composition.

[0220] The supplementation of one or more GAAs are to be understood as one or more GAAs being added on top of the already existing amino acids in the second PN composition.

[0221] In an embodiment, the combination PN composition further comprises one or more carbohydrates in an amount of 0.5% to 10% by weight, such as 0.5% to 5% by weight, such as 1% to 5% by weight, preferably 1% to 3% by weight,85583PC01

[0222] 24

[0223] more preferably 1% to 2% by weight, most preferably 1.4% by weight of the combination PN composition.

[0224] The combination PN composition of the invention may comprise a reduced amount of carbohydrate, such as glucose, while adding an additional amount of glucogenic amino acids and galactose. Hence a GAA and galactose supplementation is combined with reduced glucose levels. In that way, the glucose content of the final combination PN composition may be provided via the second (commercial) PN composition, wherein the default glucose content in the commercial PN is reduced to optimal glucose levels.

[0225] In an embodiment, the one or more carbohydrates are selected from the group consisting of glucose, fructose, dextrose, galactose, or any combination thereof. As seen in the Examples, the PN composition comprises glucose, however in principle any type of carbohydrate suitable for a PN composition may be present in the PN composition, such as monosaccharides and / or disaccharides.

[0226] In an embodiment, one or more carbohydrate is glucose.

[0227] As seen in the Examples, the PN composition comprises glucose.

[0228] While GAAs and galactose can be slowly converted into glucose, and although not strictly necessary, a small amount of glucose may still be present in the combination PN composition in order to further prevent hypoglycaemia in a subject.

[0229] The reduced amount of glucose having a concentration in the combination PN composition as described above can be obtained via:

[0230] • the presence of glucose in the first PN composition having a concentration as defined above,

[0231] • the presence of glucose in the second PN composition (commercial PN), wherein the concentration of glucose can be adjusted as to obtain a reduced concentration of glucose, or

[0232] • the presence of glucose in both the first and second PN composition, where in the concentrations of glucose are such that when the first and second PN composition are mixed, a reduced concentration of glucose is obtained.

[0233] As seen in the Examples, glucose was removed from the PN glucose chamber to reach a final glucose content of 1.4%. The reduction of glucose causes a85583PC01

[0234] 25

[0235] decreased risk of hyperglycemia, which can trigger excessive glycolysis-induced resistance response. Hence, by reducing the amount of glucose in the PN composition enhanced disease resistance and tolerance, in turn maximized infection survival is obtained.

[0236] In an embodiment, the second PN composition comprises glucose in an amount of 0% to 20% by weight, preferably 0% to 10% by weight, more preferably 0% to 5% by weight, most preferably 0% by weight of the second PN composition.

[0237] Hence, the content of glucose in the commercial second PN composition may preferably be low or 0% in order to easily control the glucose concentration of the final combination PN composition, which is administered to a subject.

[0238] For example, the second PN composition may comprise 0% by weight of glucose, wherein glucose can be supplemented to the second PN composition via the first PN composition.

[0239] As explained above, the glucose content of the final combination PN composition may also be provided via the second (commercial) PN composition. A third option is obtaining an optimal glucose content of the final combination PN composition via both the first and the second PN compositions comprising glucose i.e. the mixing of both the first and second PN compositions results in a desired reduced glucose content of the combination PN composition.

[0240] In an embodiment, the PN composition according to the invention further comprises:

[0241] - water in an amount of 60% to 85% by weight of the PN composition; - one or more additional amino acids in an amount of 1% to 6% by weight of the PN composition;

[0242] - one or more fats in an amount of 0% to 4% by weight the PN composition,

[0243] - one or more vitamins; and / or

[0244] - one or more electrolytes.

[0245] In principle, the PN composition may comprise any further components suitable for a PN composition.

[0246] The amount of vitamin and / or electrolyte in the PN composition preferably complies with the officially recommended daily vitamin and electrolyte intake.85583PC01

[0247] 26

[0248] In an embodiment, the one or more additional amino acids are one or more ketogenic amino acids, such as leucine and lysine.

[0249] Hence, the one or more additional amino acids is not a GAA.

[0250] In an embodiment, the ketogenic amino acid is a natural, artificial, or modified amino acid analogue or substitute.

[0251] In principle, the one or more additional amino acids may be any type of amino acid suitable for a PN composition.

[0252] In an embodiment, the one or more vitamins are selected from the group consisting of Vitamin Bl (Thiamine), Vitamin B2 (Riboflavin), Vitamin B3 (Niacin), Vitamin B5 (Pantothenic acid), Vitamin B6 (Pyridoxine), Vitamin B7 (Biotin), Vitamin B9 (Folic acid), Vitamin B12 (Cobalamin), Vitamin C (Ascorbic acid), Vitamin A (Retinol), Vitamin D (Cholecalciferol), Vitamin E (o-Tocopherol), Vitamin K (Phylloquinone), or any combination thereof.

[0253] In principle, the one or more vitamins may be any vitamin suitable for a PN composition.

[0254] In an embodiment, the one or more electrolytes are selected from the group consisting of sodium, potassium, magnesium, calcium, selenium, zinc, copper, manganese, phosphate, sulphate, chloride, fluoride, iodide, acetate, citric acid, malate, lactate, or any combination thereof.

[0255] In principle, the one or more electrolytes may be any electrolyte suitable for a PN composition.

[0256] In an embodiment, the one or more fats are selected from the group consisting of long-chain triglycerides, such as soybean oil, olive oil, medium-chain triglycerides, such as coconut oil, omega-3 fatty acids, such as fish oil, or any combination thereof.

[0257] In principle, the one or more fats may be any type of fat suitable for a PN composition.

[0258] In an embodiment, the one or more fat is a fat emulsion.

[0259] In an embodiment, the combination PN composition is in liquid or powder form.85583PC01

[0260] 27

[0261] Preferably, the combination PN composition is liquid form.

[0262] In principle, the combination PN composition can be in any form, such as a liquid or a powder form. If the PN composition is in powder form, it may be hydrated using a liquid, such as water.

[0263] A method for producing a stock PN composition

[0264] An aspect of the invention relates to a method for producing a (stock) PN composition according to the invention comprising the steps of:

[0265] 1) providing one or more glucogenic amino acids (GAAs) in an amount of at least 0.5% by weight of the PN composition;

[0266] 2) providing galactose in an amount of at least 1% by weight of the PN composition;

[0267] 3) optionally, providing one or more additional components;

[0268] 4) mixing the one or more GAAs, galactose, and the optional one or more additional components; and

[0269] 5) obtaining the (stock) PN composition.

[0270] An additional aspect of the invention relates to a method for producing a PN composition according to the invention comprising the steps of:

[0271] a) providing a PN mixture / composition comprising one or more glucogenic amino acids (GAA) and one or more carbohydrates, such as glucose; b) adjusting or supplementing the amount of the one or more GAAs and obtaining an amount of the one or more GAAs of at least 0.2% by weight, such as at least 2% by weight, such as at least 3% by weight, of the PN composition;

[0272] c) adjusting a one or more carbohydrates for obtaining an amount the one or more carbohydrates of of 0.5% to 10% by weight, such as 0.5% to 5% by weight, such as 1% to 5% by weight, preferably 1% to 3% by weight, more preferably 1% to 2% by weight, most preferably 1.4% by weight of the PN composition;

[0273] d) obtaining the PN composition.

[0274] In an embodiment, the galactose is provided in an amount of 5% to 50% by weight, such as 10% to 50% by weight, such as 10% to 45% by weight, such as 10% to 30% by weight, preferably 10% to 25% by weight, more preferably 10%85583PC01

[0275] 28

[0276] to 22% by weight, most preferably 10% to 20% by weight of the (stock) PN composition.

[0277] In an embodiment, the one or more GAAs are selected from a group consisting of alanine, arginine, aspartate, asparagine, cysteine, glycine, glutamate, glutamine histidine, hydroxyproline, proline, methionine, serine, threonine, valine, or a combination thereof, preferably the one or more GAAs are selected from a group consisting of methionine, valine, asparagine, aspartate, glutamate, or a combination thereof, more preferably the one or more GAAs are selected from a group consisting of valine, asparagine, aspartate, glutamate, or a combination thereof.

[0278] In an embodiment, the one or more GAAs comprise valine, asparagine, aspartate, and / or glutamate.

[0279] In an embodiment, the one or more GAAs are provided as a mixture.

[0280] In an embodiment, the one or more GAAs are two or more GAAs, such as three or more GAAs, preferably four or more GAAs.

[0281] In an embodiment, the one or more GAAs and one or more additional components are provided in two or more separate chambers or containers.

[0282] In an embodiment, the one or more GAAs and one or more carbohydrates are provided in two or more separate chambers or containers.

[0283] The provision of the individual components, such as galactose, fats, and / or amino acids, in separate chambers is to allow for release and mixing the content of the chambers, when necessary, such as just before administration of the PN composition. The separation of the components in chambers enhances safety as they reduce handling and thus the risks of contamination of the nutritional mixture. In addition, some component or micronutrients, such as vitamins, have a high level of instability, hence these components are preferably be added and mixed in the PN composition just before administration.85583PC01

[0284] 29

[0285] In an embodiment, the one or more GAAs are in an amount of 2% to 10% by weight, such as 2% to 9% by weight, such as 2% to 8% by weight, preferably 2.5% to 6% by weight, such as 3% to 6% by weight, such as 3% to 5% by weight, such as 0.5% to 4% by weight, such as 1% to 4% by weight, preferably 1% to 3% by weight, more preferably 0.5% to 2% by weight, most preferably 1% to 2% by weight of the (stock) PN composition.

[0286] In an embodiment, the one or more additional components are selected from the group consisting of one or more additional carbohydrates, water, one or more additional amino acids, one or more vitamins, one or more electrolytes, one or more fats, or any combination thereof.

[0287] In principle, the one or more additional components is selected from the group of components, which are usually present in a PN composition.

[0288] In an embodiment, the water is in an amount of 60% to 85% by weight of the PN composition.

[0289] In an embodiment, the one or more additional amino acids are in an amount of 1.5% to 6% by weight of the PN composition.

[0290] In an embodiment, the one or more amino acids are one or more ketogenic amino acids, such as leucine and lysine.

[0291] In an embodiment, the one or more amino acids are a natural, artificial, or modified amino acid analogue or substitute.

[0292] In an embodiment, the one or more fats are in an amount of 0% to 4% by weight, such as 1% to 4% by weight of the PN composition.

[0293] In an embodiment, the one or more fats are selected from the group consisting of long-chain triglycerides, such as soybean oil, olive oil, medium-chain triglycerides, such as coconut oil, omega-3 fatty acids, such as fish oil, or any combination thereof.

[0294] In an embodiment, the one or more fat is a fat emulsion.85583PC01

[0295] 30

[0296] In an embodiment, the one or more additional carbohydrates are provided in an amount of 0.5% to 10% by weight, such as 0.5% to 5% by weight, such as 1% to 5% by weight, preferably 1% to 3% by weight, more preferably 1% to 2% by weight, most preferably 1.4% by weight.

[0297] In an embodiment, the method further comprises a step of adding one or more additional carbohydrates in an amount of 0.5% to 10% by weight, such as 0.5% to 5% by weight, such as 1% to 5% by weight, preferably 1% to 3% by weight, more preferably 1% to 2% by weight, most preferably 1.4% by weight of the (stock) PN composition.

[0298] The stock PN composition of the invention may comprise a reduced amount of carbohydrate, such as glucose, while adding an additional amount of glucogenic amino acids. As seen in the Examples, glucose was removed from the PN glucose chamber to reach a final glucose content of 1.4% of the PN composition.

[0299] In an embodiment, the one or more additional carbohydrates are selected from the group consisting of glucose, fructose, dextrose, galactose, or any combination thereof.

[0300] In an embodiment, the one or more additional carbohydrate is glucose.

[0301] In an embodiment, the one or more additional components is a second PN composition, wherein the second PN composition comprises:

[0302] - water in an amount of 60% to 85% by weight of the second PN composition;

[0303] - one or more amino acids in an amount of 1% to 6% by weight of the second PN composition;

[0304] - one or more fats in an amount of 0% to 4% by weight the second PN composition;

[0305] - one or more vitamins; and / or

[0306] - one or more electrolytes.85583PC01

[0307] 31

[0308] A method for producing a combination PN composition

[0309] An aspect of the invention relates to a method for producing a combination PN composition according to the invention comprising the steps of:

[0310] 1) providing one or more glucogenic amino acids (GAAs) and galactose;

[0311] 2) mixing the one or more GAAs and the galactose and obtaining a first PN composition comprising one or more GAAs in an amount of at least 0.5% by weight and galactose in an amount of at least 1% by weight of the first PN composition;

[0312] 3) providing a second PN composition;

[0313] 4) mixing the first PN composition of step 2) and the second PN composition of step 3); and

[0314] 5) obtaining the combination PN composition.

[0315] A further aspect of the present invention relates to a method for producing a combination PN composition according to the invention comprising the steps of:

[0316] 1) providing a first PN composition, wherein the first PN composition is the PN composition according to the invention;

[0317] 2) providing a second PN composition;

[0318] 3) mixing the first PN composition of step 1) and the second PN composition of step 2); and

[0319] 4) obtaining the combination PN composition.

[0320] In an embodiment, the combination PN composition comprises one or more GAAs in an amount of at least 0.2% by weight, and / or galactose in an amount of at least 5% of the combination PN composition.

[0321] In an embodiment, the combination PN composition comprises one or more GAAs in an amount of 0.2% to 2% by weight, such as 0.5% to 1.5% by weight, preferably 0.5% to 1% by weight, more preferably 0.7% to 1% by weight of the combination PN composition.

[0322] In an embodiment, the combination PN composition comprises galactose in an amount of 5% to 20% by weight, preferably 5% to 15% by weight, more preferably 5% to 10% by weight.85583PC01

[0323] 32

[0324] In an embodiment, the second PN composition comprises glucose in an amount of 0% to 2% by weight, preferably 0% by weight of the second PN composition.

[0325] In an embodiment, the second PN composition is a commercial PN composition.

[0326] PN composition for use in treatment

[0327] An aspect of the invention relates to a parenteral nutrition (PN) composition according to the invention for use in the treatment and / or prevention of infection or sepsis in a subject.

[0328] The PN composition according to the invention is the stock PN composition and / or the combination PN composition as defined above.

[0329] In an embodiment, the treatment is a prophylactic treatment.

[0330] An additional aspect of the present invention relates to a parenteral nutrition (PN) composition according to the invention for use in preventing or delaying sepsis in a subject.

[0331] As seen in the Examples, the PN composition according to the present invention indeed prevents or delays sepsis. GAA supplementation and optionally combined with glucose restriction effectively limits glycolysis, enhances gluconeogenesis to prevent severe hypoglycemia, as well as completely protected against sepsis.

[0332] Another aspect of the invention relates to a parenteral nutrition (PN) composition according to the invention for use in administration to a subject for optimizing the immune function.

[0333] An aspect of the invention relates to a parenteral nutrition (PN) composition according to the invention for use in optimizing the immune function of a subject. As seen in the Examples, the PN composition according to the invention optimizes the immune function by improving disease tolerance instead of resistance, prioritizing the maintenance of normal organ function over a strong immune response to clear the pathogens and thereby decreases the severity and risk of sepsis.

[0334] Another aspect of the invention relates to a parenteral nutrition (PN) composition according to the invention for use in administration to a preterm infant.85583PC01

[0335] 33

[0336] The PN composition can be administered to infants, preferably preterm infants, because improved glucose homeostatic control and balanced defence strategies can be lifesaving during neonatal infection. Low immunity is often seen in newborns and premature infants because at birth, an infant's immune system is not fully developed, hence the PN composition of the invention can improve energy metabolism and glucose homeostatic control, which lead to the prevention of excessive inflammation and tissue damage. The PN composition of the invention has sepsis preventive effects of glucogenic amino acid supplementation and optionally glucose restriction is strongly tied to a rewiring of the host energy metabolism.

[0337] In clinical settings, sepsis delaying or preventing effects of the PN composition of the invention can provide an important window of opportunity for other adjunct therapies (e.g., antibiotics or inotropes) in infected preterm newborns prior to clinical deterioration to septic shock.

[0338] In an embodiment, the subject has a low immunity compared to a corresponding healthy subject.

[0339] Low immunity is a condition where the body's immune system is not functioning as effectively as it should compared to a corresponding normal healthy subject. This can make a subject with low immunity more susceptible to infections and illnesses. Hence, the PN composition of the invention can improve energy metabolism and glucose homeostatic control, which lead to the prevention of excessive inflammation and tissue damage.

[0340] In an embodiment, the subject has a dysfunctional immune system or dysfunctional immune function.

[0341] In an embodiment, the subject has an infection or symptoms of infection.

[0342] As seen in the Examples, the PN composition indeed works in a subject having an infection or symptoms of infection.

[0343] However, if the PN composition is administered to a subject without an infection, said subject will not suffer any adverse effects of the PN composition. Thus, the PN composition of the invention is safe for administration in a subject without an infection.85583PC01

[0344] 34

[0345] In an embodiment, the subject has a condition of dysregulated glucose homeostasis.

[0346] In principle, the PN composition of the invention can be administered to a subject having any condition, wherein the immune system or immune function is dysfunctional or impaired.

[0347] In an embodiment, the condition of dysregulated glucose homeostasis is selected from the group consisting of type 1-diabetes, type 2-diabetes, Addison's disease, immunodeficiency, malnutrition, or any combination thereof.

[0348] In an embodiment, the subject is a mammal, preferably a human, such as a human infant, preferably a human preterm infant, a human adult, such as a human elderly, or a patient under intensive care.

[0349] As seen in the Examples, the subject can be a human preterm infant.

[0350] The immune system of elderly people and ICU patents tends to become less effective, hence their immune system becomes slower to respond, which increases the risk of infections. Therefore, the PN composition can be used in a human elderly person and / or patients under intensive care.

[0351] In principle, the PN composition according to the invention can be administered to any (human) subject regardless of age and condition, hence the PN composition may even be administered to a healthy non-infected subject. Example 11 shows that a PN composition comprising GAAs and galactose does not adversely affect early clinical stability or growth in non-infected healthy subjects, such as preterm piglets, despite inducing a substantially lower and more stable blood glucose profile.

[0352] In an embodiment, the subject is a human adult, a human adolescent, and / or a human child.

[0353] In a further embodiment, the subject has a gastrointestinal disease or a gastrointestinal condition, such as intestinal insufficiency, intestinal failure, and / or short bowel. Hence, the subject may suffer from gastrointestinal issues, wherein the gut is unable to digest or absorb water, macronutrients, micronutrients, and electrolytes sufficient to sustain life, and thus require parenteral nutrition.85583PC01

[0354] 35

[0355] In an embodiment, the preterm infant is suspected of having an infection, preferably the preterm infant has an infection.

[0356] As seen in the Examples, a neonatal sepsis model of preterm piglets infected with Staphylococcus epidermidis has been used. Staphylococcus epidermidis is one of the most common pathogen causing neonatal sepsis in preterm infants.

[0357] In an embodiment, the infection is selected from the group consisting of bacterial, viral, fungal or any combination thereof.

[0358] In principle, the infection can be any type of infection, which the immune system must fight off. PN composition according to the present invention modifies the host response, and not the pathogen metabolism / growth. Therefore, it is possible that the PN composition can be widely applied to several other types of infections. Thus, without being bound by theory, it is expected that the PN composition of the invention will have the same effect of improving energy metabolism and glucose homeostatic control, which lead to the prevention of excessive inflammation and tissue damage, if the subject is infected with a bacterial, viral, and / or fungal infection.

[0359] In a preferred embodiment, the infection is a bacterial infection.

[0360] As seen in the Examples, the infection is a bacterial infection o Staphylococcus epidermidis.

[0361] In an embodiment, the bacterial infection is selected from the group consisting of coagulase-negative Staphylococcus, such as Staphylococcus epidermidis, E.coH, and group B streptococcus.

[0362] In principle, the bacterial infection can be any type of bacteria, since the immune response to bacterial infections are similar, if not identical.

[0363] In an embodiment, the bacterial infection is Staphylococcus epidermidis.

[0364] As seen in the Examples, the infection is a bacterial infection of Staphylococcus epidermidis.

[0365] In an embodiment, the PN composition is administered intravenously or intraarterially, such as via the umbilical artery.85583PC01

[0366] 36

[0367] In an embodiment, the PN composition is administered in an amount of 0.2g to 10g, such as 0.8g to 10g, such as 0.8g to 8g, such as 0.8g to 6g, such as 0.8g to 4g, such as lg to 6g, such as lg to 2g, such as 1.5g to 6g of one or more GAAs per kg per day.

[0368] In an embodiment, the PN composition is administered in an amount of 0.8g to 15g, such as 0.8g to 10g, such as 0.8g to 8g, such as 0.8g to 7g, such as 0.8g to 6g, such as 0.8g to 4g, such as lg to 6g, such as 1.5g to 6g of galactose per kg per day.

[0369] In an embodiment, the PN composition is administered to an infant in an amount of

[0370] • 0.2g to 3g of one or more GAAs per kg per day, and / or

[0371] • 0.8g to 15g of galactose per kg per day.

[0372] In an embodiment, wherein the PN composition is administered to an adult in an amount of

[0373] • 0.2g to 6g of one or more GAAs per kg per day, and / or

[0374] • 0.8g to 15g of galactose per kg per day.

[0375] In an embodiment, the PN composition is administered in an amount of:

[0376] - 0.2g to 1.5g valine per kg per day, preferably 0.5g valine per kg per day; - 0.2g to 1.5g asparagine per kg per day, preferably 0.5g asparagine per kg per day;

[0377] - 0.2g to 1.5g aspartate per kg per day, preferably 0.5g aspartate per kg per day; and / or

[0378] - 0.2g to 1.5g glutamate per kg per day, preferably 0.5g glutamate per kg per day.

[0379] Use of a PN composition

[0380] An aspect of the invention relates to use of the (stock) PN composition according to the invention for mixing or combining with a second PN composition.

[0381] In an embodiment, the second PN composition is a commercial PN composition.85583PC01

[0382] 37

[0383] An aspect of the invention relates to use of a parenteral nutrition (PN) composition comprising one or more glucogenic amino acids (GAAs) in an amount of at least 2% by weight of the PN composition.

[0384] A further aspect of the invention relates to use of a parenteral nutrition (PN) composition comprising one or more glucogenic amino acids (GAAs) in an amount of at least 2% by weight of the PN composition for preventing or delaying sepsis in a subject.

[0385] As seen in the Examples, the PN composition according to the present invention indeed prevents or delays sepsis. GAA supplementation and optionally combined with glucose restriction effectively limits glycolysis, enhances gluconeogenesis to prevent severe hypoglycemia, as well as completely protected against sepsis.

[0386] An additional aspect of the invention relates to use of a parenteral nutrition (PN) composition according to the invention for preventing or delaying sepsis in a subject.

[0387] An aspect relates to use of a parenteral nutrition (PN) composition comprising one or more glucogenic amino acids (GAAs) in an amount of at least 2% by weight of the PN composition for optimizing the immune function of a subject.

[0388] As seen in the Examples, the PN composition according to the invention optimizes the immune function by improving disease tolerance instead of resistance, prioritizing the maintenance of normal organ function over a strong immune response to clear the pathogens and thereby decreases the severity and risk of sepsis.

[0389] In an embodiment, the PN composition comprises one or more GAAs are in an amount of 2% to 10% by weight, such as 2% to 9% by weight, such as 2% to 8% by weight, preferably 2.5% to 6% by weight, more preferably 3% to 6% by weight, most preferably 3% to 5% by weight of the PN composition.

[0390] Another aspect relates to use of a parenteral nutrition (PN) composition according to the invention for optimizing the immune function of a subject.85583PC01

[0391] 38

[0392] An additional aspect of the invention relates to use of a parenteral nutrition (PN) composition according to the invention for treating and / or preventing infection or sepsis in a subject.

[0393] In an embodiment, the subject has a low immunity compared to a corresponding healthy subject.

[0394] In an embodiment, the subject has an infection or symptoms of infection.

[0395] In an embodiment, the subject has a condition of dysregulated glucose homeostasis.

[0396] In an embodiment, the condition of dysregulated glucose homeostasis is selected from the group consisting of type 1-diabetes, type 2-diabetes, Addison's disease, immunodeficiency, malnutrition, or any combination thereof.

[0397] In an embodiment, the subject is a mammal, preferably a human, such as a human infant, preferably a human preterm infant, a human adult, such as a human elderly, or a patient under intensive care.

[0398] In an embodiment, the subject is a human adult, a human adolescent, and / or a human child.

[0399] In a further embodiment, the subject has a gastrointestinal disease or a gastrointestinal condition, such as intestinal insufficiency, intestinal failure, and / or short bowel. Hence, the subject may suffer from gastrointestinal issues, wherein the gut is unable to digest or absorb water, macronutrients, micronutrients, and electrolytes sufficient to sustain life, and thus require parenteral nutrition.

[0400] In an embodiment, the preterm infant is suspected of having an infection, preferably the preterm infant has an infection.

[0401] As seen in the Examples, a neonatal sepsis model of preterm piglets infected with Staphylococcus epidermidis has been used. Staphylococcus epidermidis is one of the most common pathogen causing neonatal sepsis in preterm infants.85583PC01

[0402] 39

[0403] In an embodiment, the infection is selected from the group consisting of bacterial, viral, fungal or any combination thereof.

[0404] In an embodiment, the infection is a bacterial infection.

[0405] In an embodiment, the bacterial infection is selected from the group consisting of coagulase-negative Staphylococcus, such as Staphylococcus epidermidis, E.coli, and group B streptococcus.

[0406] In an embodiment, the bacterial infection is Staphylococcus epidermidis.

[0407] In an embodiment, the PN composition is administered intravenously or intraarterially, such as via the umbilical artery.

[0408] In an embodiment, the PN composition is administered in an amount of 0.2g to 10g, such as 0.8g to 10g, such as 0.8g to 8g, such as 0.8g to 6g, such as 0.8g to 4g, such as lg to 6g, such as lg to 2g, such as 1.5g to 6g of one or more GAAs per kg per day.

[0409] In an embodiment, the PN composition is administered in an amount of 0.8g to 15g, such as 0.8g to 10g, such as 0.8g to 8g, such as 0.8g to 7g, such as 0.8g to 6g, such as 0.8g to 4g, such as lg to 6g, such as 1.5g to 6g of galactose per kg per day.

[0410] In an embodiment, the PN composition is administered to an infant in an amount of

[0411] • 0.2g to 3g of one or more GAAs per kg per day, and / or

[0412] • 0.8g to 15g of galactose per kg per day.

[0413] In an embodiment, wherein the PN composition is administered to an adult in an amount of

[0414] • 0.2g to 6g of one or more GAAs per kg per day, and / or

[0415] • 0.8g to 15g of galactose per kg per day.

[0416] In an embodiment, the PN composition is administered in an amount of:85583PC01

[0417] 40

[0418] - 0.2g to 1.5g valine per kg per day, preferably 0.5g valine per kg per day; - 0.2g to 1.5g asparagine per kg per day, preferably 0.5g asparagine per kg per day;

[0419] - 0.2g to 1.5g aspartate per kg per day, preferably 0.5g aspartate per kg per day; and / or

[0420] - 0.2g to 1.5g glutamate per kg per day, preferably 0.5g glutamate per kg per day.

[0421] A kit of parts

[0422] An aspect of the invention relates to a kit of parts comprising

[0423] • the PN composition according to the invention,

[0424] • a second PN composition, and

[0425] • optionally, instructions for use.

[0426] Method for optimizing the immune function

[0427] An aspect of the invention relates to a method for optimizing the immune function of a subject, the method comprises using a parenteral nutrition (PN) composition comprising one or more glucogenic amino acids (GAAs) in an amount of at least 2% by weight.

[0428] An aspect of the invention relates to a method for optimizing the immune function of a subject comprising administering the (stock) PN composition or combination PN composition according to the invention to the subject in need thereof.

[0429] Another aspect of the invention relates a method of treating and / or preventing infection or sepsis in a subject comprising administering the (stock) PN composition or combination PN composition according to the invention to the subject in need thereof.

[0430] In an embodiment, the (stock) PN composition comprises:

[0431] • one or more GAAs in an amount of 2% to 10% by weight, such as 2% to 9% by weight, such as 2% to 8% by weight, preferably 2.5% to 6% by weight, such as 3% to 6% by weight, such as 3% to 5% by weight, such as 0.5% to 4% by weight, such as 1% to 4% by weight, preferably 1% to 3%85583PC01

[0432] 41

[0433] by weight, more preferably 0.5% to 2% by weight, most preferably 1% to 2% by weight of the PN composition, and / or

[0434] • galactose in an amount of 5% to 50% by weight, such as 10% to 50% by weight, such as 10% to 45% by weight, such as 10% to 30% by weight, preferably 10% to 25% by weight, more preferably 10% to 22% by weight, most preferably 10% to 20% by weight of the PN composition.

[0435] In an embodiment, the combination PN composition comprises:

[0436] • one or more GAAs in an amount of 0.2% to 2% by weight, such as 0.5% to 1.5% by weight, preferably 0.5% to 1% by weight, more preferably 0.7% to 1% by weight of the combination PN composition, and / or

[0437] • galactose in an amount of 5% to 20% by weight, preferably 5% to 15% by weight, more preferably 5% to 10% by weight of the combination PN composition.

[0438] In an embodiment, the subject has a low immunity compared to a corresponding healthy subject.

[0439] In an embodiment, the subject has an infection or symptoms of infection.

[0440] In an embodiment, the subject has a condition of dysregulated glucose homeostasis.

[0441] In an embodiment, the condition of dysregulated glucose homeostasis is selected from the group consisting of type 1-diabetes, type 2-diabetes, Addison's disease, immunodeficiency, malnutrition, or any combination thereof.

[0442] In an embodiment, the subject is a mammal, preferably a human, such as a human infant, preferably a human preterm infant, a human adult, such as a human elderly, or a patient under intensive care.

[0443] In an embodiment, the subject is a human adult, a human adolescent, and / or a human child.85583PC01

[0444] 42

[0445] In a further embodiment, the subject has a gastrointestinal disease or a gastrointestinal condition, such as intestinal insufficiency, intestinal failure, and / or short bowel. Hence, the subject may suffer from gastrointestinal issues, wherein the gut is unable to digest or absorb water, macronutrients, micronutrients, and electrolytes sufficient to sustain life, and thus require parenteral nutrition.

[0446] In an embodiment, the preterm infant is suspected of having an infection, preferably the preterm infant has an infection.

[0447] In an embodiment, the infection is selected from the group consisting of bacterial, viral, fungal or any combination thereof.

[0448] In an embodiment, the infection is a bacterial infection.

[0449] In an embodiment, the bacterial infection is selected from the group consisting of coagulase-negative Staphylococcus, such as Staphylococcus epidermidis, E.coli, and group B streptococcus.

[0450] In an embodiment, the bacterial infection is Staphylococcus epidermidis.

[0451] In an embodiment, the PN composition is administered intravenously or intraarterially, such as via the umbilical artery.

[0452] In an embodiment, the PN composition is administered in an amount of 0.2g to 10g, such as 0.8g to 10g, such as 0.8g to 8g, such as 0.8g to 6g, such as 0.8g to 4g, such as lg to 6g, such as lg to 2g, such as 1.5g to 6g of one or more GAAs per kg per day.

[0453] In an embodiment, the PN composition is administered in an amount of 0.8g to 15g, such as 0.8g to 10g, such as 0.8g to 8g, such as 0.8g to 7g, such as 0.8g to 6g, such as 0.8g to 4g, such as lg to 6g, such as 1.5g to 6g of galactose per kg per day.

[0454] In an embodiment, the PN composition is administered to an infant in an amount of85583PC01

[0455] 43

[0456] • 0.2g to 3g of one or more GAAs per kg per day, and / or

[0457] • 0.8g to 15g of galactose per kg per day.

[0458] In an embodiment, wherein the PN composition is administered to an adult in an amount of

[0459] • 0.2g to 6g of one or more GAAs per kg per day, and / or

[0460] • 0.8g to 15g of galactose per kg per day.

[0461] In an embodiment, the PN composition is administered in an amount of:

[0462] - 0.2g to 1.5g valine per kg per day, preferably 0.5g valine per kg per day; - 0.2g to 1.5g asparagine per kg per day, preferably 0.5g asparagine per kg per day;

[0463] - 0.2g to 1.5g aspartate per kg per day, preferably 0.5g aspartate per kg per day; and / or

[0464] - 0.2g to 1.5g glutamate per kg per day, preferably 0.5g glutamate per kg per day.

[0465] Further aspects of the invention

[0466] An aspect of the invention relates to a method for administering the (stock) PN composition or combination PN composition according to the invention to a subject in need thereof.

[0467] A further aspect relates to a method for administering a parenteral nutrition (PN) composition comprising one or more GAAs in an amount of at least 2% by weight, such as at least 3% by weight of the PN composition.

[0468] In an embodiment, the subject is a preterm infant.

[0469] Another aspect relates to a method for optimizing the immune function of a subject, comprising administering a parenteral nutrition (PN) composition comprising one or more GAAs in an amount of at least 2% by weight of the PN composition.85583PC01

[0470] 44

[0471] Another aspect relates to a method for preventing or delaying sepsis in a subject, comprising administering a parenteral nutrition (PN) composition comprising one or more GAAs in an amount of at least 2% by weight of the PN composition.

[0472] In an embodiment, the PN composition comprises one or more GAAs are in an amount of 2% to 10% by weight, such as 2% to 9% by weight, such as 2% to 8% by weight, preferably 2.5% to 6% by weight, more preferably 3% to 6% by weight, most preferably 3% to 5% by weight of the PN composition.

[0473] In an embodiment, the subject is a mammal, preferably a human, such as a human infant, preferably a human preterm infant, or a human elderly.

[0474] In an embodiment, the subject is a human adult, a human adolescent, and / or a human child.

[0475] In a further embodiment, the subject has a gastrointestinal disease or a gastrointestinal condition, such as intestinal insufficiency, intestinal failure, and / or short bowel. Hence, the subject may suffer from gastrointestinal issues, wherein the gut is unable to digest or absorb water, macronutrients, micronutrients, and electrolytes sufficient to sustain life, and thus require parenteral nutrition.

[0476] It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention. Thus, for example the embodiments / claims relating to the method of the invention also apply to the embodiments relating to the use-embodiments / claims. Hence, individual features mentioned in different claims, may possibly be advantageously combined, and the mentioning of these features in different claims does not exclude that a combination of features is not possible and advantageous.

[0477] Although the present invention has been described in connection with the specified embodiments, it should not be construed as being in any way limited to the presented examples. The scope of the present invention is to be interpreted in the light of the accompanying claim set. In the context of the claims, the terms "comprising" or "comprises" do not exclude other possible elements or steps. Also,85583PC01

[0478] 45

[0479] the mentioning of references such as "a" or "an" etc. should not be construed as excluding a plurality. The use of reference signs in the claims with respect to elements indicated in the figures shall also not be construed as limiting the scope of the invention.

[0480] All patent and non-patent references cited in the present application are hereby incorporated by reference in their entirety.

[0481] The invention will now be described in further details in the following non-limiting examples.

[0482] Examples

[0483] Example 1 - Preparation of a neonatal sepsis model

[0484] Aim of study

[0485] The aim of the study is to prepare and obtain a suitable neonatal sepsis model, which represents preterm newborn infants.

[0486] Materials and methods

[0487] Preterm piglets

[0488] Seventy-four crossbred preterm piglets (Landrace x Large White x Duroc) were delivered by cesarean section at day 106 (90% gestation, term at day 117) from three healthy pregnant sows (21, 23, and 27 piglets per sow). The anesthesia and surgical procedures of sows are described in detail elsewhere. After delivery, the newborn piglets were immediately transferred to preheated (37°C) newborn incubators with a supplementary oxygen supply (1-2 L / min). When needed, animals were resuscitated by Doxapram and Flumazenil (0.1 ml / kg for each, intramuscular injection), tactile stimulation, and manual noninvasive positive pressure ventilation, applied as necessary until achieving respiratory stability. Furthermore, each piglet was equipped with a vascular catheter inserted into the dorsal aorta via the transected umbilical cord, facilitating the administration of PN, bacterial inoculation, and blood sampling.

[0489] S. epidermidis culture preparation

[0490] Originating from frozen stock, the Staphylococcus epidermidis (S. epidermidis) bacteria (isolated from a septic infant) were cultured in tryptic soy broth85583PC01

[0491] 46

[0492] overnight. Following this incubation, bacterial density was accurately gauged using spectroscopic techniques. Based on these measurements, a working solution (109CFU / kg) was formulated by diluting the culture with a precise volume of sterile saline.

[0493] Infecting piglets with S. epidermidis

[0494] Approximately two hours after the cesarean section, all animals were randomly stratified based on birth weight and gender into groups receiving one of the types of PN: rGLU, and rGLU-GAAs. Animals were then inoculated with live

[0495] S. epidermidis (SE) or control saline via an interatrial infusion over three minutes. During the post-inoculation period, animals reaching predefined humane endpoint (lethal sepsis criteria, including arterial blood pH of < 7.1 and clinical signs of deep lethargy, discoloration, and tachypnea) were euthanized for blood sampling and tissue collection. To standardize sample collection times and improve animal welfare, the experimental duration was adjusted for the second and third litters to 15 h. This modification was prompted by initial findings where animals in the first litter received intensive care for up to 20 h post-inoculation.

[0496] Blood samples were collected at 3, 6, and 12 h post-bacterial challenge and at the study end via the arterial catheter for blood gas analysis and hematology, as well as plasma collection and storage for cytokine measurements. At 3 and 6 h and the study ended, blood samples were drawn through the jugular vein puncture for bacterial enumeration. Serum and plasma samples at euthanasia were used for serum biochemical analysis and plasma metabolomic analysis.

[0497] At humane endpoint or 15 h of post-inoculation, all piglets were deeply anesthetized with a Zoletil mixture (0.1 ml / kg), which included Zoletil 50 (125 mg tiletamine, 125 mg zolazepam), xylazine (6.25 ml xylazine 20 mg / ml), ketamine (1.25 ml ketamine 100 mg / ml), and butorphanol (2.5 ml butorphanol 10 mg / ml), and were subsequently euthanized with an intracardiac injection of barbiturate. The liver was collected and preserved (frozen) during necropsy for liver transcriptomic and metabolomic analysis.

[0498] Results and conclusion85583PC01

[0499] 47

[0500] The neonatal sepsis model in the form of preterm piglets infected with S. epidermidis was successfully obtained in order to explore nutritional strategies that can optimize the host defense via controlling glycolysis-induced inflammation, in turn protecting against lethal sepsis.

[0501] ic amino acid on

[0502]

[0503] Aim of study

[0504] The aim of the study is to investigate the effect of glucose restriction and presence of glucogenic amino acid in PN in a neonatal sepsis model in preterm piglets.

[0505] Materials and methods

[0506] The neonatal sepsis model was prepared as explained in Example 1.

[0507] Parenteral nutrition

[0508]

[0509] Specialized formulations of parenteral nutrition (PN) were developed from the Kabiven G19% infusion formula (Fresenius-Kabi). Kabiven G19% infusion formula comprises:

[0510] • 3.3% amino acids including alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine;

[0511] • 3.9% lipids including soya oil (CAS No.:8001-22-7);

[0512] • 9.7% glucose monohydrate; and

[0513] • 0.7% electrolytes calcium chloride dihydrate, magnesium sulfate heptahydrate, potassium chloride, sodium acetate trihydrate, and sodium glycerophosphate.

[0514] Kabiven G19% is provided as a three chamber bag and an overpouch. Each chamber consists of glucose, amino acid and electrolyte solution, and fat emulsion, respectively.

[0515] For PN with standard glucose (sGLU) provision, the glucose chamber was emptied, and a specific volume of 50% glucose solution was added into the glucose chamber to reach 10% glucose PN with infusion rate of 6 mL / kg / h, equivalent to 14.4g / kg / d glucose, similar to PN used for preterm infants. For glucose- restricted85583PC01

[0516] 48

[0517] (rGLU) PN, a precise volume of glucose was removed from the PN glucose chamber to reach a final glucose content of 1.4% (2 g / kg / d with an infusion rate of 6 mL / kg / h). For PN with glucose restriction and supplementation of four glucogenic amino acids (rGLU-GAAs), a precise volume of four glucogenic amino acids solution was added in the glucose-restricted PN solution to reach the infusion rate of 0.5 g / kg / d for each amino acid (aspartate, glutamate, asparagine, valine).

[0518] Blood gas, hematologv, inflammatory markers, serum biochemistry, and Dlasma ATP measurement

[0519] Blood samples at 3, 6, 12, and 15 h underwent routine blood gas analysis utilizing the GEM Premier 3000 (Instrumentation Laboratory, USA) for blood pH, pCC , oxygen saturation, base excess, glucose, and lactate measurement. Hematological assessments were conducted with the ADVIA 2120i Hematology System (Siemens, Germany). For the quantification of plasma cytokines, TNF-o, IL-6, and IL-10 were analyzed using porcine-specific DuoSet enzyme-linked immunosorbent assays (R&D Systems). Serum biochemistry evaluations were executed using the ADVIA 1800 Chemistry System (Siemens, Germany). Additionally, extracellular ATP level was measured by the ATP Colorimetric Assay Kit (Sigma-Aldrich).

[0520] Statistics

[0521] All statistical analyses were executed via R version 4.3.2 unless otherwise stated. For discerning differences at specific intervals (3, 6, 12, and 15 h), continuous data underwent analysis via a linear mixed-effects model, incorporating group, gender, and birth weight as fixed factors and litter as a random factor, using Ime4 package. Another linear mixed-effects model was employed to probe further disparities spanning the entire experimental duration. This model integrated group, time, their interaction, gender, and birth weight as fixed factors, with litter and pig ID as random factors, using Ime4 package. Normal distribution, variance homogeneity of residuals, and fitted values were assessed. The data that did not conform to a normal distribution were logarithmic transformed. If transformation did not achieve approximate log-normal distribution, the non-parametric Mann-Whitney U test was used instead. To examine different strategies in the infection response, reaction norm analysis was performed using blood pH as a readout for health at 3, 6, and 15 h and plotted against the pathogen burdens at the same85583PC01

[0522] 49

[0523] time point by linear regression. Extra sum-of-squares F Test was used to compare slopes. Statistical significance was defined as P-value < 0.05.

[0524] Results

[0525] A restricting of glucose in PN and with further supplementation of strictly glucogenic amino acids (GAAs) during neonatal infection of S. epidermidis (aspartate, asparagine, glutamate, and valine, SE-rGLU-GAAs, Figure 1A) was tested. Glucogenic amino acids are capable of entering the TCA cycle, providing the necessary metabolites for both gluconeogenesis and energy generation through OxPhos.

[0526] Survival curves of the animals throughout the experiment shows 100% survival of SE-rGLU-GAAs animals (Figure IB).

[0527] The SE-rGLU-GAAs animals maintained stable blood glucose levels of around 2 mM, in contrast to most infected controls being hyperglycemic with blood glucose >8 mM at the end of the study (Figure 1C). It is unknown what the cut-off for hypoglycemia in newborn pigs is, but glucose levels in rGLU-GAAs animals were not associated with any clinical symptoms of hypoglycemia (e.g., seizure, tachypnea, paleness, lethargy, convulsion, and hypothermia). Clinical parameters also indicated gradual deterioration of the infected controls from 12h until the end of the study, indicated by severe drops of pH, spO2, base excess, and hemoglobin and elevation of pCO2 and lactate, suggesting both respiratory and metabolic acidosis (Figure 1D-1I). In contrast, the combination of glucose restriction and GAA supply prevented all these disturbances. rGLU-GAAs animals exhibited a slight yet significant and transient rise in lactate levels at the early phase of infection (6 h). This increase suggests an enhanced conversion of the administered GAAs into pyruvate, which is then metabolized into lactate, likely through the TCA cycle. Moreover, the SE-rGLU-GAAs group at the end of the study exhibited increased blood urea nitrogen (BUN) and ATP production, suggesting the GAAs were metabolized, possibly through the TCA cycle and OxPhos (Data not shown). Biochemical parameters measured at euthanasia also indicate that glucose restriction in combination with GAAs protected against injuries to the liver (lower AST and ALT) and kidneys (lower creatinine), as well as prevented either impairment in hepatic synthesis of negative acute-phase reactants or vascular injury and lipid dysregulation (higher albumin and cholesterol) (Data not shown).85583PC01

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[0529] Conclusion

[0530] An enhanced conversion of the administered GAAs into pyruvate was found, which is then metabolized into lactate, likely through the TCA cycle. Glucose restriction in combination with GAAs protected against injuries to the liver (lower AST and ALT) and kidneys (lower creatinine), as well as prevented either impairment in hepatic synthesis of negative acute-phase reactants or vascular injury and lipid dysregulation.

[0531]

[0532] amino acid on

[0533]

[0534] host disease resistance and tolerance

[0535] Aim of study

[0536] The aim of this study is to examine bacterial clearance, immune, and inflammatory status to explore the associated host defence strategies.

[0537] Materials and methods

[0538] See Example 1 and 2

[0539] Results

[0540] Blood bacterial loads in SE-rGLU-GAA animals were reduced (significantly at 3 h and by tendency at 6 h) compared to infected controls, indicating an improved host resistance eliminating pathogens during the early phase of infection (Figure 2A). This was associated with better preservation of blood leukocyte subsets and prevention of thrombocytopenia by the intervention (Figure 2B-2F). Notably, rGLU-GAAs led to a significant reduction of plasma levels of the pro-inflammatory cytokines TNF-o and IL-6 and an elevation of anti-inflammatory cytokine IL-10 (Figure 2G-2I), consistently from the early phase of infection (3-6 h) until the end of the study, suggesting well-controlled inflammatory responses. These resistance phenotype and inflammatory status likely contribute to the substantial protective effects of rGLU-GAA treatment against organ injuries.

[0541] To evaluate whether rGLU-GAA treatment altered disease tolerance, reaction norm analysis was employed, plotting blood pH against bacterial burdens over time during infection. At 3 h, both infected groups showed similar slopes (Data not shown). However, by 6 h, SE-rGLU-GAAs animals still maintained a shallow slope, indicating stable health, in contrast to the infected controls, which showed85583PC01

[0542] 51

[0543] a steep decrease in health when bacterial burdens increased (Data not shown).

[0544] This indicates enhanced disease tolerance in the intervention group. At euthanasia, although the slopes of the reaction norms were similar between the two groups (Data not shown), the low blood pH suggests that infected controls were in a state of immunoparalysis, whereas SE-rGLU-GAAs animals still maintained tolerance response. Strikingly, combined glucose restriction and GAA supplementation exerted transient resistant response in early phase of infection and maintained consistent tolerance phenotype during the whole study period to prevent lethal sepsis and organ injuries.

[0545] Conclusion

[0546] Combining glucose restriction and GAA supplementation effectively limited glycolysis, enhanced gluconeogenesis to prevent severe hypoglycemia as seen in animals with glucose restriction, as well as completely protected against sepsis. The metabolism of GAA via gluconeogenesis helps maintain a necessary glucose supply of glucose for newborn vital organs (such as the brain) and acts as a counterbalance to glycolysis, thus indirectly affecting inflammation. Even though preterm infants and piglets are insulin resistant, they possess the required enzymes for normal gluconeogenic activities. Thus, glucose homeostatic control during neonatal infection in preterm infants is achieved by restricting PN glucose supply and supplementing with GAAs.

[0547] This intervention did not completely inhibit pro-inflammatory responses, as the treated animals were capable of mounting sufficient resistance response to better eliminate invading bacteria during the first six hours of infection relative to the infected controls. Strikingly, tolerance mechanisms were also enhanced by the intervention at six hours and maintained until the end of the study. The results show simultaneous enhancement of both disease tolerance and resistance as well as no sign of declining health over the infection course and thereby elimination of infection.

[0548] and inflammation

[0549]

[0550] Aim of study

[0551] The aim of the study is to test whether protection against sepsis in SE-rGLU-GAA animals is mediated by reduced glycolysis due to glucose restriction and increased85583PC01

[0552] 52

[0553] gluconeogenesis due to the supplementation of glucogenic amino acids. The effects are explored via hepatic transcriptomics

[0554] Materials and methods

[0555] See Examples 1-3

[0556] Hepatic transcriptome profiling and analysis

[0557] RNeasy mini kit (QIAGEN, US) was used to extract liver RNA from all piglets. Gene expression was profiled by utilizing whole-transcriptome shotgun sequencing. Library preparation and sequencing were carried out by NOVOGENE services (Cambridge, UK). We used 1000 ng of RNA and the VAHTS mRNA-Seq V3 Library Prep Kit for Illumina (Vazyme, Nanjing, PRC) to prepare RNA-Seq libraries. The libraries were sequenced on the Illumina NovaSeq 6000 platform, generating 150 bp paired-end reads. Quality and adapter trimming of raw reads were performed using TrimGalore (Babraham Bioinformatics, Cambridge, UK). The resulting clean reads, approximately 26 million per sample, were aligned to the porcine genome Sscrofall.V) using Tophat2. Gene annotation information for the porcine genome was sourced from Ensembl (release 99). The script htseq-count was employed to generate a gene-count matrix.

[0558] Differentially expressed gene (DEG) analysis was performed using the DESeq2 package version 1.38.3. We excluded lowly expressed genes, defined as having a raw count of at least 10 in 4, 5, and 6 samples in Experiment 1, Experiment 2, and Experiment 3, respectively. Litter was added to adjust the model. To robustly estimate the fold change (FC), we applied the IfcShrink function with ashr estimator. A false discovery rate (FDR) cut-off of 0.05 was implemented to obtain DEGs. Genes were ranked by the FC (in log scale) before pathway analyses. The gene set enrichment analysis (GSEA) module in the cl uster Pro filter package version 4.0.2 was utilized with Sus scrofa Kyoto Encyclopedia of Genes and Genomes (KEGG) knowledgebases to elucidate pathway-level disturbances. Down-or up-regulated pathways were defined based on a normalized enrichment score (NES) with negative or positive values, respectively. The FDR cut-off of 0.05 was considered to determine significant pathways. To create a comprehensive visualization of specific genes within GSEA-enriched pathways, DEGs were identified based on the KEGG, Biological Processes aspect of Gene Ontology (GO: BP), Reactome, and Hallmark knowledgebases, with additional insights from the literature. The relative expression of DEGs was then visualized through85583PC01

[0559] 53

[0560] heatmaps generated using the ComplexHeatmap package version 2.15.1. The analysis of liver transcriptomics data was performed in R 4.2.3.

[0561] Results

[0562] The PCA plot revealed distinct clusters corresponding to four experimental groups, indicating the impact of both intervention and infection on hepatic gene expression levels (Data not shown). Notably, infection exerted a less pronounced effect in GAA-treated animals than GLU controls. GSEA results between the two infected groups revealed that most metabolic pathways were upregulated in SE-rGLU-GAAs animals, including OxPhos, TCA cycle, carbohydrate metabolism (pyruvate metabolism, glycolysis / gluconeogenesis), as well as multiple amino acids (valine, aspartate, glutamate, and arginine) and lipid (fatty acid) associated metabolic pathways (Figure 3A and Table not shown). In contrast, several adaptive immune pathways (Thl / Th2, Thl7 cell differentiation, and IL-17 signaling) and pro-inflammatory pathways (TNF, Hippo, NF-kappa B, HIF-1 signaling) were suppressed. Importantly, between uninfected groups, all metabolic pathways were upregulated in CON-rGLU-GAAs, whereas no immunity and inflammation-related pathways were enriched (Figure 3B and Table not shown).

[0563] DEGs analysis revealed that SE-rGLU-GAAs animals exhibited 3518 up-regulated and 3721 down-regulated DEGs, compared to SE-sGLU animals. Specifically focusing on the genes involved in glycolysis / gluconeogenesis (96 genes), 54 genes were regulated between the two infected groups. Most genes strictly involved in gluconeogenesis (14 out of 16) were upregulated, whereas most genes strictly involved in glycolysis (26 out of 35) were downregulated in SE-rGLU-GAAs animals (Data not shown). This indicates that glycolysis was inhibited by glucose restriction while GAA supply markedly enhanced gluconeogenesis. Additionally, 22 genes involved in aspartate, asparagine, glutamate, and valine metabolism were upregulated, and 9 genes were downregulated (Data not shown). We further found regulated genes associated with various energy metabolism-related pathways, including the TCA cycle (22 up / 2 down), fatty acid p-oxidation (44 up / 4 down), ketogenesis (3 up / 0 down), and OxPhos (146 up / 15 down) (Data not shown). In contrast, most DEGs in immune-related pathways were significantly downregulated in SE-rGLU-GAAs animals, including the RIG-I-like receptor signaling (27 down / 4 up), Thl and Th2 cell differentiation (26 down / 9 up), Thl785583PC01

[0564] 54

[0565] cell differentiation (36 down / 13 up), IL-17 signaling (38 down / 7 up), TNF signaling (52 down / 9 up), Hippo signaling (65 down / 20 up), NF-kappa B signaling (41 down / 15 up), and Wnt signaling (54 down / 36 up) (Data not shown).

[0566] Conclusion

[0567] Collectively, these findings support that enhanced disease tolerance by combining a restricted glucose PN with GAAs was associated with inhibited hepatic glycolysis but improved gluconeogenesis, mitochondrial OxPhos, TCA cycle, amino acid metabolism, and fatty acid p-oxidation.

[0568] Example 5 - Hepatic metabolomics confirms metabolic rewiring by rGLU-GAAs treatment

[0569] Aim of study

[0570] The aim of the study is to confirm metabolic regulations found by hepatic transcriptomics using hepatic untargeted metabolomics from the same tissues and animals.

[0571] Materials and methods

[0572] See Examples 1-4

[0573] Heoatic metabolome profiling and analysis

[0574] Untargeted metabolomics analysis was conducted using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) through the services provided by Creative Proteomics (Shirley, NY, USA). The methodology involved the initial step of thawing and weighing 50 mg samples, followed by the addition of 800 pL of pre-cooled methanol. The samples were then vortexed for 180 s at 65 Hz and sonicated for 30 min at 4°C. Subsequently, each sample was incubated at -20°C for 1 h, vortexed for 30 s, and kept at 4°C for an additional 30 min. The samples were then centrifuged at 12,000 rpm and 4°C for 15 min. The resulting supernatant was transferred to new tubes, stored at -20°C for another hour, and centrifuged under the same conditions. Following this, 200 pL of the supernatant and 5 pL of DL-o-Chlorophenylalanine (0.14 mg / mL) were combined in a vial for subsequent instrumental analysis. The experimental analysis utilized an ACQUITY UPLC system (Waters, Milford, MA, USA) combined with a Q-Exactive-Plus Hybrid Quadrupole-Orbitrap mass-spectrometer (Thermo, Waltham, MA, USA). The85583PC01

[0575] 55

[0576] column employed was an ACQUITY UPLC HSS T3 column measuring 100 mm x 2.1 mm with a particle size of 1.8 pm (Waters, Milford, MA, USA). The mobile phase consisted of water with 0.05% formic acid as mobile phase A and acetonitrile as mobile phase B. A gradient elution program was utilized as follows: starting at 5% B for 0-1 min, increasing to 95% B from 1-12.5 min, holding at 95% B from 12.5-13.5 min, decreasing back to 5% B from 13.5-13.6 min, and finally returning to 5% B from 13.6-16 min. The flow rate was maintained at 0.3 mL / min with a column temperature of 40 °C. The autosampler temperature was set to 4 °C throughout the analysis. Mass spectrometry parameters for both positive (POS) and negative (NEG) ion modes included a heater temperature of 300 °C, sheath gas flow rate of 45 arb, auxiliary gas flow rate of 15 arb, sweep gas flow rate of 1 arb, spray voltage of 3.0 kV for POS and 3.2 kV for NEG, capillary temperature of 350 °C, and S-Lens RF levels of 30% for POS and 60% for NEG.

[0577] There were 5614 and 5067 features in positive and negative ion modes were detected. Among them, 1098 putativelly annotated features, assigned by the vendor and cross-examined by our team, were eventually used for subsequent analyses. The low repeatability annotated features were excluded, whose relative standard deviation (RSD) was > 25% in the pooled QC samples. Prior to statistical analysis, data was median-normalized and loglO-transformed using MetaboAnalystR package version 4.0.0 in R version 4.2.3. A linear mixed-effects model was conducted, incorporating group, gender, and birth weight as fixed factors and litter as a random factor, using Ime4 packages. An FDR cut-off of 0.1 was applied to derive molecules with differential abundance (MDAs). MDAs-based pathway analysis, which integrated hypergeometric test and out-degree centrality, was performed in the MetaboAnalyst 6.0 (https: / / www.metaboanalyst.ca) with Sus scrofa KEGG knowledgebase. Pathways with a P-value cut-off of 0.05 and more than one significant hit were considered statistically significant.

[0578] Results

[0579] A PCA of 1014 identified metabolites revealed similar metabolic clusters among experimental groups (Data not shown). Between SE-rGLU-GAAs and SE-sGLU animals, 77 metabolites were found to be significantly altered (Table not shown). Pathway analysis revealed that most of the metabolites with differential abundance (MDAs) were associated with arginine biosynthesis and carbohydrate85583PC01

[0580] 56

[0581] metabolism, including glycolysis / gluconeogenesis (Figure 4 and Table not shown). Specifically focusing on MDAs, carbohydrate-derived metabolites, and lactic acid were reduced by SE-rGLU-GAAs (Data not shown), supporting the finding of attenuated glycolysis from transcriptomics. In particular, glycerol-3-phosphate, a key substrate for di hydroxyacetone phosphate (DHAP) conversion by glycerol-3-phosphate dehydrogenase 1 (GPD1) in gluconeogenesis, was also decreased by the intervention (Data not shown), in line with the elevation of hepatic GPD1 expression (Data not shown). Further, the decreased level of arginine in SE-rGLU-GAAs animals was also potentially due to its conversion into glucose via gluconeogenesis or metabolism via the urea cycle, which was supported by increased ARG1 levels from transcriptomics data (Data not shown). As a result, citrulline, together with several other genes in the urea cycle ASL, ARG1, OTC, and ASS1 (Data not shown), were all upregulated by the intervention. Lastly, most fatty acids, carnitines (involved in fatty acid transport into mitochondria before oxidation), and arachidonic acid-related metabolites were upregulated (9 out of 12) in SE-rGLU-GAAs animals, supporting the finding of increased fatty acid oxidation and OxPhos from transcriptomic data. Overall, hepatic metabolomic data were largely in line with transcriptomic data, all pointing to suppressed glycolysis as well as enhanced gluconeogenesis, fatty acid oxidation, Oxphos, and amino acid metabolism by rGLU-GAAs intervention.

[0582] Conclusion

[0583] The hepatic metabolomic data were largely in line with transcriptomic data. The tolerance phenotype exerted by glucose restricted and GAAs was associated with improved hepatic energy metabolic pathways, including TCA cycle, OxPhos, fatty acid oxidation, and ketogenesis. These pathways have been shown to play critical roles in providing alternative energy for vital organs (e.g., brain and heart) and protecting against inflammation-induced tissue damage during sepsis.

[0584] Example 6 - Plasma metabolomics confirms systemic metabolism modulated bv rGLU-GAAs

[0585] Aim of study

[0586] The aim of the study is to investigate systemic metabolic modulations induced by rGLU-GAAs during infection, using metabolomics of plasma samples.85583PC01

[0587] 57

[0588] Materials and methods

[0589] See Examples 1-5

[0590] Plasma metabolome orofiling and analysis

[0591] The UPLC-MS method was used via a service from Creative Proteomics (Shirley, NY, USA) for plasma untargeted analysis. Briefly, samples were first thawed, and then 100 pL was transferred into a 1.5 mL tube. Next, 300 pL of pre-cooled methanol was added and vortexed for 30 s. Samples were subsequently stored at -20 °C for 1 h and then centrifuged at 9660 ref and 4 °C for 15 min. The supernatant was transferred to new tubes, stored at -20 °C for 1 h, and centrifuged under the same conditions. Subsequently, 120 pL of the supernatant was mixed with and 3 pL of DL-o-Chlorophenylalanine (0.5 mg / mL) in a vial before injecting into the instrument. The same UPLC-MS apparatus, along with the stationary and mobile phases and gradient elution program, were employed under identical conditions as those used in the hepatic metabolomics analysis.

[0592] A total of 754 putatively annotated features in POS and NEG ion modes were used for downstream analyses. We used a cut-off RSD of 25% in the pooled QC samples to exclude low repeatability annotated features. The median normalization and loglO-transformation were applied before statistical analysis. For SE-rGLU-GAAs vs. SE-sGLU groups analysis, a linear mixed-effects model was utilized with group, gender, and birth weight as fixed factors and litter as a random factor using the Ime4 packages. For SE-rGLU-GAAs vs. SE-rGLU groups analysis, another linear model was utilized, adjusted for gender and birth weight (no litter adjustment because all animals were in the same litter), using the Umma method in MetaboAnalystR package version 4.0.0 in R version 4.2.3. We used FDR cut-off of 0.1 to determine the MDAs. The integrated hypergeometric test and out-degree centrality modules were performed in the MetaboAnalyst 6.0 (https: / / www.metaboanalyst.ca) using the Sus scrofa KEGG knowledgebase. Significant pathways (P-value cut-off of 0.05 and more than one significant hit) were subjected to biological interpretation.

[0593] Results

[0594] Different from the hepatic metabolome, PCA from the plasma metabolome showed a clear separation between SE-rGLU-GAAs vs. infected controls (Data not shown). Numerous significantly altered metabolites were found, with 12285583PC01

[0595] 58

[0596] increased and 160 decreased in SE-rGLU-GAAs animals (Table not shown). MDA-based pathway analysis revealed several enriched pathways involved in metabolism of amino acids (both glucogenic and ketogenic amino acid biosynthesis and metabolism), carbohydrates (butanoate, glyoxylate and dicarboxylate metabolism, TCA cycle), and unsaturated fatty acids (Figure 5 and Table not shown). Of note, all MDAs involved in glycolysis, pyruvate metabolism, and pentose phosphate pathways were downregulated in SE-rGLU-GAAs vs. the infected control, further confirming the inhibition of glycolysis, likely via glucose restriction. For TCA cycle-related MDAs, cis-aconitic acid and citric acid were downregulated, but 2-oxoglutaric acid was upregulated. This again confirms the enhancement of gluconeogenesis via the TCA cycle induced by GAAs, as all four supplemented GAAs (asparagine, aspartate, valine, and glutamate) can be catabolized together with 2-oxoglutaric acid prior to entering the TCA cycle without the involvement of cis-aconitic acid and citric acid. Moreover, nicotinate and nicotinamide metabolism are crucial for synthesizing NAD+ and NADP+, which are key for ATP production. It was found that nicotinic acid was upregulated, while nicotinamide was downregulated in SE-rGLU-GAAs animals. The rGLU-GAAs supply during infection also elevated multiple glucogenic and ketogenic amino acids, ketone bodies, carnitine, and fatty acid derivatives, again suggesting improved fatty acid p-oxidation and ketogenesis (Data not shown).

[0597] Collectively, the plasma metabolomic data further strengthens the hepatic transcriptomic and metabolomic findings. The sepsis preventive effects of glucose restriction and glucogenic amino acid supplementation were strongly tied to a rewiring of the host energy metabolism, resulting in reduced glycolysis and enhanced gluconeogenesis, amino acids metabolism, ketogenesis, and fatty acid P-oxidation (Data not shown).

[0598] Conclusion

[0599] Collectively, the plasma metabolomic data further strengthens the hepatic transcriptomic and metabolomic findings. The sepsis preventive effects of glucose restriction and glucogenic amino acid supplementation were strongly tied to a rewiring of the host energy metabolism.85583PC01

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[0601] Example 7 - Glucogenic amino acid supply during glucose restriction elevates blood glucose and enhances both host resistance and tolerance Aim of study

[0602] The aim of the study is to explore the impacts of GAA supplementation on a glucose-restricted background on glycemia and host defence against infection. Thus, the separate effect of GAAs from glucose restriction compared to glucose restriction plus GAA supplementation was investigated.

[0603] Materials and methods

[0604] See Examples 1 and 2

[0605] Results

[0606] A total of 2 / 7 animals with glucose restriction alone succumbed to sepsis, whereas all 6 rGLU-GAAs animals survived (Figure 6A-6B).

[0607] Importantly, glucose- restricted animals were severely hypoglycemic with mean blood glucose < 1 mM , while the GAA-supplemented animals had stable blood glucose levels around 2 mM during the 15 h of the study (Figure 6C), clearly indicating induction of gluconeogenesis by GAAs. GAAs also prevented respiratory acidosis (higher blood pH, lower pCC , and unaltered lactate, Figure 6D-6F), while increased BUN levels suggest increased amino acid catabolism (Figure 6G).

[0608] Importantly, GAAs enhanced bacterial clearance at both 3 and 6 h postinoculation, indicating improved disease resistance responses (Figure 6H). This was associated with better blood leukocyte replenishment and reduced pro-inflammatory responses at the later phase of the experiment (Figure 6 I-L). Subsequent reaction norm analysis showed similar slopes at 3-6 h postinoculation between the two groups, but supplementation of GAA led to improved tolerance at the end of the study (Data not shown). Together, this experiment confirmed that GAAs directly contributed to the clinical benefits of the combined glucose restriction and GAA intervention shown above, most likely via enhancement of both disease resistance and tolerance, as well as improved hepatic gluconeogenesis. Importantly, current findings indicate that avoidance of severe hypoglycemia also contributed to sepsis prevention. The higher glucose levels in SE-rGLU-GAAs vs. SE-rGLU animals were not exogenous but stemmed from gluconeogenesis induced by supplying GAA during glucose restriction.

[0609] Conclusion85583PC01

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[0611] The study confirms that GAAs directly contributed to the clinical benefits of the combined glucose restriction and GAA intervention.

[0612] restriction elevates

[0613]

[0614] tion

[0615] Aim of study

[0616] The aim of the study is to confirm the metabolic and inflammatory impacts of GAA supply via hepatic transcriptomics and plasma metabolomics.

[0617] Materials and methods

[0618] See Examples 1-5

[0619] Results

[0620] PCA of the hepatic transcriptome revealed distinct clustering between SE-rGLU-GAAs and SE-rGLU groups (Data not shown). DEG analysis revealed 189 upregulated and 136 downregulated genes in SE-rGLU-GAAs vs. SE-rGLU animals. From GSEA analysis, GAAs upregulated multiple metabolic pathways, including OxPhos, TCA cycle, pyruvate metabolism, glycolysis / gluconeogenesis, amino acid, and lipid metabolic pathways (Figure 7 and Table not shown). Within these pathways, most of the DEGs (46 out of 47) were significantly upregulated in SE-rGLU-GAAs animals, whereas LDHA, which converts pyruvate to lactate during anaerobic glycolysis, was the only gene downregulated (Data not shown).

[0621] Conversely, a series of immune pathways and immune DEGs (23 out of 28) were downregulated (Data not shown).

[0622] For plasma metabolomics, PCA also revealed distinct clustering patterns between the two groups (Data not shown). Most amino acids, acylcarnitines, ketone body, keto acid, fatty acids, and other organic acids were upregulated in SE-rGLU-GAAs animals (Data not shown), including the four amino acids from the GAA supply. The increased levels of ketone body (3- hydroxy butyrate, BHB) and keto acid (methyl acetoacetic acid) support GAAs supply improved ketogenesis. Under hypoglycemia, ketogenesis also occurs in the liver via fatty acid p-oxidation to generate ketone bodies as an alternative energy source.

[0623] It was clear that SE-rGLU-GAAs vs. SE-sGLU comparison (Examples 4-6) had much more profound changes in hepatic transcriptome and plasma metabolome than SE-rGLU-GAAs vs. SE-rGLU comparison (Example 7). This implies the metabolic rewiring impacts were mainly mediated by glucose restriction.85583PC01

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[0625] Regardless, the additive effects of GAAs in the background of glucose restriction on disease resistance and tolerance, as well as gluconeogenesis and systemic inflammation were critical to both sepsis prevention and glucose homeostasis.

[0626] Conclusion

[0627] The metabolic rewiring impacts were mainly mediated by glucose restriction. Regardless, the additive effects of GAAs in the background of glucose restriction on disease resistance and tolerance, as well as gluconeogenesis and systemic inflammation were critical to both sepsis prevention and glucose homeostasis.

[0628]

[0629] alucose

[0630] Aim of study

[0631] The aim of the study is to investigate the effect of galactose compared to glucose in PN in a neonatal sepsis model in preterm piglets.

[0632] Materials and methods

[0633] The neonatal sepsis model was prepared as explained in Example 1.

[0634] Parenteral nutrition preparation

[0635] Specialized formulations of parenteral nutrition (PN) were developed from the Kabiven G19% infusion formula (Fresenius-Kabi). Kabiven G19% infusion formula comprises:

[0636] • 3.3% amino acids including alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine;

[0637] • 3.9% lipids including soya oil (CAS No.:8001-22-7);

[0638] • 9.7% glucose monohydrate; and

[0639] • 0.7% electrolytes calcium chloride dihydrate, magnesium sulfate heptahydrate, potassium chloride, sodium acetate trihydrate, and sodium glycerophosphate.

[0640] Kabiven G19% is provided as a three chamber bag and an overpouch. Each chamber consists of glucose, amino acid and electrolyte solution, and fat emulsion, respectively.85583PC01

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[0642] For PN with standard glucose (sGLU) provision, the glucose chamber was emptied, and a specific volume of 50% glucose solution was added into the glucose chamber to reach 10% glucose PN with infusion rate of 6 mL / kg / h, equivalent to 14.4g / kg / d glucose, similar to PN used for preterm infants. For PN with galactose supply with standard glucose equivalent level (sGAL), glucose chamber was emptied, and galactose solution was added to reach 10% galactose PN (14.4g / kg / d).

[0643] Blood gas, hematologv, inflammatory markers, serum biochemistry, and Dlasma ATP measurement

[0644] Blood samples at 3, 6, 12, and 15 h underwent routine blood gas analysis utilizing the GEM Premier 3000 (Instrumentation Laboratory, USA) for blood pH, pCC , oxygen saturation, base excess, glucose, and lactate measurement. Hematological assessments were conducted with the ADVIA 2120i Hematology System (Siemens, Germany). For the quantification of plasma cytokines, TNF-o, IL-6, and IL-10 were analyzed using porcine-specific DuoSet enzyme-linked immunosorbent assays (R&D Systems). Serum biochemistry evaluations were executed using the ADVIA 1800 Chemistry System (Siemens, Germany). Additionally, extracellular ATP level was measured by the ATP Colorimetric Assay Kit (Sigma-Aldrich).

[0645] Statistics

[0646] All statistical analyses were executed via R version 4.3.2 unless otherwise stated. For discerning differences at specific intervals (3, 6, 12, and 15 h), continuous data underwent analysis via a linear mixed-effects model, incorporating group, gender, and birth weight as fixed factors and litter as a random factor, using Ime4 package. Another linear mixed-effects model was employed to probe further disparities spanning the entire experimental duration. This model integrated group, time, their interaction, gender, and birth weight as fixed factors, with litter and pig ID as random factors, using Ime4 package. Normal distribution, variance homogeneity of residuals, and fitted values were assessed. The data that did not conform to a normal distribution were logarithmic transformed. If transformation did not achieve approximate log-normal distribution, the non-parametric Mann-Whitney U test was used instead. To examine different strategies in the infection response, reaction norm analysis was performed using blood pH as a readout for health at 3, 6, and 15 h and plotted against the pathogen burdens at the same85583PC01

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[0648] time point by linear regression. Extra sum-of-squares F Test was used to compare slopes. Statistical significance was defined as P-value < 0.05.

[0649] Results

[0650] It was explored how alternative carbohydrate supply with identical energy intake modulated glucose homeostasis and sepsis outcomes, by substituting galactose for glucose in the PN given to infected preterm piglets. Galactose is a monosaccharide bound to glucose in the natural structure of lactose in breastmilk. After breastfeeding, lactose is cleaved by lactase in the gut into galactose and glucose prior to absorption. Despite its safety, direct use of galactose as nutrients for infants has not been reported. Galactose metabolism primarily starts with Leloir pathway prior to entering glycolysis, and it was hypothesized that the slow conversion of galactose (compared to glucose) into glucose-6-phosphate can reduce systemic glycolytic activity during neonatal infection, in turn reducing host inflammation and sepsis risk.

[0651] Preterm piglets were delivered at 90% gestational age, infected with S. epidermidis (SE) or saline control (CON), reared with standard dose of glucose (sGLU) or galactose (sGAL), and intensively monitored until 15 h post-inoculation (Figure 8A). Compared to SE-sGLU animals, SE-sGAL animals showed lower mortality due to sepsis (Figure 8B), which was predefined as arterial blood pH of < 7.1 plus deep lethargy, apnea or hypoperfusion. This was accompanied by higher blood pH (Figure 8C) and lower carbon dioxide pressure (pCC ) (Figure 8D) in SE-sGAL vs. SE-sGLU animals over time, suggesting that galactose supply prevented respiratory acidosis. During the first 6h post-inoculation, galactose-supplemented animals also showed a slight elevation of lactate (Figure 8E), potentially indicating a stronger glycolysis-related immune response to resist the infection. Importantly, glucose-supplemented animals displayed highly variable blood glucose levels, and most of these animals were hyperglycemic (glucose level > 8 mM) at euthanasia, likely derived from both high glucose supply and disrupted glycemic control during infection (Figure 8F).

[0652] In contrast, both infected and uninfected animals with galactose supply were normoglycemic, with gradually increasing blood glucose levels from 2.4 to around 4 mM over time, indicating the ability to continuously convert exogenous galactose into glucose.85583PC01

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[0654] In the sepsis model, the improved clinical status during infection is often accompanied by preservation of circulating leukocytes, improved antiinflammatory response, and tolerance mechanisms at the later phase of infection. Here, SE-sGAL animals also showed less depletion of blood leukocytes and other hematologic parameters (neutrophils over time, Figure 8G, and total leukocytes at 6h and monocytes and hemoglobin at 15h post-inoculation, Figure 8H-8J), and an increase in plasma IL-10 (Figure 8K). In contrast, the two infected groups had similar rates of bacterial clearance (Figure 8L) and plasma levels of the inflammatory cytokines TNF-o and IL-6 (Figure 8M-8N). These data imply that galactose supply decreased the severity of sepsis, likely via improved disease tolerance instead of resistance, prioritizing the maintenance of normal organ function over a strong immune response to clear the pathogens.

[0655] To further confirm the tolerance phenotype across different stages of infection, a reaction norm analysis was employed by plotting changes in host health against pathogen burdens for infected groups. Here, improved tolerance would be indicated by a shallower slope, showing the capacity to maintain health despite increasing pathogen burdens. Blood pH was used as a readout for general health, and reaction norms were generated at 3, 6, and 15 h post-inoculation (Data not shown). It was found that both SE-sGAL and SE-sGLU animals displayed similar phenotypes at 3 h. However, at 6 h post-inoculation, SE-sGAL animals showed a significantly shallower slope, whereas the infected controls dropped in blood pH with increasing levels of blood bacteria. At the end of the study, the difference between slopes was less pronounced, but there was a tendency for decreased health in the SE-sGAL group when bacterial levels increased, as some animals became septic. In contrast, animals in SE-sGLU reached a paralysis state with consistently low health across various levels of blood bacteria. In summary, these reaction norm analyses indicate that improved tolerance strategy in galactose-treated animals during the first 6 h of infection appeared to delay the sepsis progression.

[0656] Further, plasma markers of organ dysfunctions also supported the clinical impacts of galactose supplementation. At euthanasia, all infected animals showed lower levels of plasma albumin, cholesterol, and alkaline phosphatase (Data not shown), but galactose supplementation alleviated drops in these markers, suggesting either less impairment in liver capacity to synthesize the negative85583PC01

[0657] 65

[0658] acute-phase reactants, or decreased vascular permeability as well as lipid-dysregulated metabolism. On the other hand, other liver injury markers, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (Data not shown), as well as kidney injury marker creatinine (Data not shown), were not different between the SE-sGAL and SE-sGLU groups. In summary, clinical parameters indicate that substituting glucose for galactose in PN maintained normoglycemia and enhanced disease tolerance, thereby delaying sepsis progression.

[0659] Conclusion

[0660] It was found that replacing glucose with galactose improves glucose homeostasis and disease tolerance to decrease sepsis severity. The supply of galactose, instead of glucose, successfully prevented hyperglycemia, and at the same time enhanced disease tolerance phenotype during early phase of infection and overall anti-inflammatory response over the infection course. These together led to reduced risk of sepsis, relative to infected controls. Important markers of liver and kidney injuries (liver enzymes, albumin, cholesterol, creatinine) were minimally affected by the galactose intervention.

[0661] In clinical settings, sepsis delaying or preventing effects of the galactose intervention may provide important window of opportunity for other adjunct therapies (e.g., antibiotics or inotropes) in infected preterm newborns prior to clinical deterioration to septic shock.

[0662] Considering the effect of GAAs in PN as shown in Examples 2-7, it is expected that a PN composition comprising both galactose and GAAs will provide an improved glucose homeostasis and disease tolerance to decrease sepsis. The effect can even be expected to be synergistic.

[0663] connected to

[0664]

[0665] Aim of study

[0666] The aim of the study is to investigate the metabolic and inflammatory pathways connected to the improved clinical outcomes following galactose supply.

[0667] Materials and methods85583PC01

[0668] 66

[0669] The neonatal sepsis model was prepared as explained in Example 1.

[0670] Hepatic transcriptome profiling and analysis

[0671] RNeasy mini kit (QIAGEN, US) was used to extract liver RNA from all piglets. Gene expression was profiled by utilizing whole-transcriptome shotgun sequencing. Library preparation and sequencing were carried out by NOVOGENE services (Cambridge, UK). We used 1000 ng of RNA and the VAHTS mRNA-Seq V3 Library Prep Kit for Illumina (Vazyme, Nanjing, PRC) to prepare RNA-Seq libraries. The libraries were sequenced on the Illumina NovaSeq 6000 platform, generating 150 bp paired-end reads. Quality and adapter trimming of raw reads were performed using TrimGalore (Babraham Bioinformatics, Cambridge, UK). The resulting clean reads, approximately 26 million per sample, were aligned to the porcine genome (Sscrofall.l) using Tophat2.31 Gene annotation information for the porcine genome was sourced from Ensembl (release 99). The script htseq-count32 was employed to generate a gene-count matrix.

[0672] Differentially expressed gene (DEG) analysis was performed using the DESeq2 package version 1.38.3.33 We excluded lowly expressed genes, defined as having a raw count of at least 10 in 4, 5, and 6 samples in Experiment 1, Experiment 2, and Experiment 3, respectively. Litter was added to adjust the model. To robustly estimate the fold change (FC), we applied the IfcShrink function with ashr estimator. A false discovery rate (FDR) cut-off of 0.05 was implemented to obtain DEGs. Genes were ranked by the FC (in log scale) before pathway analyses. The gene set enrichment analysis (GSEA) module in the clusterProfilter package version 4.0.235 was utilized with Sus scrofa Kyoto Encyclopedia of Genes and Genomes (KEGG) knowledgebases to elucidate pathway-level disturbances. Down-or up-regulated pathways were defined based on a normalized enrichment score (NES) with negative or positive values, respectively. The FDR cut-off of 0.05 was considered to determine significant pathways. To create a comprehensive visualization of specific genes within GSEA-enriched pathways, DEGs were identified based on the KEGG, Biological Processes aspect of Gene Ontology (GO: BP), Reactome, and Hallmark knowledgebases, with additional insights from the literature. The relative expression of DEGs was then visualized through heatmaps generated using the ComplexHeatmap package version 2.15.1.36 The analysis of liver transcriptomics data was performed in R 4.2.3.85583PC01

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[0674] Results

[0675] Galactose can be converted to glucose in the liver and subsequently stored as glycogen or released directly into the bloodstream to ensure normoglycemia and physiological organ functions. Therefore, hepatic transcriptomics were performed to explore metabolic and inflammatory pathways connected to the improved clinical outcomes following galactose supply. Principal component analysis (PCA) revealed distinct clusters of transcriptomic profiles of infected animals from uninfected ones, whereas the two infected groups showed only marginal separation (Data not shown). To investigate the differentially regulated pathways between SE-sGAL and SE-sGLU animals, gene set enrichment analysis (GSEA) was employed. SE-sGAL animals exhibited upregulation of various metabolic pathways, including energy metabolism (OxPhos), carbohydrate metabolism (citrate cycle, pyruvate metabolism, glycolysis / gluconeogenesis), as well as multiple amino acids and lipid metabolic pathways (Figure 9A and Table not shown). In addition, SE-sGAL animals demonstrated downregulation of various innate and adaptive immune pathways, including Thl / Th2, Thl7, and IL-17 signaling. Between the uninfected animals, CON-sGAL animals likewise showed an increase of similar pathways related to carbohydrate metabolism, but also enhanced immune pathways and attenuated OxPhos (Figure 9B and Table not shown). It was also found that SE-sGAL animals exhibited 39 up-regulated and 12 down-regulated genes relative to SE-sGLU animals. Of note, differentially expressed genes (DEGs) belong to glycolysis (GCKR and ENO4), pyruvate metabolism (PKLR and TKFC), pentose phosphate pathway (TALDO1), gluconeogenesis (FBP1), TCA cycle (PDHB), OxPhos (NDUFC2), mitochondrial function (AK1 and SLC25A1), amino acids metabolism (GLDC, HOGA1, and DDC), and lipid metabolism (EPHX1, ACSM4, ACADSB, ACSM5, PHYH, and DHCR7) were all upregulated, while inflammation and immune-related DEGs (BMF, IKBKE, and IRAK3) were all downregulated (Data not shown).

[0676] Conclusion

[0677] The hepatic transcriptomic data further supports the conclusions from clinical data that galactose supply improved energy metabolism and glucose homeostatic control, leading to the prevention of excessive inflammation and tissue damage.85583PC01

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[0679] Example 11 - Galactose supplemented with glucogenic amino acids (GAA) in parenteral nutrition (PN)

[0680] Aim of study

[0681] The aim of the study is to investigate whether replacement of glucose with galactose supplemented with glucogenic amino acids (GAA) in parenteral nutrition (PN) improves metabolic stability, immune responses, and infection outcomes in preterm piglets infected with Staphylococcus epidermidis.

[0682] Materials and methods

[0683] Twenty-six preterm piglets were delivered by cesarean section at gestational day 106 (approximately 90% of term). Piglets received intramuscular doxapram (0.1 mL, 20 mg / mL) to stimulate respiration and flumazenil (0.1 mL, 0.1 mg / mL) to reverse maternal anesthesia. Additional respiratory support and pharmacological stimulation were provided as needed. Following stabilization, sex and birth weight were recorded, and piglets were fitted with an umbilical arterial catheter using aseptic technique. The catheter was advanced to the dorsal aorta, secured to the skin, and connected to an infusion line for continuous parenteral nutrition (PN). Piglets were housed individually in temperature-controlled incubators with supplemental oxygen as required during the first hours after birth.

[0684] Parenteral nutrition regimens

[0685] Immediately after catheterization, piglets were randomized according to sex and birth weight to receive one of the two PN types:

[0686] 1. Standard glucose-based PN (control) (n = 14):

[0687] A modified Kabiven-based formulation (Fresenious Kabi, Germany) containing 10% glucose, lipids, amino acids, electrolytes, sterile water, and heparin.

[0688] 2. Galactose + glucogenic amino acid PN (Galactose+GAA) (n = 12):

[0689] A similar PN where the carbohydrate component in the standard glucose- based PN was replaced with a concentrated solution of galactose and glucogenic amino acids (GAAs, equal parts valine, glutamate, aspartate and asparagine). This resulted in a final carbohydrate concentration of 5% with additional 0.7% of GAA's.85583PC01

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[0691] Both formulations were infused the umbilical arterial catheter throughout the study period at a rate of 6 mL / kg / hour. The reduction in total carbohydrate concentration resulted in a reduced total energy intake in the Galactose+GAA group (399 kJ / kg / day) compared to those receiving standard glucose-based PN (508 kJ / kg / day). Total intake of energy, carbohydrates and amino acids shown in Table 1:

[0692]

[0693] Table 1: Different PN Regimens. Daily parenteral nutrient provision in preterm piglets receiving standard glucose-based PN or galactose supplemented with glucogenic amino acids (Galactose+GAA). Values are expressed per kg bodyweight per day.

[0694] Passive immunization

[0695] To reduce the risk of spontaneous infection, partial passive immunization was provided by intra-arterial infusion of maternal sow plasma (8 mL / kg bodyweight) over 30 minutes on day 1 after birth.

[0696] Bacterial inoculation

[0697] On day 3 of life, piglets were inoculated with Staphylococcus epidermidis. A standardized bacterial suspension was prepared from frozen stock, cultured overnight, quantified by optical density, and adjusted to the target concentration. Piglets received S. epidermidis at a dose of 1 x 109CFU / kg, administered over 3 minutes through the umbilical arterial catheter.

[0698] Clinical monitoring and supportive care85583PC01

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[0700] Piglets were continuously monitored by trained personnel throughout the experiment. Clinical assessments included major milestones (ability to stand, walk, open eyes and passage of meconium), activity level, skin color, peripheral temperature, respiratory effort, heart rate, abdominal distension, and signs of bleeding or diarrhea. Body temperature was measured frequently, particularly during the first 24 hours. Incubator temperature and ventilation were adjusted to manage hypo- or hyperthermia.

[0701] Humane endpoints and euthanasia

[0702] Animals were euthanized immediately if they developed severe clinical signs of sepsis. This was defined as severe acidosis (pH < 7.1) with or without marked lethargy, respiratory failure or circulatory collapse. Surviving animals were euthanized 48 hours after bacterial inoculation. For euthanasia, piglets were sedated with intramuscular Zoletil (0.2 mL / kg, ScanVet, Denmark), followed by cardiac blood collection. Animals were then euthanized with an intracardiac dose of pentobarbital (2-3 mL / kg, ScanVet, Denmark). Tissue collection was performed immediately after death according to predefined protocols.

[0703] Blood samDling

[0704] Arterial blood gas measurements were obtained on days 1 and 2 at 09:00 and 21:00 o'clock. Following S. epidermidis inoculation, blood samples were collected at 0, 6, 12, and 24 hours via the arterial catheter for blood gas, plasma, hematology, and molecular analyses. Additional venous blood samples were obtained at 3, 6, and 24 hours post-inoculation for bacteriological culture.

[0705] Laboratory analyses

[0706] Blood bacterial density was quantified by culture of venous blood samples collected at predefined time points after Staphylococcus epidermidis inoculation. Serial 10-fold dilutions were prepared in sterile buffer and plated on blood agar plates, which were incubated overnight at 37 °C. Colony-forming units (CFU) were counted and expressed as CFU per milliliter of blood. Plasma cytokine concentrations were measured in EDTA plasma using commercially available porcine kits (R&D systems, USA) according to the manufacturer's instructions. Cytokine values below the detection limit were assigned half the lowest detectable concentration. Arterial blood gases, as wells as pH, glucose and lactate, were measured immediately after sampling using a bedside blood gas analyzer (ABL800 FLEX, Radiometer, Copenhagen, Denmark). Hematological parameters, including85583PC01

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[0708] total leukocyte and platelet counts, were measured in EDTA-anticoagulated blood using an automated hematology analyzer (ADVIA 2120i, Siemens, Germany). Statistics

[0709] Statistical analyses were performed using Stata version 14.2 (StataCorp, USA). Repeated measures data were analyzed using linear mixed-effects models with animal ID included as a random effect. Non-repeated outcomes were analyzed using generalized linear models as appropriate. Model assumptions were assessed by inspection of residuals, and variables were log-transformed when necessary. A P value < 0.05 was considered statistically significant.

[0710] Results

[0711] Blood glucose levels and clinical condition before onset of infection.

[0712] All animals remained clinically stable until bacterial inoculation on day 3, with no differences between groups in time to standing, walking, or eye opening (Figure 1OA). Body weight trajectories did not differ between groups (Figure 1OB). From birth until onset of infection, blood glucose concentrations were significantly lower in piglets receiving Galactose+GAA PN compared with glucose-based PN (Figure IOC, P < 0.001). Galactose+GAA-supplemented animals maintained stable glucose levels around 3.5 mmol / L, whereas glucose-fed controls showed marked variability, ranging from approximately 6 to 12 mmol / L. Despite these differences, no signs of hypoglycemia were observed. Before infection, there were no differences between groups in acid-base status, including pH and lactate (Figure 10D-E).

[0713] Together, these findings indicate that Galactose+GAA supplementation does not adversely affect early clinical stability or growth in non-infected preterm piglets, despite inducing a substantially lower and more stable blood glucose profile.

[0714] Supplementation of Galactose and GAA improve clinical responses to infection Following S. epidermidis inoculation, 11 of 14 piglets in the glucose control group reached humane endpoints, whereas none of the Galactose+GAA-supplemented animals (0 / 12) deteriorated to euthanasia criteria. Although this difference only showed tendency to statistical significance in a log-rank survival analysis (Figure 1OF, P = 0.09), it suggested a potential for improvements in clinical outcomes. After infection onset, blood lactate concentrations were significantly lower in the85583PC01

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[0716] Galactose+GAA group compared with controls (Figure 1OE, P < 0.05), with the largest differences observed between 3 and 12 hours post-inoculation. Blood pH did not differ between groups during infection (Figure 1OD). Blood glucose concentrations remained significantly lower in Galactose+GAA-supplemented animals at all post-infection time points (Figure IOC, P < 0.05). During infection, glucose levels decreased to approximately 2.5 mmol / L in this group without evidence of hypoglycemic symptoms.

[0717] These findings suggest that Galactose+GAA supplementation suppresses lactate accumulation during neonatal infection while maintaining blood glucose within a low but clinically tolerated range.

[0718] Galactose+GAA supplementation improves bacterial clearance and modulates inflammation

[0719] Blood bacterial densities were significantly lower in Galactose+GAA-supplemented piglets at 48 hours after infection compared with glucose-fed controls (Figure 11A). From 3 to 24 hours post-inoculation, bacterial clearance from the bloodstream was consistently more efficient in the Galactose+GAA group, indicating improved antibacterial host responses. Plasma cytokine profiles differed markedly between groups. Galactose+GAA-supplemented animals exhibited higher levels of interleukin-10 across the post- infection period (Figure 11C, P < 0.001), with significant differences at all time points except euthanasia, where IL-10 levels were lower than in controls (P < 0.05). In parallel, interleukin-6 concentrations were significantly lower throughout the infection course in Galactose+GAA animals (Figure 11D, P < 0.001), most prominently at 3, 6, and 12 hours after infection onset. Tumor necrosis factor-o levels were transiently higher in Galactose+GAA-supplemented animals at 6 and 12 hours post- infection. This pattern suggests preserved early pro-inflammatory signaling alongside enhanced anti-inflammatory regulation.

[0720] Total leukocyte subsets did not differ between groups after infection (Figure HE). However, thrombocyte counts were significantly higher in Galactose+GAA-supplemented animals at euthanasia (Figure HF), consistent with reduced systemic inflammation.85583PC01

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[0722] Replacement of glucose with a combination of galactose and GAA in PN profoundly altered metabolic and immunological responses to neonatal S. epidermidis infection in preterm piglets. Galactose+GAA supplementation resulted in lower and more stable blood glucose concentrations, attenuated lactate accumulation, improved bacterial clearance, and a cytokine profile indicative of enhanced immune regulation preventing aggressive inflammation.

[0723] The marked reduction in blood lactate during infection suggests a shift away from excessive glycolytic flux. Despite lower circulating glucose concentrations, Galactose+GAA-supplemented animals showed no signs of hypoglycemia or impaired clinical stability, supporting the concept that alternative carbohydrate substrates can safely sustain systemic energy demands in preterm neonates. Immunologically, Galactose+GAA supplementation was associated with lower IL-6 and higher IL-10 responses, indicating attenuation of excessive inflammatory signaling while preserving early TNF-o responses critical for bacterial control. This balanced immune response likely contributed to improved bacterial clearance and reduced clinical deterioration. Although the survival difference only showed tendency to significant difference, the absence of clinical deterioration in Galactose+GAA-treated animals, together with consistent improvements across metabolic, bacteriological, and inflammatory endpoints, supports a biologically meaningful protective effect.

[0724] This experiment builds directly on previous work demonstrating that glucose availability and hepatic glycolytic flux are key determinants of poor clinical outcome in neonatal Staphylococcus epidermidis sepsis. A reduction in hepatic glycolytic flux leads to reduction in pro-inflammatory immune responses and reduction in infection related organ injury, all while preserving antibacterial defenses. Consistent with this, Galactose+GAA-supplemented piglets displayed stable normoglycemia and substantially attenuated lactate production during infection.

[0725] An aspect of this experiment is the combination of glucogenic amino acids alongside galactose in PN. Galactose differs fundamentally from glucose in its metabolism. Whereas glucose rapidly enters glycolysis, galactose must first be processed through the Leloir pathway before entering glycolysis. It was shown85583PC01

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[0727] that parenteral supplementation of galactose indeed limits hepatic glycolytic flux during neonatal infections, while supplementation of GAA increased endogenous glucose production and supported mitochondrial metabolism. The GAA selected for this experiment can feed directly into the tricarboxylic acid cycle, thereby sustaining mitochondrial metabolism under inflammatory conditions. For its effects on clinical outcomes, it was shown that substitution of PN glucose with galactose alone delayed, but did not prevent, clinical deterioration during experimental infection, while substitution of glucose with GAA completely prevented clinical deterioration but led to hypoglycaemia. It is therefore speculated or expected that the combined supplementation of galactose and GAA creates a synergistic metabolic environment in which immune and non-immune tissues are supplied with energy substrates that favor oxidative phosphorylation over aerobic glycolysis. Such a shift would be expected to reduce lactate accumulation, preserve mitochondrial function, and support anti-inflammatory responses, all features that were all observed in the present study.

[0728] In the two days prior to bacterial inoculation, no differences were observed in clinical state or growth of the animals, despite clear differences in blood glucose levels. This is in line with previous observations where substitution of PN glucose for galactose in preterm neonates without infection has been shown to be safe and feasible. Although the Galactose+GAA PN provided a lower total energy intake than the standard glucose-based PN, the absence of differences in early growth, clinical stability, or acid-base balance before infection suggests that the observed improvements during sepsis are unlikely to be driven by caloric restriction per se, but rather by differences in substrate metabolism.

[0729] Conclusion

[0730] In conclusion, replacement of glucose with galactose supplemented with GAA in PN markedly altered metabolic and immune responses to Staphylococcus epidermidis infection in preterm piglets. This nutritional strategy was associated with lower and more stable blood glucose concentrations, reduced lactate accumulation, improved bacterial clearance, and a more regulated inflammatory response, without compromising clinical stability. Although the Galactose+GAA formulation provided a lower total energy intake, the consistency of effects across metabolic, inflammatory, and bacteriological endpoints suggests that qualitative85583PC01

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[0732] differences in substrate utilization, rather than caloric delivery, underpin the observed benefits. These findings support the concept that PN composition can actively shape neonatal immunometabolism and influence infection outcomes, and they provide a strong rationale for further mechanistic studies and translational evaluation of galactose- and amino acid-based PN strategies in vulnerable human newborns.

Claims

85583PC0176Claims1. A parenteral nutrition (PN) composition comprising:a) one or more glucogenic amino acids (GAAs) in an amount of at least 0.5% by weight of the PN composition, andb) galactose in an amount of at least 1% by weight of the PN composition.

2. The PN composition according to claim 1, wherein the one or more GAAs are in an amount of 0.5% to 4% by weight, such as 1% to 4% by weight, preferably 1% to 3% by weight, more preferably 0.5% to 2% by weight, most preferably 1% to 2% by weight of the PN composition.

3. The PN composition according to any of claims 1 or 2, wherein the the one or more GAAs are selected from a group consisting of alanine, arginine, aspartate, asparagine, cysteine, glycine, glutamate, glutamine histidine, hydroxyproline, proline, methionine, serine, threonine, valine, or a combination thereof, preferably the one or more GAAs are selected from a group consisting of methionine, valine, asparagine, aspartate, glutamate, or a combination thereof, more preferably the one or more GAAs are selected from a group consisting of valine, asparagine, aspartate, glutamate, or a combination thereof.

4. The PN composition according to claim 3, wherein valine, asparagine, aspartate, and glutamate are in a ratio of 1:1:1:1.

5. The PN composition according to any of claims 3-4, wherein the amount of valine, asparagine, aspartate, and / or glutamate is 0.5% to 4% by weight, such as 1% to 4% by weight, preferably 1% to 3% by weight, more preferably 0.5% to 2% by weight, most preferably 1% to 2% by weight of the PN composition.

6. The PN composition according to any of the preceding claims, the one or more GAAs are present in an amount of 66% to 100% by weight, such as 67% to 100% by weight, such as 68% to 100% by weight, such as 80% to 100% by weight, preferably 100% by weight relative to the total amount of amino acids of the PN composition.85583PC01777. The PN composition according to any of the preceding claims, the one or more GAAs are a natural, artificial, or modified amino acid analogue or substitute.

8. The PN composition according to any of the preceding claims, wherein the galactose is in an amount of 5% to 50% by weight, such as 10% to 50% by weight, such as 10% to 45% by weight, such as 10% to 30% by weight, preferably 10% to 25% by weight, more preferably 10% to 22% by weight, most preferably 10% to 20% by weight of the PN composition.

9. The PN composition according to any of the preceding claims, wherein the PN composition is a solution, such as a liquid solution.

10. The PN composition according to any of the preceding claims, wherein the PN composition comprises glucose in an amount of 0% to 50% by weight, such as 0% to 40% by weight, such as 0% to 30% by weight, such as 0% to 25% by weight, such as 0% to 20% by weight, preferably 0% to 10% by weight, more preferably 0% to 5% by weight, most preferably 0% by weight of the PN composition.

11. A combination parenteral nutrition (PN) composition comprising:a) a first PN composition, wherein the first PN composition is the PN composition according to any of the preceding claims, andb) a second PN composition.

12. The combination PN composition according to claim 11, wherein the second PN composition comprises glucose in an amount of 0% to 20% by weight, preferably 0% to 10% by weight, more preferably 0% to 5% by weight, most preferably 0% by weight of the second PN composition.

13. The combination PN composition according to any of claims 11 or 12, wherein the second PN composition comprises:- water in an amount of 60% to 85% by weight of the second PN composition,- one or more amino acids in an amount of 1% to 6% by weight of the second PN composition,85583PC0178- one or more fats in an amount of 0% to 4% by weight the second PN composition,- one or more vitamins, and / or- one or more electrolytes.

14. The combination PN composition according to any of claims 11-13, wherein the combination PN composition comprises one or more GAAs in an amount of at least 0.2% by weight, and / or galactose in an amount of at least 5% of the combination PN composition.

15. The combination PN composition according to claim 14, wherein the combination PN composition comprises one or more GAAs in an amount of 0.2% to 2% by weight, such as 0.5% to 1.5% by weight, preferably 0.5% to 1% by weight, more preferably 0.7% to 1% by weight of the combination PN composition.

16. The combination PN composition according to claim 14, wherein the combination PN composition comprises galactose in an amount of 5% to 20% by weight, preferably 5% to 15% by weight, more preferably 5% to 10% by weight of the combination PN composition.

17. A method for producing a PN composition according to any of claims 1-16 comprising the steps of:1) providing one or more glucogenic amino acids (GAAs) in an amount of at least 0.5% by weight of the PN composition;2) providing galactose in an amount of at least 1% by weight of the PN composition;3) optionally, providing one or more additional components;4) mixing the one or more GAAs, galactose, and the optional one or more additional components; and5) obtaining the PN composition.

18. The method according to claim 17, wherein the one or more additional components are selected from the group consisting of one or more additional85583PC0179carbohydrates, water, one or more additional amino acids, one or more vitamins, one or more electrolytes, one or more fats, or any combination thereof.

19. The method according to claim 17, wherein the one or more additional components is a second PN composition.

20. The method according to claim 19, wherein the second PN composition comprises:- water in an amount of 60% to 85% by weight of the second PN composition,- one or more amino acids in an amount of 1% to 6% by weight of the second PN composition,- one or more fats in an amount of 0% to 4% by weight the second PN composition,- one or more vitamins, and / or- one or more electrolytes.

21. The method according to any of claims 17-20, wherein the galactose is provided in an amount of 5% to 50% by weight, such as 10% to 50% by weight, such as 10% to 45% by weight, such as 10% to 30% by weight, preferably 10% to 25% by weight, more preferably 10% to 22% by weight, most preferably 10% to 20% by weight of the PN composition.

22. The method according to any of claims 17-21, wherein the one or more GAAs are in an amount of 2% to 10% by weight, such as 2% to 9% by weight, such as 2% to 8% by weight, preferably 2.5% to 6% by weight, such as 3% to 6% by weight, such as 3% to 5% by weight, such as 0.5% to 4% by weight, such as 1% to 4% by weight, preferably 1% to 3% by weight, more preferably 0.5% to 2% by weight, most preferably 1% to 2% by weight of the PN composition.

23. The method according to any of claims 17-22, wherein the the one or more GAAs are selected from a group consisting of alanine, arginine, aspartate, asparagine, cysteine, glycine, glutamate, glutamine histidine, hydroxyproline, proline, methionine, serine, threonine, valine, or a combination thereof, preferably the one or more GAAs are selected from a group consisting of methionine, valine,85583PC0180asparagine, aspartate, glutamate, or a combination thereof, more preferably the one or more GAAs are selected from a group consisting of valine, asparagine, aspartate, glutamate, or a combination thereof.

24. The method according to any of claims 17-23, wherein the one or more GAAs are a natural, artificial, or modified amino acid analogue or substitute.

25. A method for producing a combination PN composition according to any of claims 11-16 comprising the steps of:1) providing one or more glucogenic amino acids (GAAs) and galactose;2) mixing the one or more GAAs and the galactose and obtaining a first PN composition comprising one or more GAAs in an amount of at least 0.5% by weight and galactose in an amount of at least 1% by weight of the first PN composition;3) providing a second PN composition;4) mixing the first PN composition of step 2) and the second PN composition of step 3); and5) obtaining the combination PN composition.

26. A method for producing a combination PN composition according to any of claims 11-16 comprising the steps of:1) providing a first PN composition, wherein the first PN composition is the PN composition according to any of claims 1-10;2) providing a second PN composition;3) mixing the first PN composition of step 1) and the second PN composition of step 2); and4) obtaining the combination PN composition.

27. A parenteral nutrition (PN) composition according to any of claims 1-16 for use in the treatment and / or prevention of infection or sepsis in a subject.

28. The PN composition for use according to claim 27, wherein the treatment is a prophylactic treatment.85583PC018129. The PN composition for use according to any of claims 27-28, wherein the subject has a condition of dysregulated glucose homeostasis, such as type 1-diabetes, type 2-diabetes, Addison's disease, immunodeficiency, malnutrition, or any combination thereof.

30. The PN composition for use according to any of claims 27-29, wherein the subject has a dysfunctional immune system or dysfunctional immune function.

31. The PN composition for use according to any of claims 27-30, wherein the subject is a mammal, preferably a human, such as a human infant, preferably a human preterm infant, a human adult, such as a human elderly, or a patient under intensive care.

32. The PN composition for use according to any of claims 27-31, wherein the infection is selected from the group consisting of bacterial, viral, fungal or any combination thereof, preferably the infection is a bacterial infection.

33. The PN composition for use according to claim 32, the bacterial infection is selected from the group consisting of coagulase-negative Staphylococcus, such as Staphylococcus epidermidis, E.coli, and group B streptococcus.

34. The PN composition for use according to any of claims 27-33, wherein the PN composition is administered in an amount of 0.2g to 10g, such as 0.8g to 10g, such as 0.8g to 8g, such as 0.8g to 6g, such as 0.8g to 4g, such as lg to 6g, such as lg to 2g, such as 1.5g to 6g of one or more GAAs per kg per day.

35. The PN composition for use according to any of claims 27-34, wherein the PN composition is administered in an amount of 0.8g to 15g, such as 0.8g to 10g, such as 0.8g to 8g, such as 0.8g to 7g, such as 0.8g to 6g, such as 0.8g to 4g, such as lg to 6g, such as 1.5g to 6g of galactose per kg per day.

36. The PN composition for use according to any of claims 27-35, wherein the PN composition is administered to an infant in an amount of• 0.2g to 3g of one or more GAAs per kg per day, and / or• 0.8g to 15g of galactose per kg per day.85583PC018237. The PN composition for use according to any of claims 27-35, wherein the PN composition is administered to an adult in an amount of• 0.2g to 6g of one or more GAAs per kg per day, and / or• 0.8g to 15g of galactose per kg per day.

38. The PN composition for use according to any of claims 27-37, the PN composition is administered in an amount of:- 0.2g to 1.5g valine per kg per day, preferably 0.5g valine per kg per day; - 0.2g to 1.5g asparagine per kg per day, preferably 0.5g asparagine per kg per day;- 0.2g to 1.5g aspartate per kg per day, preferably 0.5g aspartate per kg per day; and / or- 0.2g to 1.5g glutamate per kg per day, preferably 0.5g glutamate per kg per day.

39. The PN composition for use according to any of claims 27-38, the PN composition is administered intravenously or intraarterially, such as via the umbilical artery.

40. Use of the PN composition according to any of claims 1-10 for mixing or combining with a second PN composition.

41. A kit of parts comprising• the PN composition according to any of claims 1-10,• a second PN composition, and• optionally, instructions for use.

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