Uses of lactobacillus reuteri in therapy of premature infant subjects

Abstract

The present invention relates to treating, preventing or reducing a respiratory complication in a premature infant subject. In particular, the present invention provides Lactobacillus reuteri for use in treating or preventing or reducing a respiratory complication in a premature infant subject. The present invention also provides methods of treating or preventing or reducing a respiratory complication in a premature infant subject, comprising administering to said subject an effective amount of Lactobacillus reuteri. The present invention also relates to reducing risk of mortality in a premature infant subject. The present invention provides Lactobacillus reuteri for use in reducing risk of mortality in a premature infant subject. The present invention also provides methods of reducing risk of mortality in a premature infant subject, comprising administering to said subject an effective amount of Lactobacillus reuteri.

Description

[0001] Uses of Lactobacillus reuteri in therapy of premature infant subjects

[0002] The present invention relates generally to the field of live bacteria therapeutics. More specifically, this invention relates to uses and methods that employ certain bacteria for treating, preventing or reducing respiratory complications in premature infant subjects. The invention also relates to uses and methods that employ certain bacteria to reduce risk of mortality in premature infant subjects.

[0003] Improvements in neonatal intensive care have significantly improved the survival of premature infants. In this regard, 50 years ago only 5-10% of the smallest babies survived, whereas today the survival rate is much higher (-80%). Factors including the use of mechanical ventilation and pharmaceuticals such as surfactants have significantly contributed towards this improvement in survival rate. However, further improvements in the survival rate for premature infants are of course desirable.

[0004] Lactobacillus reuteri (also known as Limosilactobacillus reuteri) is a heterofermentative lactic acid bacterium and is frequently found in the gastrointestinal tract of humans and other animals. L. reuteri is considered an indigenous organism of the human gastrointestinal tract and is, for example, present on the mucosa of the gastric corpus, gastric antrum, duodenum, and ileum. Different L. reuteri strains have the ability to colonize the intestine, act as a diarrhea therapeutic agent, modulate the gut motility, function as an inhibitor of bacterial pathogens, immunologically modulate the gastrointestinal mucosa, function as an anti-inflammatory agent in the stomach, etc.

[0005] The present inventors have conducted a clinical study, the objective of which was to show that a Lactobacillus reuteri strain can improve the health of premature infants by preventing necrotizing enterocolitis (NEC) and improving premature infants’ gastrointestinal function (measured as time to sustained feeding tolerance, SFT). NEC is a serious intestinal disease that is primarily seen in premature infants. Despite showing positive trends, there was no statistical significance for the two primary endpoints in this clinical study (NEC and SFT). Despite there being no statistical significance for the two primary endpoints of this study, there was, surprisingly, a statistically significant reduction in all-cause mortality in the Lactobacillus reuteri strain-treated group as compared to in the placebo treated group. This corresponded to a significant risk reduction of 27% which means that in reality 23 infants’ lives were spared by the administration of the Lactobacillus reuteri strain. Thus, the inventors have found that the survival rate of premature infants is improved by the administration of the Lactobacillus reuteri strain. The inventors also surprisingly found that administration of the Lactobacillus reuteri strain led to a reduction in the primary cause of death being respiratory complications. This is particularly surprising given that Lactobacillus reuteri strains are known to colonize the intestine and the aim of the study was primarily to assess effects on a gastrointestinal disease (NEC) and on gastrointestinal function (measured as SFT).

[0006] The inventors’ findings show that the Lactobacillus reuteri strain is useful for treating, preventing or reducing respiratory complications in premature infant subjects. The inventors’ findings also show that the Lactobacillus reuteri strain can reduce the risk of mortality in premature infant subjects, for example mortality due to a respiratory complication.

[0007] Thus, in one aspect, the present invention provides Lactobacillus reuteri for use in treating or preventing or reducing (e.g. reducing the severity of) a respiratory complication in a premature infant subject.

[0008] In one aspect, the present invention provides a Lactobacillus reuteri strain that is the active ingredient of IBP-9414 for use in treating or preventing or reducing (e.g. reducing the severity of) a respiratory complication in a premature infant subject. The Lactobacillus reuteri strain that is the active ingredient of IBP-9414 is the Lactobacillus reuteri strain PTA-121141 (deposited at the ATCC as described elsewhere herein).

[0009] Thus, alternatively viewed, in one aspect, the present invention provides Lactobacillus reuteri strain PTA-121141 for use in treating or preventing or reducing (e.g. reducing the severity of) a respiratory complication in a premature infant subject.

[0010] Lactobacillus reuteri strain PTA-121141 was deposited at the American Type Culture Collection (ATCC) (10801 University Boulevard, Manassas, Virginia 20110-2209, United States of America) on 26 March 2014. The deposit was made at the ATCC under the Budapest Treaty. PTA-121141 is the accession number of the deposited strain. References herein to “Lactobacillus reuteri strain PTA-121141”, “Lactobacillus reuteri strain ATCC PTA-121141”, “PTA-121141”, “Lactobacillus reuteri PTA-121141” and “a Lactobacillus reuteri strain that is the active ingredient of IBP-9414” (or analogous expressions) are references to this deposited strain.

[0011] Lactobacillus reuteri, for example Lactobacillus reuteri strain PTA-121141, can be grown under any suitable conditions for the growth of Lactobacilli and the skilled person would be familiar with such conditions. Lactobacillus reuteri, for example Lactobacillus reuteri strain PTA-121141, can, for example, be grown in MRS (DeMan, Rogosa and Sharpe) broth at 37°C. MRS broth is widely commercially available (e.g. Oxoid). Unless otherwise clear from the context, references herein to “a Lactobacillus reuteri strain in accordance with the present invention” (or similar expressions) are references to Lactobacillus reuteri strain PTA-121141. Lactobacillus reuteri strain PTA-121141 is a preferred Lactobacillus reuteri strain for use in accordance with the present invention, although as is evident from the discussion below, in certain aspects of the present invention the use of Lactobacillus reuteri strain PTA-121141 is not essential.

[0012] Lactobacillus reuteri (e.g. a Lactobacillus reuteri strain in accordance with the present invention) for use in accordance with the present invention are live Lactobacillus reuteri (i.e. live bacteria are used). “Live” Lactobacillus reuteri means metabolizing Lactobacillus reuteri or dormant bacteria that can be activated to metabolize. A “metabolizing Lactobacillus reuteri" is viable, meaning it grows and replicates utilizing suitable substrate components, for example resulting in the production of metabolites. A “dormant Lactobacillus reuteri" means a non-metabolizing bacteria that does not replicate but is live and can be activated (or reactivated) to a metabolizing bacteria (e.g. by exposure to suitable to a suitable substrate(s) or nutrient(s) for growth). Examples of such dormant Lactobacillus reuteri are frozen or freeze-dried or lyophilised (or otherwise dried, e.g. spray-dried) Lactobacillus reuteri.

[0013] “Activation” of Lactobacillus reuteri means the process of changing the state of a Lactobacillus reuteri from being dormant to the state of metabolizing bacteria. Thus, in some embodiments, live Lactobacillus reuteri are Lactobacillus reuteri that have been activated (or reactivated) into a metabolizing form, or that are in the process of being activated (or reactivated) into a metabolizing form.

[0014] Typically, the respiratory complication is a respiratory complication that is associated with (or characteristic of) premature birth. Preferably, the respiratory complication is a respiratory complication that can cause (or lead to) death or that can increase the risk of death.

[0015] In some embodiments, the respiratory complication may be one or more selected from the group consisting of respiratory distress, respiratory failure, chronic respiratory failure, bronchopulmonary dysplasia, apnea, pulmonary hemorrhage, pulmonary hypertension, pneumonitis, pneumonia, hypoxia, respiratory depression, bradypnea, hypercapnia, respiratory acidosis, pulmonary failure, pneumothorax and pulmonary (interstitial) fibrosis.

[0016] In some embodiments, the respiratory complication may be one or more selected from the group consisting of respiratory distress, respiratory failure, chronic respiratory failure, bronchopulmonary dysplasia, apnea, pulmonary hemorrhage, pulmonary hypertension, pneumonitis, and pneumonia. In some embodiments, the respiratory complication may be one or more selected from the group consisting of respiratory distress, respiratory failure, pulmonary hemorrhage and pneumonia.

[0017] In a preferred embodiment, the respiratory complication is respiratory distress.

[0018] Alternatively viewed, in one aspect, the present invention provides a Lactobacillus reuteri strain in accordance with the present invention for use in treating or preventing or reducing (e.g. reducing the severity of) a respiratory condition (or respiratory disorder or respiratory disease) in a premature infant subject. Typically, the respiratory complication is a respiratory condition (or respiratory disorder or respiratory disease) that is associated with (or characteristic of) premature birth.

[0019] In one aspect, the present invention provides a Lactobacillus reuteri strain in accordance with the present invention for use in treating or preventing or reducing (e.g. reducing the severity of) a condition (or disorder or disease) in a premature infant subject, wherein said condition (or disorder or disease) is selected from the group consisting of respiratory distress, respiratory failure, chronic respiratory failure, bronchopulmonary dysplasia, apnea, pulmonary hemorrhage, pulmonary hypertension, pneumonitis, pneumonia, hypoxia, respiratory depression, bradypnea, hypercapnia, respiratory acidosis, pulmonary failure, pneumothorax and pulmonary (interstitial) fibrosis.

[0020] In another aspect, the present invention provides a method of reducing risk of mortality in a premature infant subject, said method comprising administering to said subject an effective amount of Lactobacillus reuteri.

[0021] In another aspect, the present invention provides a method of reducing risk of mortality in a premature infant subject, said method comprising administering to said subject an effective amount of the Lactobacillus reuteri strain that is the active ingredient of IBP-9414.

[0022] Alternatively viewed, the present invention provides a method of reducing risk of mortality in a premature infant subject, said method comprising administering to said subject an effective amount of a Lactobacillus reuteri strain in accordance with the present invention.

[0023] Alternatively viewed, the present invention provides a method of reducing risk of death in a premature infant subject, said method comprising administering to said subject an effective amount of a Lactobacillus reuteri strain in accordance with the present invention.

[0024] Alternatively viewed, the present invention provides a method of preventing death in a premature infant subject, said method comprising administering to said subject an effective amount of a Lactobacillus reuteri strain in accordance with the present invention. Discussion herein in connection with methods of reducing risk of mortality or reducing risk of death may also be applied to methods of preventing death of the present invention. An effective amount (e.g. a therapeutically or prophylactically effective amount) can be readily determined, e.g. based on a clinical assessment. Certain exemplary doses and dosage regimes are described elsewhere herein.

[0025] Typically, methods of reducing risk of mortality (or death) or methods of preventing death are methods of reducing risk of mortality (or death) or methods of preventing death of infants, preferably neonates. Alternatively viewed, typically methods of reducing risk of mortality (or death) or methods of preventing death are methods of reducing risk of infant mortality (or infant death) or methods of preventing infant death of infants, preferably methods of reducing risk of neonatal mortality (or neonatal death) or methods of preventing neonatal death. Preferably, methods of reducing risk of mortality (or death) or methods of preventing death are methods of reducing risk of neonatal mortality (or death) or methods of preventing neonatal death. In some embodiments, methods of reducing risk of mortality (or death) or methods of preventing death are methods of reducing risk of mortality (or death) or methods of preventing death within the first 6 months, the first 5 months, the first 4 months, the first 3 months, the first 2 months or the first 1 month after birth. In some embodiments, methods of reducing risk of mortality (or death) or methods of preventing death are methods of reducing risk of mortality (or death) or methods of preventing death within the first 24 weeks, 20 weeks, 16 weeks, 12 weeks, 8 weeks, 4 weeks, 3 weeks, 2 weeks or 1 week after birth. In some embodiments, methods of reducing risk of mortality (or death) or methods of preventing death are methods of reducing risk of mortality (or death) or methods of preventing death within the first 14 days after birth or within the first 28 days after birth.

[0026] In some embodiments, methods of reducing risk of mortality (or risk of death) or methods of preventing death are methods of reducing risk of mortality (or risk of death), or methods of preventing death associated with (or due to or caused by) a respiratory complication (or a respiratory condition, disease or disorder). Preferred respiratory complications are described elsewhere herein.

[0027] Thus, in some embodiments of methods of reducing risk of mortality (or risk of death) or methods of preventing death of the present invention, the subject has, or is at risk of having or at risk of developing, a respiratory complication (or respiratory condition, disorder or disease).

[0028] In some embodiments, methods and uses of the present invention further comprise administering citrate to said premature infant subject. Citrate may be administered prior to, simultaneously with (or substantially simultaneously with), or subsequent to administration of Lactobacillus reuteri. However, typically and preferably, citrate is administered simultaneously with Lactobacillus reuteri. Preferably, citrate and Lactobacillus reuteri are in the same composition (or formulation) and thus are preferably administered in the same composition. Typically, Lactobacillus reuteri (e.g. a Lactobacillus reuteri strain in accordance with the present invention) is administered to the premature infant subject in the form of a composition (or formulation). Typically, said composition (or formulation) is a pharmaceutical composition (or pharmaceutical formulation).

[0029] Thus, compositions (or formulations) comprising Lactobacillus reuteri (e.g. a Lactobacillus reuteri strain in accordance with the present invention) for use in accordance with the invention constitute a further aspect of the present invention. Compositions (or formulations) comprising Lactobacillus reuteri (e.g. a Lactobacillus reuteri strain in admixture with one or more further components (or ingredients), for example a suitable diluent(s), carrier(s) and / or excipient(s), for use in the methods and uses in accordance with the invention, constitute preferred embodiments of the present invention. Such compositions (or formulations) are preferably pharmaceutically acceptable. Suitable diluents, excipients and carriers (e.g. pharmaceutically acceptable diluents, excipients and carriers) are known to the skilled person.

[0030] Other components or excipients may also be present in compositions (or formulations) for use in accordance with the present invention, for example components which are useful for the stabilization or other properties of the composition or formulation (or preparation), e.g. any appropriate cryoprotectants or stabilizers.

[0031] Thus, in one aspect, the present invention provides a composition (or formulation) comprising Lactobacillus reuteri (e.g. a Lactobacillus reuteri strain in accordance with the present invention) for use in the methods and uses of the present invention. Typically, said composition comprises one or more diluent, carrier and / or excipient, preferably one or more pharmaceutically acceptable diluent, carrier or excipient.

[0032] In preferred embodiments, compositions (or formulations) for use in the methods and uses of the invention comprise citrate.

[0033] Thus, in preferred embodiments, compositions (or formulations) for use in the methods and uses of the invention comprise (i) Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention) and (ii) citrate.

[0034] Thus, in preferred embodiments, methods and uses of the present invention comprise administering to said premature infant subject a pharmaceutical composition comprising (i) Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention) and (ii) citrate.

[0035] Thus, in preferred embodiments, compositions (or formulations) for use in accordance with the methods and uses of the present invention comprise one or more source of citrate, e.g. citrate ions, for example citrate salts such as sodium citrate (e.g. trisodium citrate dihydrate), or other metal salts of citrate or citric acid. Preferred salts may be those which have a minimal influence on pH, e.g. those which can be present in buffered solutions at around neutral pH, e.g. pH 6.0 to 7.5. In some embodiments, a source of citrate is sodium citrate (e.g. trisodium citrate dihydrate).

[0036] In some embodiments, compositions (or formulations) for use in the methods and uses of the invention comprise Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention), citrate (or a source of citrate) and one more additional components (e.g. one or more other excipients).

[0037] In some embodiments, compositions (or formulations) for use in the methods and uses of the invention further comprise a sugar (e.g. lactose, e.g. provided in the form of lactose monohydrate).

[0038] In some embodiments, compositions (or formulations) for use in the methods and uses of the invention further comprise citrate (or a source of citrate) and a sugar (e.g. lactose, e.g. provided in the form of lactose monohydrate).

[0039] In some embodiments, compositions (or formulations) for use in accordance with the methods and uses of the present invention further comprise one or more (or all) of the components (or ingredients or excipients) selected from the group consisting of a sugar (e.g. lactose, e.g. provided in the form of lactose monohydrate), gelatin (preferably hydrolysed gelatin), monosodium glutamate, maltodextrin and ascorbic acid.

[0040] In some embodiments, compositions (or formulations) for use in accordance with the methods and uses of the present invention further comprise one or more (or all) of the components (or ingredients or excipients) selected from the group consisting of lactose (e.g. provided in the form of lactose monohydrate), gelatin (preferably hydrolysed gelatin), monosodium glutamate, maltodextrin and ascorbic acid.

[0041] In some embodiments, compositions (or formulations) for use in accordance with the methods and uses of the present invention further comprise one or more (or all) of the components (or ingredients or excipients) selected from the group consisting of lactose monohydrate, hydrolysed gelatin, monosodium glutamate, maltodextrin and ascorbic acid.

[0042] Thus, in some embodiments, compositions (or formulations) for use in accordance with the methods and uses of the present invention comprise (i) Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention) and (ii) one or more (or all) of the components (or ingredients or excipients) selected from the group consisting of a sugar (e.g. lactose, e.g. provided in the form of lactose monohydrate), gelatin (preferably hydrolysed gelatin), monosodium glutamate, maltodextrin and ascorbic acid.

[0043] In some embodiments, compositions (or formulations) for use in accordance with the methods and uses of the present invention comprise (i) Lactobacillus reuteri (e.g.

[0044] Lactobacillus reuteri strain in accordance with the present invention) and (ii) one or more (or all) of the components (or ingredients or excipients) selected from the group consisting of lactose (e.g. provided in the form of lactose monohydrate), gelatin (preferably hydrolysed gelatin), monosodium glutamate, maltodextrin and ascorbic acid.

[0045] In some embodiments, compositions (or formulations) for use in accordance with the methods and uses of the present invention comprise (i) Lactobacillus reuteri (e.g.

[0046] Lactobacillus reuteri strain in accordance with the present invention) and (ii) one or more (or all) of the components (or ingredients or excipients) selected from the group consisting of lactose monohydrate, hydrolysed gelatin, monosodium glutamate, maltodextrin and ascorbic acid.

[0047] In some embodiments, compositions (or formulations) for use in accordance with the methods and uses of the present invention comprise (i) Lactobacillus reuteri (e.g.

[0048] Lactobacillus reuteri strain in accordance with the present invention), (ii) citrate (or a source of citrate) and (iii) one or more (or all) of the components (or ingredients or excipients) selected from the group consisting of a sugar (e.g. lactose, e.g. provided in the form of lactose monohydrate), gelatin (preferably hydrolysed gelatin), monosodium glutamate, maltodextrin and ascorbic acid.

[0049] In some embodiments, compositions (or formulations) for use in accordance with the methods and uses of the present invention comprise (i) Lactobacillus reuteri (e.g.

[0050] Lactobacillus reuteri strain in accordance with the present invention), (ii) citrate (or a source of citrate) and (iii) one or more (or all) of the components (or ingredients or excipients) selected from the group consisting of lactose (e.g. provided in the form of lactose monohydrate), gelatin (preferably hydrolysed gelatin), monosodium glutamate, maltodextrin and ascorbic acid.

[0051] In some embodiments, compositions (or formulations) for use in accordance with the methods and uses of the present invention comprise (i) Lactobacillus reuteri (e.g.

[0052] Lactobacillus reuteri strain in accordance with the present invention), (ii) citrate (or a source of citrate) and (iii) one or more (or all) of the components (or ingredients or excipients) selected from the group consisting of lactose monohydrate, hydrolysed gelatin, monosodium glutamate, maltodextrin and ascorbic acid.

[0053] In some embodiments, compositions (or formulations) for use in accordance with the present invention comprise (i) Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention) and (ii) one or more (or all) of the components (or ingredients or excipients) selected from the group consisting of citrate (or a source of citrate), a carbon source (such as a sugar, e.g. lactose, e.g. provided in the form of lactose monohydrate), gelatin (preferably hydrolysed gelatin), monosodium glutamate, maltodextrin and ascorbic acid. Preferably, said citrate is provided in the form of trisodium citrate di hydrate. In some embodiments, compositions (or formulations) for use in accordance with the present invention comprise (i) Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention) and (ii) one or more (or all) of the components (or ingredients or excipients) selected from the group consisting of trisodium citrate dihydrate, lactose monohydrate, hydrolysed gelatin, monosodium glutamate, maltodextrin and ascorbic acid.

[0054] Preferred carbon sources include carbohydrates such as sugars. Lactose (e.g. in the form of lactose monohydrate) is a preferred sugar for use. However, any other appropriate sugar can be used (for supporting growth of the bacteria), for example sucrose, fructose, glucose or galacto-oligosaccharides (GOS).

[0055] In some embodiments, compositions (or formulations) for use in accordance with the methods and uses of the present invention may further comprise mannitol.

[0056] In some embodiments, compositions (or formulations) for use in accordance with the methods and uses of the present invention may further comprise a buffer or buffer solution, e.g. to allow an appropriate pH to be retained.

[0057] In some embodiments, compositions (or formulations) for use in accordance with the method and uses of the present invention may comprise (or be formulated with or be reconstituted with) any appropriate solution for administration to the subject, e.g. a human. For example, the compositions (or formulations) may comprise (or be formulated with or be reconstituted with) water (e.g. distilled water or sterile water or sterile distilled water) or other aqueous solution, some kind of formulation which mimics or replicates conditions in the intestine (e.g. simulated intestinal substrate (SIS) medium, or similar), breast milk, formula milk, an enteral formula, or any other solution suitable for enteric administration (e.g. administration as an enteral feeding product).

[0058] In some embodiments, compositions (or formulations) for use in accordance with the methods and uses of the present invention may further comprise water (e.g. sterile water).

[0059] In some embodiments, compositions (or formulations) for use in accordance with the methods and uses of the present invention may further comprise a milk-based product, for example breast milk or formula milk. However, in some embodiments, compositions (or formulations) for use in accordance with the present invention lack milk proteins. In some embodiments, compositions (or formulations) for use in accordance with the present invention do not comprise a milk-based product. Thus, in some embodiments, compositions (or formulations) for use in accordance with the present invention do not comprise breast milk. In some embodiments, compositions (or formulations) for use in accordance with the present invention do not comprise formula milk. In some embodiments, compositions (or formulations) for use in accordance with the methods and uses of the present invention are used (administered to a subject) within 12 hours, within 6 hours or within 3 hours of having been prepared. For example, in some embodiments, compositions (or formulations) that have been prepared by reconstituting Lactobacillus reuteri (e.g. by reconstituting lyophilized or freeze dried or spray dried Lactobacillus reuteri) are used (administered to a subject) within 12 hours, within 6 hours or within 3 hours of having been reconstituted (e.g. suspended or resuspended).

[0060] In some embodiments, the composition (or formulation) is a suspension of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention). The suspension is typically suitable for enteral (e.g. oral or via feeding tube) administration. In some embodiments, the suspension is suitable for enteral administration using (or via) an orogastric tube, a nasogastric tube or a gastroduodenal tube. Other components as described herein may also be present in said composition.

[0061] There may be one dose of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention), or there may be more than one dose (e.g. 2 or 3 doses) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention), administered to the subject per day.

[0062] In preferred embodiments, one dose of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention) is administered to the subject per day.

[0063] In some embodiments, 1x105to 1x1011or 1x105to 1x1O10, for example 1x106to 1x1011, 1x106to 1x1O10, 1x106to 1x109, or 1x106to 1x108, or 1x108to 1x1O10, or 1x108to 1x1011CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention) may be administered to the subject per day.

[0064] In some embodiments, 1x108to 1x1011CFUs or 1x109to 1x1011CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention) may be administered to the subject per day.

[0065] In some embodiments, 1x108to 1x101°CFUs, or 1x109to 1x101°CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention) may be administered to the subject per day.

[0066] In some embodiments, 1x109to 1x101°CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention) may be administered to the subject per day.

[0067] In some embodiments, around 1x108or around 1x109or around 1x1O10or around 1x1011CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention) may be administered to the subject per day. In some embodiments, around 1x109CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention) may be administered to the subject per day.

[0068] In some embodiments, at least 1x105, at least 1x106, at least 1x107, at least 1x108, at least 1x109, or at least 1x1O10CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention) may be administered to the subject per day.

[0069] In some embodiments, the above amounts (or numbers) of CFUs may be (or may equate to) amounts (or numbers) of CFUs per dose of a composition (or formulation) administered (or to be administered) to the subject.

[0070] As mentioned above, in some embodiments, one dose of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention) is administered to the subject per day.

[0071] Thus, in some embodiments, one dose is administered to the subject per day, said one dose containing 1x105to 1x1011or 1x105to 1x1O10, for example 1x106to 1x1011, 1x106to 1x1O10, 1x106to 1x109, or 1x106to 1x108, or 1x108to 1x1O10, or 1x108to 1x1011CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention).

[0072] In some embodiments, one dose is administered to the subject per day, said one dose containing 1x108to 1x1011CFUs or 1x109to 1x1011CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention), preferably 1x109to 1x1011CFUs.

[0073] In some embodiments, one dose is administered to the subject per day, said one dose containing 1x108to 1x101°CFUs, or 1x109to 1x1O10CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention), preferably 1x109to 1x1010CFUs.

[0074] In some embodiments, one dose is administered to the subject per day, said one dose containing around 1x108or around 1x109or around 1x1O10CFUs or around 1x1011CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention), preferably around 1x109CFUs.

[0075] In some embodiments, one dose is administered to the subject per day, said one dose containing at least 1x105, at least 1x106, at least 1x107, at least 1x108, at least 1x109, or at least 1x1O10CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention).

[0076] In some embodiments, a (or each) dose contains 1x105to 1x1011or 1x105to 1x1O10, for example 1x106to 1x1011, 1x106to 1x1O10, 1x106to 1x109, 1x106to 1x108, or 1x108to 1x1O10, or 1x108to 1x1011CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention). A dose (or doses) containing such a number of CFUs may be used irrespective of the dosing schedule being followed.

[0077] In some embodiments, a (or each) dose contains 1x108to 1x1011CFUs or 1x109to 1x1011CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention). A dose (or doses) containing such a number of CFUs may be used irrespective of the dosing schedule being followed.

[0078] In some embodiments, a (or each) dose contains 1x108to 1x1010CFUs or 1x109to 1x1010CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention). A dose (or doses) containing such a number of CFUs may be used irrespective of the dosing schedule being followed.

[0079] In some embodiments, a (or each) dose contains 1x109to 1x1010CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention). A dose (or doses) containing such a number of CFUs may be used irrespective of the dosing schedule being followed.

[0080] In some embodiments, a (or each) dose contains around 1x108or around 1x109or around 1x101°or around 1x1011CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention). A dose (or doses) containing such a number of CFUs may be used irrespective of the dosing schedule being followed.

[0081] In some embodiments, a (or each) dose contains around 1x109CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention). A dose (or doses) containing such a number of CFUs may be used irrespective of the dosing schedule being followed.

[0082] In some embodiments, a (or each) dose contains at least 1x105, at least 1x106, at least 1x107, at least 1x108, at least 1x109, or at least 1x1O10CFUs. A dose (or doses) containing such a number of CFUs may be used irrespective of the dosing schedule being followed.

[0083] In other embodiments, other numbers (amounts) of CFUs may be used and / or other dosage regimes may be employed. Any effective (e.g. therapeutically effective or prophylactically effective) numbers (amounts) of CFUs may be used, and the skilled person could establish this. Any suitable dosages or dosage regime may be used, and the skilled person could establish these.

[0084] A dose (or doses) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention) may be administered in any appropriate (or suitable) volume of a composition (or formulation). Suitable composition (or formulation) volumes per dose may be, for example, 0.1ml to 5ml, for example 0.25ml to 5ml, 0.5ml to 5ml, 1ml to 5ml, 0.1ml to 2ml, for example 0.25ml to 2ml, 0.5ml to 2ml, 1ml to 2ml, 0.1ml to 1ml, for example 0.25ml to 1ml, or 0.5ml to 1ml. Suitable composition (or formulation) volumes per dose may be, for example, about 0.1ml, about 0.25ml, about 0.5ml, about 1ml, about 2ml or about 5ml.

[0085] In some embodiments, a composition volume per dose is about 1ml (or 1ml) or about 0.5ml (or 0.5ml).

[0086] In some embodiments, a composition volume per dose is about 1ml (or 1ml). In some preferred embodiments, a composition volume per dose is about 0.5ml (or 0.5ml).

[0087] As described above, in preferred embodiments, one dose per day is administered to the subject.

[0088] Thus, in some embodiments, an amount (or number) of CFUs as discussed above may be that amount (or number) of CFUs in a dose that has a volume as described above.

[0089] In some embodiments, a dose of 1x108to 1x1011or 1x109to 1x1011CFUs may be administered in a composition volume of 0.1ml to 5ml.

[0090] In some embodiments, a dose of 1x108to 1x101° or 1x109to 1x101° CFUs may be administered in a composition volume of 0.1ml to 5ml.

[0091] In some embodiments, a dose of around 1x108or around 1x109or around 1x101°or around 1x1011CFUs (preferably around 1x109) may be administered in a composition volume of 0.1 ml to 5ml.

[0092] In some embodiments, a dose of 1x108to 1x1011or 1x109to 1x1011or1x108to 1x101° or 1x109to 1x101° CFUs may be administered in a composition volume of about 1ml. In some embodiments, a dose of around 1x108or around 1x109or around 1x101°CFUs or around 1x1011(preferably around 1x109) may be administered in a composition volume of about 1ml.

[0093] In some embodiments, a dose of 1x108to 1x1011or 1x109to 1x1011or1x108to 1x101° or 1x109to 1x101° CFUs may be administered in a composition volume of about 0.5ml. In some embodiments, a dose of around 1x108or around 1x109or around 1x101° or around 1x1011CFUs (preferably around 1x109) may be administered in a composition volume of about 0.5ml.

[0094] In some embodiments, a dose of (or containing) at least 1x105, at least 1x106, at least 1x107, at least 1x108, at least 1x109, or at least 1x101° CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention) may be administered in a composition volume as described above (e.g. 0.1ml to 5ml).

[0095] In some embodiments, the above amounts (or numbers) of CFUs may be (or may equate to) amounts (or numbers) of CFUs per 0.5ml dose of composition (or formulation) administered (or to be administered) to the subject per day. In some preferred embodiments 1x109CFUs in a composition (or formulation) volume of 0.5ml is administered to the subject per day.

[0096] In some embodiments, the above amounts (or numbers) of CFUs may be (or may equate to) amounts (or numbers) of CFUs per 1ml (or per ml) of composition (or formulation) administered (or to be administered) to the subject per day.

[0097] In preferred embodiments, where amounts (or numbers) of CFUs for administration per day are discussed herein and / or number of dose(s) administered per day are discussed herein, those amounts (or numbers) of CFUs and / or number of dose(s) are administered to the subject every day during the period of therapy (or every day in the period from the day the first dose is administered until the day the final dose is administered). Thus, in some embodiments, there is a daily dose administered.

[0098] In other embodiments, where amounts (or numbers) of CFUs for administration per day are discussed herein and / or where number of dose(s) administered per day are discussed herein, those amounts (or numbers) of CFUs and / or number of dose(s) are administered to the subject on at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the days during the period of therapy (or on at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the days in the period from the day the first dose is administered until the day the final dose is administered).

[0099] As described above, compositions (or formulations) for use in accordance with the present invention preferably further comprise citrate (e.g. provided in the form of trisodium citrate di hydrate).

[0100] Any suitable amount or concentration of citrate may be used. Thus, any appropriate amount of citrate (or source of citrate, e.g. provided in the form of trisodium citrate dihydrate) may be present in compositions (or formulations or preparations) for use in accordance with the present invention. In some embodiments, the concentration of citrate (e.g. provided in the form of trisodium citrate dihydrate) is 0.01 or 0.05 mg / ml to 100 mg / ml, e.g. 0.05 mg / ml to 1, 2, 3, 5, 10, 20, 30 or 50 mg / ml, or e.g. 0.1 mg / ml to 1, 2, 3, 5, 10, 20, 30 or 50 mg / ml. In some embodiments, the concentration of citrate (e.g. provided in the form of trisodium citrate dihydrate) is 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 mg / ml to 100 mg / ml. In some embodiments, the concentration of citrate (e.g. provided in the form of trisodium citrate dihydrate) is 0.01 mg / ml or 0.1 mg / ml or 1 mg / ml to 50mg / ml. In some embodiments, the concentration of citrate (e.g. provided in the form of trisodium citrate dihydrate) is 0.01mg / ml or 0.1mg / ml or 1mg / ml to 10mg / ml. In some embodiments, the concentration of citrate is 0.01 mg / ml or 0.1 mg / ml to 1 mg / ml. In some embodiments, the concentration of citrate (e.g. provided in the form of trisodium citrate dihydrate) is at least 0.01 , at least 0.05, at least 0.1 , at least 0.5 or at least 1 mg / ml. In some embodiments, the concentration of citrate (e.g. provided in the form of trisodium citrate dihydrate) is up to 100mg / ml, up to 50mg / ml, up to 10mg / ml or up to 1mg / ml. In some embodiments, the concentration of citrate (e.g. provided in the form of trisodium citrate dihydrate) may be (or may be about) 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 mg / ml. These values can also be appropriate for equivalent mgs / dose given to the subject. Thus, in some embodiments, the amount (in mgs) of citrate per dose (of a composition or formulation for use in accordance with the present invention) may be a value (or number value) set out in this paragraph.

[0101] In some embodiments, Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention) is (or has been) lyophilized, freeze-dried, frozen or spray dried. In some preferred embodiments, Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention) is (or has been) lyophilized. Thus, in some embodiments, the composition (or formulation) for use in accordance with the invention may be (or may have been) lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized). Any of the amounts (or numbers) of CFUs of Lactobacillus reuteri that are described elsewhere may be present in a lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation), for example in a vial (e.g. a glass vial) of a lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation).

[0102] Lyophilization, freeze-drying, freezing or spray-drying may be done in any appropriate way, and the person skilled in the art is familiar with appropriate techniques and methods for this. For example, individual components in (or to be in) a final product (e.g. a final lyophilized product) can be combined together in a liquid formulation and then lyophilized or dried. Alternatively, individual dry (e.g. lyophilized) components, including the dry or lyophilized Lactobacillus reuteri can be mixed or blended together to form a dry formulation. Dry formulations can then be reconstituted to form an appropriate Lactobacillus reuteri containing composition or formulation for administration to the subjects.

[0103] Typically of course, dry compositions (e.g. lyophilized, freeze-dried or spray dried compositions) are reconstituted (e.g. suspended or resuspended) to form an appropriate Lactobacillus reuteri containing (final) composition (or formulation or suspension), or dose of a composition, for administration to a subject.

[0104] Reconstitution may be done in any appropriate way and the person skilled in the art is familiar with suitable methods and agents. Reconstitution (or suspension or resuspension) of the Lactobacillus reuteri may be done with water (e.g. distilled water or sterile water or sterile distilled water) or other aqueous solution, some kind of formulation which mimics or replicates conditions in the intestine (e.g. simulated intestinal substrate (SIS) medium, or similar), breast milk, formula milk, an enteral formula, or any other solution suitable for enteric administration (e.g. administration as an enteral feeding product). In some embodiments, a milk-based product is not used.

[0105] In a lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation), Lactobacillus reuteri are typically dormant. In some cases, once reconstituted into a composition for administration to a subject, some growth (or activation or reactivation) of the Lactobacillus reuteri may occur (or may already have occurred) before the composition reaches the intestine of the subject, e.g. during its reconstitution and administration to the subject. In other cases, growth (or activation or reactivation) of the Lactobacillus reuteri may commence once the Lactobacillus reuteri reaches the intestine of the subject. As described elsewhere herein, activation (or reactivation) of Lactobacillus reuteri means the process of changing the state of a Lactobacillus reuteri from being dormant to the state of metabolizing bacteria.

[0106] In some embodiments, other components may also be present in the lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation), i.e. in addition to the Lactobacillus reuteri. For example, in some embodiments, one or more (or all) of the other components of compositions (or formulations) for use in accordance with the invention as described herein are present in the lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation). Thus, in some embodiments, the lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation) further comprises one or more (or all) components (or ingredients) selected from the group consisting of citrate (or a source of citrate), a carbon source (such as a sugar, e.g. lactose, e.g. provided in the form of lactose monohydrate), gelatin (preferably hydrolysed gelatin), monosodium glutamate, maltodextrin and ascorbic acid. Preferably, said citrate is provided in the form of trisodium citrate dihydrate.

[0107] In some preferred embodiments, the lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation) further comprises citrate (or a source of citrate, preferably trisodium citrate dihydrate).

[0108] In some preferred embodiments, the lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation) further comprises citrate (or a source of citrate, preferably trisodium citrate dihydrate) and a carbon source (preferably a sugar such as lactose, e.g. provided in the form of lactose monohydrate).

[0109] In some embodiments, as alternative to, or in addition to, one or more (or all) of the other components of compositions (or formulations) for use in accordance with the invention as described herein being present in the lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation) together with the Lactobacillus reuteri, these may not be present in the lyophilized, freeze-dried, frozen or spray dried composition and instead a solution comprising one or more (or all) of these other components (preferably comprising at least citrate, or preferably comprising at least citrate and a carbon source (e.g. a sugar such as lactose)) may be used to reconstitute (e.g. suspend or resuspend) the Lactobacillus reuteri (prior to administration to a subject).

[0110] In some embodiments, a dose (or unit) of a lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation), or a dose of composition (for administration to a subject) prepared by reconstituting a lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation), contains 1x105to 1x1011or 1x105to 1x1O10, for example 1x106to 1x1011, 1x106to 1x1O10, 1x106to 1x109, or 1x106to 1x108, or 1x108to 1x1O10, or 1x108to 1x1011CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention).

[0111] In some embodiments, a dose (or unit) of a lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation), or a dose of composition (for administration to a subject) prepared by reconstituting a lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation), contains 1x108to 1x1011CFUs or 1x109to 1x1011CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention).

[0112] In some embodiments, a dose (or unit) of a lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation), or a dose of composition (for administration to a subject) prepared by reconstituting a lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation), contains 1x108to 1x1010CFUs or 1x109to 1x1010CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention).

[0113] In some embodiments, a dose (or unit) of a lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation), or a dose of composition (for administration to a subject) prepared by reconstituting a lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation), contains 1x109to 1x1010CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention).

[0114] In some embodiments, a dose (or unit) of a lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation), or a dose of composition (for administration to a subject) prepared by reconstituting a lyophilized, freeze- dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation), contains around 1x108or around 1x109or around 1x1O10or around 1x1011CFUs (colony forming units) of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention), preferably around 1x109CFUs.

[0115] In some embodiments, a dose (or unit) of a lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation), or a dose of composition (for administration to a subject) prepared by reconstituting a lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation), contains at least 1x105, at least 1x106, at least 1x107, at least 1x108, at least 1x109, or at least 1x1O10CFUs (colony forming units) of Lactobacillus reuteri (e.g.

[0116] Lactobacillus reuteri strain in accordance with the present invention).

[0117] In some embodiments, a dose (or unit) of a lyophilized, freeze-dried, frozen or spray dried (preferably lyophilized) composition (or formulation or preparation) to be reconstituted for administration to a subject (i.e. prior to reconstitution for administration to a subject) may contain any appropriate amount of citrate (or source of citrate, e.g. trisodium citrate dihydrate). In some embodiments, the amount of citrate (e.g. trisodium citrate dihydrate) in such a dose (or unit) is 0.01 or 0.05 mg to 100 mg, e.g. 0.05 mg to 1, 2, 3, 5, 10, 20, 30 or 50 mg, or e.g. 0.1 mg to 1 , 2, 3, 5, 10, 20, 30 or 50 mg. In some embodiments, the amount of citrate (e.g. trisodium citrate dihydrate) in such a dose (or unit) is 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 mg to 100 mg. In some embodiments, the amount of citrate (e.g. trisodium citrate dihydrate) in such a dose (or unit) is 0.01 mg or 0.1mg or 1mg to 50mg. In some embodiments, the amount of citrate (e.g. trisodium citrate dihydrate) in such a dose (or unit) is 0.01mg or 0.1mg or 1mg to 10mg. In some embodiments, the amount of citrate in such a dose (or unit) is 0.01 mg or 0.1 mg to 1mg. In some embodiments, the amount of citrate (e.g. trisodium citrate dihydrate) in such a dose (or unit) is at least 0.01, at least 0.05, at least 0.1, at least 0.5, at least 1mg. In some embodiments, the amount of citrate (e.g. trisodium citrate dihydrate) in such a dose (or unit) may be (or may be about) 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 mg.

[0118] In the compositions (or formulations or preparations) of the invention it is preferred that components used are in a pure or substantially pure form suitable for pharmaceutical administration to mammals, preferably humans. Thus the individual components are preferably of pharmaceutical grade which can be present or included in a known or precise amount rather than for example being present or added as part of a mixture of unknown amounts or complex amounts of components. Thus, although compositions (or formulations or preparations) for use in the invention may be administered after mixing or reconstitution in breast milk (or other milk based products) which may contain citrate and lactose amongst many other things, it is preferred that the product formulations for administration to patients contain appropriately pure preparations of citrate ions, e.g. in (or provided in) the form of citrate salts, e.g. sodium citrate (such as trisodium citrate dihydrate) as described elsewhere herein, and lactose (e.g. lactose monohydrate) or other sugars. Thus, in preferred embodiments of the invention, the compositions (or formulations) are reconstituted (or diluted) in water or are aqueous formulations and do not for example involve reconstitution with a milk based product such as breast milk or formula milk. Thus, preferred compositions (or formulations) of the invention lack milk proteins. Alternatively viewed, preferred compositions (or formulations) of the invention contain or are exposed to citrate and / or lactose which is not provided by a milk-based product such as breast milk or formula milk.

[0119] Typically and preferably, methods and uses in accordance with the present invention comprise administering more than one dose of Lactobacillus reuteri (e.g. Lactobacillus reuteri strain in accordance with the present invention) . In other embodiments, only a single dose is administered.

[0120] In some embodiments, a first dose of Lactobacillus reuteri is administered to the subject <1 week, <96h, <72h or <48h after birth of the subject. In preferred embodiments, a first dose of Lactobacillus reuteri is administered to the subject < 48h after birth.

[0121] In preferred embodiments, one or more further doses are administered subsequent to the first dose.

[0122] In some embodiments, one or more doses of Lactobacillus reuteri are administered to the subject every day during the period of therapy (every day in the period from the day the first dose is administered until the day the final dose is administered).

[0123] In preferred embodiments, one dose (a single dose) of Lactobacillus reuteri is administered to the subject every day during the period of therapy (every day in the period from the day the first dose is administered until the day the final dose is administered).

[0124] Thus, in some embodiments, there is a (or one) daily dose administered.

[0125] In other embodiments, one or more doses of Lactobacillus reuteri are administered to the subject on at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the days during the period of therapy (on at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the days in the period from the day the first dose is administered until the day the final dose is administered).

[0126] In some embodiments, the period of therapy may be up to 20 weeks, up to 15 weeks, up to 14 weeks, up to 13 weeks, up to 12 weeks, up to 11 weeks, up to 10 weeks, up to 9 weeks, up to 8 weeks, up to 7 weeks, up to 6 weeks, up to 5 weeks, up to 4 weeks, up to 3 weeks, up to 2 weeks, up to 1 week, 6 days, 5 days, 3 days or 2 d days from the (day of) first administration (first dose) of the Lactobacillus reuteri. In some embodiments, the period of therapy may be from the day of the first administration (or dose) of the Lactobacillus reuteri until the subject reaches a post-menstrual age (PMA) of 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, or 36 weeks + 6 days.

[0127] In some embodiments, the period of therapy may be from the day of the first administration (or dose) of the Lactobacillus reuteri until the subject reaches a post-menstrual age (PMA) of 34 weeks + 6 days.

[0128] In some embodiments, the period of therapy may be from the day of the first administration (or dose) of the Lactobacillus reuteri until the subject no longer requires therapy (e.g. until a clinical decision has been taken that therapy is no longer required), e.g. no longer requires therapy for a complication or disorder or condition described herein in relation to the methods and uses of the invention and / or is no longer at risk (no longer an “at risk” subject).

[0129] Lactobacillus reuteri (or compositions or formulations comprising Lactobacillus reuteri) may be presented in conventional pharmacological forms for administration.

[0130] Conventional pharmaceutical excipients, as well as the conventional methods of production, may be employed for the preparation of these forms.

[0131] In preferred embodiments, Lactobacillus reuteri (or compositions or formulations comprising Lactobacillus reuteri) may be presented, for example, in a form suitable for enteral administration.

[0132] Thus, in some embodiments, Lactobacillus reuteri (or compositions or formulations comprising Lactobacillus reuteri) may be presented in a form suitable for administration direct to the stomach of the subject, e.g. a form suitable for administration by a feeding tube such as a mouth (orogastric) or nose (nasogastric) tube. An orogastric tube may be referred to as an OG tube. An nasogastric tube may be referred to as a NG tube. In some embodiments, Lactobacillus reuteri (or compositions or formulations comprising Lactobacillus reuteri) may be presented in a form suitable for administration by a gastroduodenal tube. In some embodiments, Lactobacillus reuteri (or compositions or formulations comprising Lactobacillus reuteri) may be presented in a form suitable for oral administration.

[0133] Lactobacillus reuteri (or compositions or formulations comprising Lactobacillus reuteri) may be administered by any suitable administration route. In preferred embodiments, administration is enteral administration. In some embodiments, administration is by (or via) a feeding tube such as a mouth (orogastric) or nose (nasogastric) tube or gastroduodenal tube. In some embodiments, an enteral syringe is used for administration (delivery) through (or via) a feeding tube such as a mouth (orogastric) or nose (nasogastric) tube. In some embodiments, administration is oral administration. Oral administration may not be possible for all subjects, but if a subject is capable, then oral administration may be used. Thus, in some cases, Lactobacillus reuteri (or compositions or formulations comprising Lactobacillus reuteri) may be administered (or delivered) directly to a subject’s mouth. In some embodiments, if the subject has no feeding tube (e.g. no NG or OG tube) Lactobacillus reuteri (or compositions or formulations comprising Lactobacillus reuteri) may be administered (or delivered) directly to a subject’s mouth. In some embodiments, administration may be rectal administration.

[0134] In some embodiments, after (e.g. immediately following) enteral administration of a dose of Lactobacillus reuteri (or compositions or formulations comprising Lactobacillus reuteri) by a feeding tube (e.g. NG tube or OG tube) the tube is flushed with a flushing agent (e.g. 0.5ml of flushing agent). The flushing agent may, for example, be breast milk, enteral formula, air or sterile water.

[0135] Subjects in accordance with the present invention are premature infant subjects. Such subjects may also be referred to as pre-term infants.

[0136] “Infant” and “baby” are used interchangeably and refer to a newborn and its first year of life (up to one year or up to twelve months old). Preferably, the infant is a newborn baby or neonate. The terms “newborn” baby and “neonate” may be used interchangeably and refer to an infant in the time period directly after birth (for example under 28 days of age or alternatively viewed for example within 28 days of having been born).

[0137] Preferably, the infant subject in accordance with the present invention is a premature neonate.

[0138] “Premature” (or “pre-term”) infant subjects are infants that are born alive (or delivered alive) before week 37 of pregnancy (or the equivalent timepoint in non-human mammals). Thus, “premature” or “pre-term” birth is a birth that takes place before week 37 of pregnancy (or the equivalent timepoint in non-human mammals). Alternatively viewed, a “premature” (or “pre-term”) infant subject is a baby born alive before 37 weeks of pregnancy are completed. “Premature” (or “pre-term”) infants may alternatively be referred to as infants having a gestational age (GA) at birth of <37 weeks.

[0139] In some embodiments of the present invention, a premature infant (preferably a premature neonate) is a premature infant born alive before 36 weeks of pregnancy are completed, before 35 weeks of pregnancy are completed, before 34 weeks of pregnancy are completed, before 33 weeks of pregnancy are completed, before 32 weeks of pregnancy are completed, before 31 weeks of pregnancy are completed, before 30 weeks of pregnancy are completed, before 29 weeks of pregnancy are completed, before 28 weeks of pregnancy are completed, before 27 weeks of pregnancy are completed, before 26 weeks of pregnancy are completed, before 25 weeks of pregnancy are completed, before 24 weeks of pregnancy are completed or before 23 weeks of pregnancy are completed.

[0140] Alternatively viewed, in some embodiments of the present invention, a premature infant (preferably a premature neonate) is a premature infant having a gestational age (GA) at birth of <36 weeks, <35 weeks, <34 weeks, <33 weeks, <32 weeks, <31 weeks, <30 weeks, <29 weeks, <28 weeks, <27 weeks, <26 weeks, <25 weeks, <24 weeks, or <23 weeks.

[0141] In some preferred embodiments, a premature infant (preferably a premature neonate) is a premature infant having a gestational age at birth of <32 weeks.

[0142] In some embodiments, a premature infant (preferably a premature neonate) is a premature infant having a gestational age at birth of <28 weeks.

[0143] In some embodiments, a premature infant (preferably a premature neonate) is a premature infant having a gestational age at birth of <25 weeks.

[0144] In some embodiments, a premature infant (preferably a premature neonate) is a premature infant born alive after at least 23 weeks, after at least 24 weeks, after at least 25 weeks, after at least 26 weeks, after at least 27 weeks, after at least 28 weeks, after at least 29 weeks, after at least 30 weeks, after at least 31 weeks, after at least 32 weeks, after at least 33 weeks, after at least 34 weeks, after at least 35 weeks or after at least 36 weeks of pregnancy have been completed.

[0145] Alternatively viewed, in some embodiments, a premature infant (preferably a premature neonate) is a premature infant having a gestational age (GA) at birth of >23 weeks, >24 weeks, >25 weeks, >26 weeks, >27 weeks, >28 weeks, >29 weeks, >30 weeks, >31 weeks, >32 weeks, >33 weeks, >34 weeks, >35 weeks or >36 weeks.

[0146] In some preferred embodiments, a premature infant (preferably a premature neonate) is a premature infant having a gestational age at birth of >23 weeks.

[0147] In some preferred embodiments, a premature infant (preferably a premature neonate) is a premature infant having a gestational age at birth of >26 weeks.

[0148] In some preferred embodiments, a premature infant (preferably a premature neonate) is a premature infant having a gestational age at birth of >29 weeks.

[0149] In some preferred embodiments, a premature infant (preferably a premature neonate) is a premature infant born alive between 23 completed weeks and 36 completed weeks of pregnancy. Thus, in some preferred embodiments, premature infants have a gestational age of 23 completed weeks (or 23 weeks + 0 days) and 36 completed weeks (or 36 weeks + 0 days) at birth.

[0150] In some preferred embodiments, a premature infant (preferably a premature neonate) is a premature infant born alive between 26 completed weeks and 36 completed weeks of pregnancy. Thus, in some preferred embodiments, premature infants have a gestational age of 26 completed weeks (or 26 weeks + 0 days) and 36 completed weeks (or 36 weeks + 0 days) at birth.

[0151] In some preferred embodiments, a premature infant (preferably a premature neonate) is a premature infant born alive between 29 completed weeks and 36 completed weeks of pregnancy. Thus, in some preferred embodiments, premature infants have a gestational age of 29 completed weeks (or 29 weeks + 0 days) and 36 completed weeks (or 36 weeks + 0 days) at birth.

[0152] In some preferred embodiments, a premature infant (preferably a premature neonate) is a premature infant born alive between 23 completed weeks and 32 completed weeks of pregnancy. Thus, in some preferred embodiments, premature infants have a gestational age of 23 completed weeks (or 23 weeks + 0 days) and 32 completed weeks (or 32 weeks + 0 days) at birth.

[0153] In some preferred embodiments, a premature infant (preferably a premature neonate) is a premature infant born alive between 23 completed weeks and 25 completed weeks of pregnancy. Thus, in some preferred embodiments, premature infants have a gestational age of 23 completed weeks (or 23 weeks + 0 days) and 25 completed weeks (or 25 weeks + 0 days) at birth.

[0154] In some preferred embodiments, a premature infant (preferably a premature neonate) is a premature infant born alive between 23 completed weeks and 28 completed weeks of pregnancy. Thus, in some preferred embodiments, premature infants have a gestational age of 23 completed weeks (or 23 weeks + 0 days) and 28 completed weeks (or 28 weeks + 0 days) at birth.

[0155] In some preferred embodiments, a premature infant (preferably a premature neonate) is a premature infant born alive between 26 completed weeks and 28 completed weeks of pregnancy. Thus, in some preferred embodiments, premature infants have a gestational age of 26 completed weeks (or 26 weeks + 0 days) and 28 completed weeks (or 28 weeks + 0 days) at birth.

[0156] In some preferred embodiments, a premature infant (preferably a premature neonate) is a premature infant born alive between 26 completed weeks and 32 completed weeks of pregnancy. Thus, in some preferred embodiments, premature infants have a gestational age of 26 completed weeks (or 26 weeks + 0 days) and 32 completed weeks (or 32 weeks + 0 days) at birth.

[0157] In some preferred embodiments, a premature infant (preferably a premature neonate) is a premature infant born alive between 29 completed weeks and 32 completed weeks of pregnancy. Thus, in some preferred embodiments, premature infants have a gestational age of 29 completed weeks (or 29 weeks + 0 days) and 32 completed weeks (or 32 weeks + 0 days) at birth.

[0158] In some embodiments, a premature infant (preferably a premature neonate) has a birth weight of <2000g. In some embodiments, a premature infant (preferably a premature neonate) has a birth weight of <1750g, <1700g, <1650g, <1600g or<1550g. In some embodiments, a premature infant (preferably a premature neonate) has a birth weight of <1500g, <1400g, <1300g, <1200g, <1100g, <1000g, <900g, <800g, <750g, <700g, <600g or <500g.

[0159] In some preferred embodiments, a premature infant (preferably a premature neonate) has a birth weight of <1500g.

[0160] In some preferred embodiments, a premature infant (preferably a premature neonate) has a birth weight of <1000g.

[0161] In some preferred embodiments, a premature infant (preferably a premature neonate) has a birth weight of <750g.

[0162] In some embodiments, a premature infant (preferably a premature neonate) has a birth weight of >400g, >500g, >600g, >700g, >750g, >800g, >900g, >1000g, >1100g, >1200g, >1300g, >1400g or>1500g.

[0163] In some embodiments, a premature infant (preferably a premature neonate) has a birth weight of up to 750g, up to 800g, up to 900g, up to 1000g, up to 1100g, up to 1200g, up to 1300g, up to 1400g or up to 1500g.

[0164] In some embodiments, a premature infant (preferably a premature neonate) has a birth weight of up to 750g. In some embodiments, a premature infant (preferably a premature neonate) has a birth weight of up to 1000g. In some embodiments, a premature infant (preferably a premature neonate) has a birth weight of up to 1500g.

[0165] In some embodiments, a premature infant (preferably a premature neonate) has a birth weight of between 400g and 2000g, between 400g and 1500g, between 400g and 1000g or between 400g and 750g. In some embodiments, a premature infant (preferably a premature neonate) has a birth weight of between 500g and 2000g, between 500g and 1500g, between 500g and 1000g or between 500g and 750g. In some embodiments, a premature infant (preferably a premature neonate) has a birth weight of between 500g and 1500g. In some embodiments, a premature infant (preferably a premature neonate) has a birth weight of between 500g and 750g.

[0166] In some embodiments, a premature infant (preferably a premature neonate) has a birth weight of between 750g and 2000g, between 750g and 1500g, or between 750g and 1000g or between 500g and 750g. In some embodiments, a premature infant (preferably a premature neonate) has a birth weight of between 750g and 1500g. In some embodiments, a premature infant (preferably a premature neonate) has a birth weight of between 750g and 1000g.

[0167] In some embodiments, a premature infant (preferably a premature neonate) has a birth weight of between 1000g and 2000g or between 1000g and 1500g, In some embodiments, a premature infant (preferably a premature neonate) has a birth weight of between 1000g and 1500g.

[0168] In some embodiments, a premature infant (preferably a premature neonate) has a birth weight as described above and a gestational age as described above. For example, in some embodiments, a premature infant (preferably a premature neonate) may have a birth weight of 500g to 1500g (i.e. between 500g and 1500g) and a gestational age (GA) of between of 23 completed weeks (or 23 weeks + 0 days) and 32 completed weeks (or 32 weeks + 0 days) at birth. Other appropriate combinations of particular birthweights and gestational ages are derivable, e.g. from the discussion herein, and any of these combinations may be used.

[0169] The term “subject” (or “patient”) as used herein includes any mammal, for example humans and any livestock, domestic or laboratory animal. Specific examples include mice, rats, pigs, cats, dogs, sheep, rabbits, cows and monkey. Preferably, however, the subject is a human subject.

[0170] In some embodiments, the subject is a male subject (preferably a human male subject). In some embodiments, the subject is a female subject (preferably a human female subject).

[0171] In some embodiments, the subject (preferably a human subject) was born (or delivered) by Caesarean section. In some embodiments, the subject (preferably a human subject) was born (or delivered) vaginally. In some embodiments of methods and uses for reducing risk of mortality (or death or) or for preventing death in a premature infant subject, the subject (preferably a human subject) was born (or delivered) by Caesarean section.

[0172] In some embodiments, subjects or patients will be those having (or suffering from or experiencing) a respiratory complication or condition or disorder, or those at risk of having a respiratory complication or condition or disorder, or at risk of developing a respiratory complication or condition or disorder.

[0173] In some embodiments of uses and methods for treating, preventing or reducing (e.g. reducing the severity of) a respiratory complication (or respiratory condition or respiratory disorder) of the present invention, subjects or patients may be those having (or suffering from or experiencing) a respiratory complication or condition or disorder, or those at risk of having a respiratory complication or condition or disorder, or at risk of developing a respiratory complication or condition or disorder.

[0174] In some embodiments, subjects or patients may be those at risk of (at particular risk of) death. Risk of death may be as determined by a clinical assessment of the subject. Such subjects are those at risk of infant death or neonatal death. Preferably, such subjects may be those at risk of neonatal death. In some embodiments, subjects or patients may be those at risk of death within the first 6 months, the first 5 months, the first 4 months, the first 3 months, the first 2 months or the first 1 month after birth. In some embodiments, subjects or patients may be those at risk of death within the first 24 weeks, 20 weeks, 16 weeks, 12 weeks, 8 weeks, 4 weeks, 3 weeks, 2 weeks or 1 week after birth. In some embodiments, subjects or patients may be those at risk of death within the first 14 days after birth or within the first 28 days after birth.

[0175] In some embodiments of uses and methods for reducing risk of mortality of the present invention, subjects or patients may be those at risk of (at particular risk of) death (e.g. as described elsewhere herein).

[0176] In some embodiments of the present invention, the subject does not have, or has not been diagnosed as having, or is not suspected of having, or does not have clinical signs of, a gastrointestinal disease (or disorder or condition). In some embodiments of the present invention, the subject does not have, or has not been diagnosed as having, or is not suspected of having, or does not have clinical signs of, necrotizing enterocolitis (NEC).

[0177] In some embodiments of uses and methods for treating, preventing or reducing (e.g. reducing the severity of) a respiratory complication (or respiratory condition or respiratory disorder) of the present invention, the subject does not have, or has not been diagnosed as having, or is not suspected of having, or does not have clinical signs of, necrotizing enterocolitis (NEC). In some embodiments of uses and methods for reducing risk of mortality (or for reducing risk of death or for improving the chance of survival) of the present invention, the subject does not have, or has not been diagnosed as having, or is not suspected of having, or does not have clinical signs of, necrotizing enterocolitis (NEC).

[0178] NEC (necrotizing enterocolitis) is a medical condition often affecting premature infants. It is characterized by variable damage to the intestinal tract, ranging from mucosal injury to full-thickness necrosis and perforation where portions of the bowel undergo necrosis due to infection and inflammation in the intestine. It occurs postnatally. Initial symptoms include feeding intolerance, increased gastric residuals, abdominal distension and bloody stools.

[0179] In some embodiments, the subjects may be subjects that are having difficulty eating or feeding. In such subjects, administration of Lactobacillus reuteri (or a composition or formulation comprising Lactobacillus reuteri) directly to the stomach may be preferred, e.g. by mouth (orogastric) or nose (nasogastric) tube. Other forms of enteral administration of Lactobacillus reuteri (or a composition or formulation comprising said Lactobacillus reuteri) may alternatively be used as discussed elsewhere herein.

[0180] In some embodiments, the subjects do not have (or do not meet) any one or more of the exclusion criteria as set out in the Example 2 herein.

[0181] Methods and uses of the invention may involve an additional step of identifying a subject in need of treatment (or identifying a subject to whom administration of Lactobacillus reuteri or a composition or formulation comprising Lactobacillus reuteri could be of benefit).

[0182] The present invention also provides a method of treating or preventing or reducing (e.g. reducing the severity of) a respiratory complication in a premature infant subject, said method comprising administering to said subject an effective amount (e.g. a therapeutically or prophylactically effective amount) of a Lactobacillus reuteri strain in accordance with the present invention. Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to this aspect of the invention.

[0183] The present invention also provides a method of treating or preventing or reducing (e.g. reducing the severity of) a respiratory condition (or respiratory disorder or respiratory disease) in a premature infant subject, said method comprising administering to said subject an effective amount (e.g. a therapeutically or prophylactically effective amount) of a Lactobacillus reuteri strain in accordance with the present invention. Typically, the respiratory complication is a respiratory condition (or respiratory disorder or respiratory disease) that is associated with (or characteristic of) premature birth. Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to this aspect of the invention.

[0184] 1 The present invention also provides a method of treating or preventing or reducing (e.g. reducing the severity of) a condition (or disorder or disease) in a premature infant subject, wherein said condition (or disorder or disease) is selected from the group consisting of respiratory distress, respiratory failure, chronic respiratory failure, bronchopulmonary dysplasia, apnea, pulmonary hemorrhage, pulmonary hypertension, pneumonitis, pneumonia, hypoxia, respiratory depression, bradypnea, hypercapnia, respiratory acidosis, pulmonary failure, pneumothorax and pulmonary (interstitial) fibrosis, wherein said method comprises administering to said subject an effective amount (e.g. a therapeutically or prophylactically effective amount) of a Lactobacillus reuteri strain in accordance with the present invention. Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to this aspect of the invention.

[0185] An effective amount can be determined based on the clinical assessment and can be readily monitored. Certain exemplary doses and dosage regimes are described elsewhere herein.

[0186] The present invention also provides the use of a Lactobacillus reuteri strain in accordance with the present invention in the manufacture of a medicament for use in treating or preventing or reducing (e.g. reducing the severity of) a respiratory complication in a premature infant subject. Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to this aspect of the invention.

[0187] The present invention also provides the use of a Lactobacillus reuteri strain in accordance with the present invention in the manufacture of a medicament for use in treating or preventing or reducing (e.g. reducing the severity of) a respiratory condition (or respiratory disorder or respiratory disease) in a premature infant subject. Typically, the respiratory complication is a respiratory condition (or respiratory disorder or respiratory disease) that is associated with (or characteristic of) premature birth. Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to this aspect of the invention.

[0188] The present invention also provides the use of a Lactobacillus reuteri strain in accordance with the present invention in the manufacture of a medicament for use in treating or preventing or reducing (e.g. reducing the severity of) a condition (or disorder or disease) in a premature infant subject, wherein said condition (or disorder or disease) is selected from the group consisting of respiratory distress, respiratory failure, chronic respiratory failure, bronchopulmonary dysplasia, apnea, pulmonary hemorrhage, pulmonary hypertension, pneumonitis, pneumonia, hypoxia, respiratory depression, bradypnea, hypercapnia, respiratory acidosis, pulmonary failure, pneumothorax and pulmonary (interstitial) fibrosis. Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to this aspect of the invention.

[0189] The present invention also provides the use of a Lactobacillus reuteri strain in accordance with the present invention for the treatment or prevention or reduction (e.g. reduction of the severity) of a respiratory complication in a premature infant subject.

[0190] Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to this aspect of the invention.

[0191] The present invention also provides the use of a Lactobacillus reuteri strain in accordance with the present invention for the treatment or prevention or reduction of a respiratory condition (or respiratory disorder or respiratory disease) in a premature infant subject. Typically, the respiratory complication is a respiratory condition (or respiratory disorder or respiratory disease) that is associated with (or characteristic of) premature birth. Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to this aspect of the invention.

[0192] The present invention also provides the use of a Lactobacillus reuteri strain in accordance with the present invention for the treatment or prevention or reduction of (e.g. reduction in the severity of) a condition (or disorder or disease) in a premature infant subject, wherein said condition (or disorder or disease) is selected from the group consisting of respiratory distress, respiratory failure, chronic respiratory failure, bronchopulmonary dysplasia, apnea, pulmonary hemorrhage, pulmonary hypertension, pneumonitis, pneumonia, hypoxia, respiratory depression, bradypnea, hypercapnia, respiratory acidosis, pulmonary failure, pneumothorax and pulmonary (interstitial) fibrosis. Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to this aspect of the invention.

[0193] In another aspect, the present invention provides a Lactobacillus reuteri strain in accordance with the present invention for use in reducing risk of mortality in a premature infant subject. In some embodiments, said use is a use in reducing risk of mortality associated with (or due to or caused by) a respiratory complication (or a respiratory condition, disease or disorder). Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to this aspect of the invention.

[0194] In another aspect, the present invention provides a Lactobacillus reuteri strain in accordance with the present invention for use in reducing risk of death in a premature infant subject. Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to this aspect of the invention. In another aspect, the present invention provides a Lactobacillus reuteri strain in accordance with the present invention for use in preventing death in a premature infant subject. Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to this aspect of the invention.

[0195] In another aspect, the present invention provides the use of a Lactobacillus reuteri strain in accordance with the present invention in the manufacture of a medicament for reducing risk of mortality in a premature infant subject. In some embodiments, said reducing risk of mortality is reducing risk of mortality associated with (or due to or caused by) a respiratory complication (or a respiratory condition, disease or disorder). Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to this aspect of the invention.

[0196] In another aspect, the present invention provides the use of a Lactobacillus reuteri strain in accordance with the present invention in the manufacture of a medicament for reducing risk of death in a premature infant subject. Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to this aspect of the invention.

[0197] In another aspect, the present invention provides the use of a Lactobacillus reuteri strain in accordance with the present invention in the manufacture of a medicament for preventing death in a premature infant subject. Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to this aspect of the invention.

[0198] In another aspect, the present invention provides the use of a Lactobacillus reuteri strain in accordance with the present invention for reducing risk of mortality in a premature infant subject. In some embodiments, said reducing risk of mortality is reducing risk of mortality associated with (or due to or caused by) a respiratory complication (or a respiratory condition, disease or disorder). Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to this aspect of the invention.

[0199] In another aspect, the present invention provides the use of a Lactobacillus reuteri strain in accordance with the present invention for reducing risk of death in a premature infant subject. Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to this aspect of the invention.

[0200] In another aspect, the present invention provides the use of a Lactobacillus reuteri strain in accordance with the present invention for preventing death in a premature infant subject. Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to this aspect of the invention. In another aspect, the present invention provides a method of increasing (or improving) the chance of survival (or survival prospects) for a premature infant subject, said method comprising administering to said subject an effective amount of a Lactobacillus reuteri strain in accordance with the present invention. Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to this aspect of the invention. For example, discussion of various features of other aspects and embodiments of the invention relating to methods of reducing risk of mortality (or death) or methods of preventing death may be applied, mutatis mutandis, to this aspect of the invention.

[0201] In another aspect, the present invention provides a Lactobacillus reuteri strain in accordance with the present invention for use in treating or preventing or reducing (e.g. reducing the severity of) a cardiopulmonary disease in a premature infant subject. In another aspect, the present invention also provides a method of treating or preventing or reducing (e.g. reducing the severity of) a cardiopulmonary disease in a premature infant subject, said method comprises administering to said subject an effective amount of a Lactobacillus reuteri strain in accordance with the present invention. In another aspect, the present invention provides the use of a Lactobacillus reuteri strain in accordance with the present invention in the manufacture of a medicament for use in treating or preventing or reducing (e.g. reducing the severity of) a cardiopulmonary disease in a premature infant subject. Cardiopulmonary diseases include, for example, cardiopulmonary failure, cardiopulmonary arrest and cardiopulmonary insufficiency. Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to aspects of the invention described in this paragraph.

[0202] In another aspect, the present invention provides Lactobacillus reuteri for use in treating or preventing or reducing (e.g. reducing the severity of) a respiratory complication (or condition or disease or disorder) in a premature infant subject. In another aspect, the present invention also provides a method of treating or preventing or reducing (e.g. reducing the severity of) a respiratory complication (or condition or disease or disorder) in a premature infant subject, said method comprises administering to said subject an effective amount of Lactobacillus reuteri. In another aspect, the present invention provides the use of Lactobacillus reuteri in the manufacture of a medicament for use in treating or preventing or reducing (e.g. reducing the severity of) a respiratory complication (or condition or disease or disorder) in a premature infant subject. In another aspect, the present invention provides Lactobacillus reuteri for use in reducing risk of mortality (or for reducing risk of death or for improving the chance of survival) in a premature infant subject. In another aspect, the present invention provides a method of reducing risk of mortality (or for reducing risk of death or for improving the chance of survival) in a premature infant subject, said method comprises administering to said subject an effective amount Lactobacillus reuteri. In another aspect, the present invention provides the use of Lactobacillus reuteri in the manufacture of a medicament for use in reducing risk of mortality (or for reducing risk of death or for improving the chance of survival) in a premature infant subject. In another aspect, the present invention provides Lactobacillus reuteri for use in treating or preventing or reducing (e.g. reducing the severity of) a cardiopulmonary disease in a premature infant subject. In another aspect, the present invention provides a method of treating or preventing or reducing (e.g. reducing the severity of) a cardiopulmonary disease in a premature infant subject, said method comprises administering to said subject an effective amount of Lactobacillus reuteri. In another aspect, the present invention provides the use of Lactobacillus reuteri in the manufacture of a medicament for use in treating or preventing or reducing (e.g. reducing the severity of) a cardiopulmonary disease in a premature infant subject. In some embodiments of aspects of the invention set out in this paragraph, the Lactobacillus reuteri may have the ability to use (or consume) citrate as a nutrient or substrate for growth (have the ability to grow in the presence of citrate), and / or the ability to use citrate as an external electron acceptor in their metabolism (have the ability to metabolize citrate), and / or the ability to use citrate in a fermentation reaction (be citrate fermenting). In some embodiments of aspects of the invention set out in this paragraph, the Lactobacillus reuteri is Lactobacillus reuteri PTA-121141. In some embodiments of aspects of the invention set out in this paragraph, the Lactobacillus reuteri is not Lactobacillus reuteri PTA-121141. Thus, in some embodiments of aspects of the invention set out in this paragraph, the Lactobacillus reuteri may be a Lactobacillus reuteri strain other than Lactobacillus reuteri PTA-121141. Discussion of various features of other aspects of the invention and embodiments thereof may be applied, mutatis mutandis, to aspects of the invention set out in this paragraph.

[0203] Preferably, the Lactobacillus reuteri is an isolated Lactobacillus reuteri strain.

[0204] Reduction or alleviation or treatment or prevention of complications, conditions, disorders, diseases etc. can be measured by any appropriate means (e.g. assay or test), examples of which would be well known to a person skilled in the art. Preferably the reduction is significant, e.g. clinically significant or statistically significant, preferably with a probability value of <0.05. Such reduction is generally determined compared to an appropriate control individual or population, for example a healthy mammal or subject (or a population thereof) or an untreated or placebo treated mammal or subject (or a population thereof), or, if appropriate, the same individual subject before treatment.

[0205] Preferably any increases or improvements described herein (for example increases or improvements in survival chances) are measurable increases, etc., (as appropriate), more preferably they are significant increases, preferably clinically significant or statistically significant increases, for example with a probability value of <0.05, when compared to an appropriate control, e.g. control value or placebo.

[0206] Preferably any of the reductions described herein (for example the reduction in risk of mortality) are measurable reductions, more preferably they are significant reductions, preferably clinically significant or statistically significant reductions, for example with a probability value of <0.05, when compared to an appropriate control, e.g. control value or placebo.

[0207] In a further aspect, the present invention provides kits comprising a Lactobacillus reuteri (e.g. a Lactobacillus reuteri strain in accordance with the present invention), or a formulation (or composition) as defined above for a use or method according to the invention. The kits may comprise further components. Each component may be provided in a separate compartment or vessel. Where convenient and practical, mixtures of components could be provided. The components may be provided in dry, e.g. freeze dried or lyophilised form, or in solution. The kits may also be provided with instructions for using the kit in accordance with the invention or with directions for how such instructions may be obtained.

[0208] Unless otherwise clear from the context, as used throughout the entire application, the terms "a" and "an" are used in the sense that they mean "at least one", "at least a first", "one or more" or "a plurality" of the referenced components or steps, except in instances wherein an upper limit is thereafter specifically stated. The operable limits and parameters of combinations, as with the amounts of any single agent, will be known to those of ordinary skill in the art in light of the present disclosure.

[0209] Where the terms “comprise”, “comprises”, “has” or “having”, or other equivalent terms are used herein, then in some more specific embodiments these terms include the term “consists of” or “consists essentially of”, or other equivalent terms. The invention will now be further described in the following non-limiting Examples.

[0210] EXAMPLE 1 - FORMULATION OF IBP-9414

[0211] Formulation of a suitable product to be used in tube-feeding a premature infant.

[0212] The IBP-9414 powder for oral suspension is a white to off white lyophilised powder provided in a clear glass vial that contains approximately 1 x 109CFU Lactobacillus reuteri ATCC PTA-121141 per dose and other components (lactose monohydrate, hydrolysed gelatin, monosodium glutamate, maltodextrin, ascorbic acid and trisodium citrate dihydrate).

[0213] EXAMPLE 2 - THE CONNECTION STUDY

[0214] The Connection study was a randomized, double blind, parallel-group, placebo-controlled study to evaluate the efficacy and safety of IBP-9414 in premature infants 500-1500g birth weight in the prevention of necrotizing enterocolitis (NEC). It was a multicenter study conducted internationally.

[0215] Study Duration: Screening visit was within 48 hours after birth of the subject. First dose of Investigational Product (IP) was administered <48 hours after birth and continued daily until the subject reached 34 weeks + 6 days Post-Menstrual Age (PMA), or until treatment was permanently discontinued if earlier. A follow-up assessment was made at 40 weeks ± 7 days PMA.

[0216] Study Objectives:

[0217] Primary Objectives

[0218] To evaluate the efficacy of IBP-9414 vs. placebo on the prevention of necrotizing enterocolitis and on sustained feeding tolerance in very low birth weight premature infants, as well as the safety of IBP-9414 vs. placebo. The study was powered on the necrotizing enterocolitis endpoint.

[0219] Secondary Objectives

[0220] To evaluate the effect of IBP-9414 on different severities of NEC, all-cause mortality, duration of hospitalization, growth and feeding tolerance in very low birth weight premature infants.

[0221] Patient Population: Preterm infants with a birth weight of 500-1500g, and a gestational age (GA) 23 weeks+0 days - 32 weeks+0 days at birth, who are <48 hours of age. Inclusion Criteria:

[0222] To be eligible for the study, subjects must fulfill the following criteria:

[0223] • Gestational age at birth of 23 weeks+0 days to 32 weeks+0 days

[0224] • Birth weight 500-1500g

[0225] • < 48 hours of age

[0226] • Written informed consent from the subject's legally authorized representative (LAR)

[0227] Exclusion Criteria:

[0228] Subjects will be excluded if one of the following criteria is fulfilled:

[0229] • Participation in any other interventional clinical trial

[0230] • Infants in extremis to whom no further intensive care is offered by attending neonatologist

[0231] • Infants with, or at a high probability for, early onset sepsis

[0232] • Infants with recognized chromosomal anomalies

[0233] • Congenital or acquired gastrointestinal disease

[0234] • Earlier or planned administration of formulas, foods or supplements that contain added live bacteria

[0235] • Infants with known positive maternal HIV status

[0236] Number of Subjects: 2117 pre-term babies received minimum one dose of either the Active Study Product or placebo and were included in the analysis. 536 male babies received I BP-9414, and 525 male babies received the placebo. 530 female babies received IBP-9414, and 526 female babies received the placebo. The study was powered for the confirmed NEC primary endpoint.

[0237] Active Study Product, Dose, and Mode of Administration:

[0238] IBP-9414 Powder for Oral Suspension was be supplied as a freeze-dried powder in a prefilled, clear, glass 2-ml vial according the formulation described in Example 1. The Active Study Product, dose 1x109CFU, was prepared by the hospital pharmacy or designee by reconstitution in sterile water (dose volume 0.5 ml) and was delivered to the NICU in a capped and labeled masked enteral syringe.

[0239] The Active Study Product was administered within 3 hours of preparation. The 0.5 ml Active Study Product in the enteral syringe was administered enterally through the nasogastric or orogastric tube. If the subject has no nasogastric or orogastric tube, the Active Study Product can be given directly into the subject’s mouth.

[0240] Investigational Product (IP) was given once daily. Reference Therapy, Dose, and Mode of Administration:

[0241] Sterile water (dose volume 0.5 ml) was used as placebo and was prepared by the hospital pharmacy or designee before delivery to the NICU in a capped and labeled masked enteral syringe, identical in appearance to the Active Study Product. The placebo was administered within 3 hours of preparation. The 0.5 ml placebo in the enteral syringe was administered enterally through the nasogastric or orogastric tube. If the subject has no nasogastric or orogastric tube, the placebo can be given directly into the subject’s mouth.

[0242] IP was given once daily.

[0243] Study Endpoints:

[0244] Primary Endpoint

[0245] There were 2 primary endpoints:

[0246] Primary Endpoint 1: Confirmed necrotizing enterocolitis (NEC)

[0247] Primary Endpoint 2: Time to sustained feeding tolerance (SFT)

[0248] Secondary Endpoints

[0249] 1. At least one clinical sign of NEC as reported by the investigator AND abdominal X-ray evidence of intestinal pneumatosis and / or portal venous gas confirmed by independent adjudication.

[0250] 2. Surgery (or autopsy) with confirmation of NEC.

[0251] 3. Death all causes

[0252] 4. Number of days of hospitalization.

[0253] 5. Weight gain in g / kg during weeks 3 and 4 of age.

[0254] 6. Number of subjects growing at >100 g / kg / week measured during weeks 3 and 4 of age.

[0255] 7. Days with clinical signs of feeding intolerance.

[0256] Results:

[0257] Upon completion of the study and subsequent analysis of the data, it was concluded that the study showed positive trends but there were no statistical significance for the two primary endpoints: prevention of necrotizing enterocolitis (NEC), active group: 8.7% vs placebo group: 10.2% (p=0.24) and sustained feeding tolerance (SFT), active group: 16 days vs placebo group: 17 days (p=0.07). However, there was a significant reduction in the secondary endpoint of all-cause deaths, active group: 6.2% vs placebo group: 8.5% (p=0.04), corresponding to a significant risk reduction of 27%, which meant that in reality 23 infants’ lives were spared by the administration of IBP-9414 in the study, see Table 1. A reduction in all-cause deaths in the cohort of subjects treated with IBP-9414 was particularly notable for those subjects born by Caesarean section (data not shown).

[0258] Furthermore, once the inventors analyzed the causes of deaths in more depth, they surprisingly found out that upon administration of IBP-9414 there were fewer cases in which the primary cause of death was respiratory complication associated deaths as compared to placebo, see Table 2. A reduction in the primary cause of death during the first 14 days after birth being respiratory complications was particularly notable (statistically significant with p-values of 0.0145 (unstratified p-value) and 0.0133 (stratified p-value)). Premature infants in the first 14 days after birth are particularly vulnerable, so this particularly notable effect of IBP-9414 in this time-frame is of great benefit. Classification of deaths was done using ICD (international classification of diseases) version 10 which is provided by World Health Organization, https: / / icd.who.int / browse10 / 2019 / en (see in particular Chapter XVI).

[0259] This means that IBP-9414 is useful and effective to prevent or treat or reduce respiratory complications in premature and pre-term infants. The study also provides support to use the IBP-9414 to prevent and reduce premature deaths, especially premature deaths that are caused by a respiratory complication.

[0260] Table 1 - All cause death (Forest Plot)

[0261] IBP-9414 Placebo Relative risk CMH N Events (%) N Events (%) (95 % Cl) p-value

[0262] All-cause death 1066 66 (6.2) 1051 89 (8.5) i - • - n 0.73 (0.54 - 0.98) 0.036

[0263] < - . - 1 - , 0.5 0.75 1 1.25 < - - >

[0264]

[0265] Favours IBP-9414 Favours placebo

[0266] Note: Events = deaths. CMH = Cochran-Mantel-Haenszel Table 2 - Primary cause of death

[0267] IBP-9414 Placebo Total (N=1066) (N=1051) (N=2117) n (%) n (%) n (%) Primary cause of death - total study period

[0268] sepsis 22 (2.06) 24 (2.28) 46 (2.17) respiratory complications 14 (1.3) 25 (2.4) 39 (1.8) GI (NEC) 9 (0.84) 15 (1.43) 24 (1.13) IVH 11 (1.03) 8 (0.76) 19 (0.90) GI (IP) 1 (0.09) 2 (0.19) 3 (0.14) shock hypotension 1 (0.09) 1 (0.10) 2 (0.09) other 8 (0.75) 13 (1.24) 21 (0.99) unknown 0 (0.00) 1 (0.10) 1 (0.05) No death in period 1000 (93.81) 962 (91.53) 1962 (92.68)

[0269] Primary cause of death during first 14 days

[0270] sepsis 18 (1.69) 12 (1.14) 30 (1.42) respiratory complications 5 (0.47) 16 (1.52) 21 (0.99) GI (NEC) 3 (0.28) 3 (0.29) 6 (0.28) IVH 11 (1.03) 6 (0.57) 17 (0.80) GI (IP) 1 (0.09) 1 (0.10) 2 (0.09) shock hypotension 0 (0.00) 1 (0.10) 1 (0.05) other 5 (0.47) 3 (0.29) 8 (0.38) No death in period 1023 (95.97) 1009 (96.00) 2032 (95.98) Note: IVH = intraventricular hemorrhage; GI (IP) = Gastrointestinal (Intestinal Perforation); NEC = necrotizing enterocolitis.

Claims

CLAIMS1. Lactobacillus reuteri for use in treating or preventing or reducing a respiratory complication in a premature infant subject.

2. The Lactobacillus reuteri for use of claim 1 , wherein said Lactobacillus reuteri is Lactobacillus reuteri strain ATCC PTA-121141.

3. The Lactobacillus reuteri for use of claim 1 or claim 2, wherein said premature infant subject is a premature human infant.

4. The Lactobacillus reuteri for use of any one of claims 1 to 3, wherein said premature infant subject has a birthweight of between about 500g and about 1500g.

5. The Lactobacillus reuteri for use of any one of claims 1 to 4, wherein the gestational age of said subject at birth was <32 weeks.

6. The Lactobacillus reuteri for use of any one of claims 1 to 5, wherein said respiratory complication is selected from the group consisting of respiratory distress, respiratory failure, chronic respiratory failure, bronchopulmonary dysplasia, apnea, pulmonary hemorrhage, pulmonary hypertension, pneumonitis, pneumonia, hypoxia, respiratory depression, bradypnea, hypercapnia, respiratory acidosis, pulmonary failure, pneumothorax and pulmonary (interstitial) fibrosis.

7. The Lactobacillus reuteri for use of any one of claims 1 to 6, wherein said Lactobacillus reuteri is administered to said premature infant subject in the form of a pharmaceutical composition.

8. The Lactobacillus reuteri for use of any one of claims 1 to 7, wherein said treating or preventing or reducing of a respiratory complication comprises administering to said premature infant subject a pharmaceutical composition comprising (i) said Lactobacillus reuteri and (ii) citrate.

9. The Lactobacillus reuteri for use of any one of claims 1 to 8, wherein said Lactobacillus reuteri is reconstituted in water before administration to said premature infant subject.

10. The Lactobacillus reuteri for use of any one of claims 1 to 9, wherein 1x108to 1x1011CFUs (colony forming units) of said Lactobacillus reuteri are administered to said subject per day.

11. The Lactobacillus reuteri for use of any one of claims 1 to 10, wherein one dose per day of said Lactobacillus reuteri is administered to said premature infant subject.

12. The Lactobacillus reuteri for use of claim 11 , wherein said one dose contains 1x108to 1x1011CFUs (colony forming units) of said Lactobacillus reuteri.

13. The Lactobacillus reuteri for use of any one of claims 1 to 12, wherein a first dose of said Lactobacillus reuteri is administered to said premature infant subject < 48 hours after birth.

14. A method of treating or preventing or reducing a respiratory complication in a premature infant subject, said method comprising administering to said subject an effective amount of Lactobacillus reuteri.

15. The method of claim 14, wherein said Lactobacillus reuteri is Lactobacillus reuteri strain ATCC PTA-121141.

16. Use of Lactobacillus reuteri in the manufacture of a medicament for use in treating or preventing or reducing a respiratory complication in a premature infant.

17. The use of claim 16, wherein said Lactobacillus reuteri is Lactobacillus reuteri strain ATCC PTA-121141.

18. The method of claim 14 or claim 15 or the use of claim 16 or claim 17, wherein said method or said use has any one or more features of any one or more of claims 2 to 13.

19. A method of reducing risk of mortality in a premature infant subject, said method comprising administering to said subject an effective amount of Lactobacillus reuteri.

20. The method of claim 19, wherein said Lactobacillus reuteri is Lactobacillus reuteri strain ATCC PTA-121141.

21. The method of claim 19 or claim 20, wherein said method is a method of reducing risk of mortality caused by a respiratory complication.

22. The method of claim 19 or claim 20, wherein said method has any one or more features of any one or more of claims 2 to 5 or 7 to 13.

23. The method of claim 21 , wherein said method has any one or more features of any one or more of claims 2 to 13.

24. Lactobacillus reuteri for use in reducing risk of mortality in a premature infant subject.

25. The Lactobacillus reuteri for use of claim 24, wherein said Lactobacillus reuteri is Lactobacillus reuteri strain ATCC PTA-121141.

26. The Lactobacillus reuteri for use of claim 24 or claim 25, wherein said use in reducing risk of mortality is a use in reducing risk of mortality caused by a respiratory complication.

27. The Lactobacillus reuteri for use of claim 24 or claim 25, wherein said use has any one or more features of any one or more of claims 2 to 5 or 7 to 13.

28. The Lactobacillus reuteri for use of claim 26, wherein said use has any one or more features of any one or more of claims 2 to 13.

28. Use of Lactobacillus reuteri in the manufacture of a medicament for reducing risk of mortality in a premature infant subject.

29. The use of claim 28, wherein said Lactobacillus reuteri is Lactobacillus reuteri strain ATCC PTA-121141.

30. The use of claim 24 or claim 25, wherein said reducing risk of mortality is reducing risk of mortality caused by a respiratory complication.

31. The use of claim 28 or claim 29, wherein said use has any one or more features of any one or more of claims 2 to 5 or 7 to 13.

32. The use of claim 30, wherein said use has any one or more features of any one or more of claims 2 to 13.

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