Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/745—Bifidobacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to at least one strain of bacteria belonging to the species Lactobacillus paracasei , preferably Lactobacillus paracasei DG ® CNCM 1-1572 and/or Lactobacillus paracasei LPC-S01 DSM 26760, and compositions thereof for use in newborn subjects in the first 4 weeks of life in the treatment, preventive and/or curative, of gastro-intestinal tract disorders of an inflammatory and/or functional nature, preferably neonatal colic or chronic enterocolitis in preterm newborns (e.g. necrotizing enterocolitis), in the treatment, preventive and/or curative, of gastro-intestinal infections by pathogenic microorganisms (e.g. viruses or bacteria), gastro-intestinal infections by parasites, allergies, immune- mediated or autoimmune disorders, and in the supportive treatment of the subject's growth.
• pathogenic microorganisms e.g. viruses or bacteria
• Microbial colonisation of the newborn intestine begins immediately after birth and is essential for the development of the mucosal barrier function, intestinal homeostasis and the maturation of the immune system.
• the term 'newborn' or 'newborn subject' refers to a mammal (human or animal subject) in the period between the moment of birth and the first 4 weeks (28 days) of life.
• the composition of the gut microbiota In the first days of a baby's life, numerous factors influence the composition of the gut microbiota: the mother's vaginal and/or skin microbiota, vaginal or caesarean delivery, breast milk (breastfeeding) or artificial milk feeding, and the administration of antibiotics and/or other drugs.
• caesarean section, artificial milk, preterm delivery and the use of antibiotics reduce the abundance and diversity of beneficial bacterial species in the microbiota, promoting a state of dysbiosis. This in turn may increase the risk of the onset of intestinal colic, necrotic enterocolitis in preterm newborns and the development of immune-mediated diseases later in life, such as allergies or inflammatory or functional bowel disease, and/or related symptoms.
• Newborns are not physiologically comparable to 'fragile' adults, nor even to very young children, because in the first weeks of life the maturation of internal organs is not complete and their consequent functionality is not comparable to that of a child or an adult, however debilitated or ill they may be.
• 'Gut microbiota' refers to all microorganisms (all bacteria, archaea, eukaryotes, and viruses) present in the gastro-enteric (or gastro-intestinal) environment.
• the microbiota performs many useful activities for the host organism. Indeed, the microbes that compose it degrade polysaccharides, aiding digestive functions, synthesize vitamins, hinder colonisation by pathogenic species, provide signals necessary for intestinal development, and contribute to modulate inflammatory and immune responses.
• the aetiology of many gastro-enteric disorders, as well as those affecting other systems, is believed to be an aberrant immunological response to gut microbes.
• the gut microbiota constitutes, together with the metabolites it produces and enteric cells, a barrier, the malfunctioning of which is implicated in the pathophysiology of many immune-mediated disorders as well as infections by pathogenic microorganisms.
• probiotic bacterial strains i.e. live microorganisms that, when administered in adequate amounts, confer health benefits
• probiotic bacterial strains i.e. live microorganisms that, when administered in adequate amounts, confer health benefits
• the beneficial effects of probiotic bacterial strains are therefore mainly attributed to normalising the permeability of the intestinal barrier, regularising the intestinal microbiota and restoring the balance between the microbiota and the immunological response.
• the technical problem which the present invention addresses and solves is to provide an effective solution for the treatment of gastro-intestinal diseases or symptoms of an inflammatory or functional nature (for example: intestinal colic, chronic enterocolitis) and gastro-intestinal infections by pathogenic micro organisms or gastro-intestinal infections by parasites in newborn subjects and/or mammalian subjects in the first months of life (from 1 month to ⁇ 12 months; a month may have 28, or 29, or 30, or 31 days). Furthermore, the present invention addresses and solves the technical problem of preventively treating the onset of allergies and/or immune-mediated or autoimmune disorders related to the gastrointestinal system ⁇ e.g. coeliac disease) in the later years of life of the treated subject. Finally, it is an object of the present invention to provide an effective solution for supporting the body growth of newborns and/or mammalian subjects in the first months of life.
• gastro-intestinal diseases or symptoms of an inflammatory or functional nature for example: intestinal colic, chronic enterocolitis
• the Applicant addresses and solves the aforementioned technical problem by providing at least one strain of bacteria belonging to the species Lactobacillus paracasei, such as Lactobacillus paracasei DG ® CNCM 1-1572 and/or Lactobacillus paracasei LPC-S01 DSM 26760, and compositions thereof (in short, compositions of the invention) that effectively and efficiently contribute to the development of an appropriate and lactobacillus-rich, in particular Lactobacillus paracasei, gut microbiota in the newborn and/or subject in the first months of life.
• Lactobacillus paracasei such as Lactobacillus paracasei DG ® CNCM 1-1572 and/or Lactobacillus paracasei LPC-S01 DSM 26760
• compositions thereof in short, compositions of the invention
• the Lactobacillus paracasei species (such as Lactobacillus paracasei strain DG ® CNCM 1-1572 and/or Lactobacillus paracasei strain LPC-S01 DSM 26760) belongs to the species commonly used as probiotic bacteria strains.
• the L. paracasei strain DG ® CNCM 1-1572 has been extensively studied for its beneficial properties.
• the effects of L. paracasei DG ® CNCM 1-1572 on newborn subjects and/or subjects from 1 month to ⁇ 12 months have never been studied to date.
• L paracasei strain DG ® CNCM 1-1572 and L paracasei strain LPC-S01 DSM 26760, mixtures and compositions thereof according to the present invention administered to subjects in the neonatal age (first 4 weeks of life) appear to be able to effectively and efficiently colonise the intestinal tract during the period of administration and/or the first months of life, leading to a balance between the intestinal microbiota and the immune system beneficial to the host, and to the strengthening of the permeability of the intestinal barrier.
• strains of bacteria, mixtures and compositions thereof of the present invention are well tolerated, have no relevant side effects, are easy to prepare and cost-effective.
• Figure 1 scheme of the clinical study
• Figures 2A to 2E quantification of L. easel DG ® in arm 1 (composition under analysis; Tx) and arm 2 (Placebo) in faecal samples collected during V1-V5 control visits;
• Figures 3A to 3D Histograms of the relative abundance of Lactobacillus spp. at time points of V1-V5 control visits;
• Figure 4 Metabolomic predominance of partial least squares-discriminant analysis (PLS-DA) showing the identified metabolites, including butanoic acid and propanoic acid.
• PLS-DA partial least squares-discriminant analysis
• the strains, mixtures or compositions thereof of the present invention are for use in nweborn subjects and/or subjects from 1 month to ⁇ 12 months in the treatment, preventive and/or curative, of neonatal colic or chronic enterocolitis in preterm newborns or necrotizing enterocolitis.
• viruses such as Rotavirus, Adenovirus entericus, Calicivirus, Astrovirus, influenza virus
• bacteria such as Salmonella, Shigella, Staphylococcus, Campylobacter, Escherichia coir,
• gastro-intestinal infections by parasites preferably caused by helminths or intestinal worms (e.g. Ossiuri, Giardia, etc.).
• allergies immune-mediated or autoimmune disorders (e.g.
• coeliac disease Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes mellitus, systemic lupus erythematosus (lupus), vasculitis, Addison's disease, polymyositis, Sjogren's syndrome, progressive systemic sclerosis, glomerulonephritis (inflammation of the kidneys), infertility, asthma, obesity, type 1 diabetes, atopic dermatitis, multiple sclerosis, cancer, and autism.
• the species Lactobacillus paracasei preferably the Lactobacillus paracasei strain DG ® CNCM 1-1572 and/or the Lactobacillus paracasei strain LPC-S01 DSM 26760, mixtures or compositions thereof as defined hereinafter of the present invention (in short, mixtures or compositions of the invention) for use in newborn subjects (first 4 weeks of life) and/
• Lactobacillus paracasei DG ® registered trademark by SOFAR S.p.A., Italy
• SOFAR S.p.A. National Collection of Cultures of Microorganisms of the Institut Pasteur in Paris under the accession number CNCM 1-1572 on 05 May 1995 by SOFAR S.p.A.
• DG ® or L paracasei DG ® CNCM 1-1572 initially the strain had the name Lactobacillus casei DG ® sub.casei ; it was later reclassified as Lactobacillus paracasei DG ® CNCM 1-1572. It is clarified that it is always and only the same strain of bacteria, regardless of the designation Lactobacillus casei DG ® or Lactobacillus paracasei DG ® .
• Lactobacillus paracasei LPC-S01 The strain of bacteria identified as Lactobacillus paracasei LPC-S01, alternatively named Lactobacillus paracasei S01, was deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbPI (DSMZ) under the accession number DSM 26760 on 20 November 2012 by SOFAR S.p.A. (in short, LPC- S01 or L paracasei LPC-S01 DSM 26760). It is clarified that it is always and only the same strain of bacteria regardless of the designation Lactobacillus paracasei S01 DSM 26760 or Lactobacillus paracasei LPC-S01 DSM 26760 adopted by the Applicant.
• DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbPI
• compositions of the invention for use in the methods or treatment of the invention comprise: (i) said mixture M of the invention comprising or, alternatively, consisting of Lactobacillus paracasei DG ® CNCM I- 1572 and/or Lactobacillus paracasei LPC-S01 DSM 26760; and, optionally, (ii) at least one food grade or pharmaceutical grade additive and/or excipient.
• said mixture M included in the composition of the invention may comprise or, alternatively, consist of:
• Lactobacillus plantarum LpIBSOI DSM 33234 Lactobacillus plantarum LpIBSOI DSM 33234, (e) Bifidobacterium bifidum BbfIBSOI DSM 32708; and a mixture thereof.
• Bifidobacterium breve BbIBSOI The strain of bacteria belonging to the species Bifidobacterium breve identified as Bifidobacterium breve BbIBSOI was deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the deposit number DSM 33231 on 31 July 2019 by SOFAR S.p.A. (in short, BbIBSOI or B. breve BbIBSOI DSM 33231).
• the strain of bacteria belonging to the species Bifidobacterium breve identified as Bifidobacterium breve BblBS02 was deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the deposit number DSM 33232 on 31 July 2019 by SOFAR S.p.A. (in short, BblBS02 or B. breve BbIBSOI DSM 33232).
• the strain of bacteria belonging to the species Bifidobacterium animalis identified as Bifidobacterium animalis subsp. lactis BIIBS01 was deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the deposit number DSM 33233 on 31 July 2019 by SOFAR S.p.A. (in short, BIIBS01 or B. animalis subsp. lactis BIIBS01 DSM 33233).
• Lactobacillus plantarum LpIBSOI The strain of bacteria belonging to the species Lactobacillus plantarum identified as Lactobacillus plantarum LpIBSOI was deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the deposit number DSM 33234 on 31 July 2019 by SOFAR S.p.A. (in short, LpIBSOI or L. plantarum LpIBSOI).
• the strain of bacteria belonging to the species Bifidobacterium bifidum identified as Bifidobacterium bifidum MIMBb23sg BbfIBSOI, or a derivative thereof, wherein said strain of bacteria was deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the deposit number DSM 32708 on 04 December 2017 by Sofar S.p.A. (in short, BbfIBSOI or Bifidobacterium bifidum BbfIBSOI DSM 32708).
• DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
• said strains of bacteria included in the composition of the invention are viable bacterial strains (probiotics).
• said bacterial strains of the invention may be a derivative of the viable strain as defined in the present invention.
• the term "derivative" of the bacterial strain is understood to mean a postbiotic or parabiotic, such as, for example, the tyndallized, sonicated, inactivated by radiation (preferably gamma radiation) bacterial strain, the lysates or homogenates of the bacterial strain, the extracts or wall fraction of the bacterial strain, the metabolites or metabolic bioproducts or exopolysaccharides (EPS) generated by the bacterial strain and/or any other derivative product of the bacterial strain known to the skilled person in the art. Said derivatives are obtained according to methodologies known to the skilled person in the art.
• the term "derivative" of the bacterial strain is meant the tyndallized, sonicated, radiation-inactivated (preferably gamma radiation) strain, the lysates or homogenates of the bacterial strain, the extracts or parietal fraction of the bacterial strain; more preferably the tyndallized strain.
• mixtures of bacterial strains (En) included in the mixture M optionally together with at least one probiotic selected from group (II) and/or an additional active component selected from group (III), wherein said mixture M is included in the composition of the invention, are given below:
• E2 DG ® and at least one strain selected from group (I);
• E3 LPC-S01 and at least one strain selected from group (I);
• E4 DG ® and/or LPC-S01, and BbfIBSOI and at least one strain selected from group (I);
• E5 DG ® and/or LPC-S01, and a mixture of BbIBSOI, BblBS02, BIIBS01 and LpIBSOI;
• E6 DG ® and/or LPC-S01, BbfIBSOI and a mixture of BbIBSOI, BblBS02, BIIBS01 and LpIBSOI
• the mixture M included in the composition of the invention in addition to the strain L. paracasei DG ® CNCM 1-1572 and/or L. paracasei LPC-S01 DSM 26760 and optionally at least one or more strains of bacteria selected from group (I) and/or at least one further active component selected from group (III) described below, may further comprise at least one prebiotic, preferably selected from group (II) consisting of: inulin, fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS), xylitol-oligosaccharide (XOS), guar gum, lactoferrin, and a mixture thereof; preferably inulin.
• group (II) consisting of: inulin, fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS), xylitol-oligosaccharide (XOS), guar gum, lactoferrin, and a mixture thereof; preferably in
• mixture M included in the composition of the invention comprises or, alternatively, consists of L. paracasei DG ® CNCM 1-1572 and/or L. paracasei LPC-S01 DSM 26760 and inulin; preferably L. paracasei DG ® CNCM 1-1572 and inulin.
• Mixture M included in the composition of the invention, in addition to the L. paracasei strain DG ® CNCM I- 1572 and/or L. paracasei LPC-S01 DSM 26760 and optionally at least one or more bacterial strains selected from group (I) and/or at least one prebiotic selected from group (II), may further comprise at least one further active component selected from group (III) consisting of:
• antioxidant substances such as glutathione, polyphenols such as resveratrol and trans-resveratrol, coenzyme Q10, astaxanthin, lycopene;
• - minerals or salts thereof e.g. zinc, selenium, magnesium, potassium;
• omega 9 monounsaturated fatty acids, such as omega 9
• polyunsaturated fatty acids such as omega 3 and omega 6
• mixture M included in the composition of the invention comprises or, alternatively, consists of L. paracasei DG ® CNCM 1-1572 and/or L. paracasei LPC-S01 DSM 26760 and group B vitamins and vitamin D; preferably L. paracasei DG ® CNCM 1-1572 and group B vitamins and vitamin D.
• food grade or pharmaceutical grade acceptable "additives and/or excipients”, optionally included in the composition of the invention together with said strains of bacteria comprise ail auxiliary substances known to the person skilled in the art for the preparation of compositions in solid form, semi-solid or liquid form, such as, for example, carriers, diluents, solvents, solubilisers, acid!fiers, thickeners, sweeteners, flavourings, colourings, sweeteners, lubricants, surfactants, preservatives, stabilisers, pH stabilising buffers, and mixtures thereof.
• ail auxiliary substances known to the person skilled in the art for the preparation of compositions in solid form, semi-solid or liquid form, such as, for example, carriers, diluents, solvents, solubilisers, acid!fiers, thickeners, sweeteners, flavourings, colourings, sweeteners, lubricants, surfactants, preservatives, stabilisers, pH stabilising buffers, and mixtures thereof
• Said mixtures M or compositions of the invention may be a pharmaceutical composition (or Live Biotherapeutic Products), a composition for a medical device, a composition for a food supplement, a food (or novel food or food for special medical purposes (FSMP)), a composition for a food supplement or food.
• a pharmaceutical composition or Live Biotherapeutic Products
• a composition for a medical device a composition for a food supplement
• a food or novel food or food for special medical purposes (FSMP)
• FSMP novel food or food for special medical purposes
• the bacterial strains, mixtures and compositions thereof of the present invention may be formulated for oral or nasal administration, preferably for oral use, in a solid form or, alternatively, in a liquid form, e.g. in the form aqueous or oil (e.g., various seed oils or sunflower seed oil) based drops.
• aqueous or oil e.g., various seed oils or sunflower seed oil
• said at least one bacterial strain or each bacterial strain is present in the mixture M or in the composition of the invention, with respect to a daily dose of the M-mixture or composition of the invention, in a concentration in the range from 10x10 6 CFU to 10x10 12 CFU, preferably from 10x10 8 CFU to 10x10 10 CFU, more preferably in a concentration of about 10x10 8 CFU or 10x10 9 CFU (CFU: Colony Forming Unit).
• a daily dose of a mixture or composition of the invention comprises: >_1x10 9 live bacteria of L. paracasei DG ® CNCM 1-1572, and seed oil.
• the above daily dosages may be administered to the subject in need thereof in a individual dose (single dose) or in repeated doses, e.g. two, three or four daily doses.
• a individual dose single dose
• repeated doses e.g. two, three or four daily doses.
• 15-5 drops oil-based
• of the composition of the invention twice a day equal to about 2 billion of strains of bacteria, preferably L. paracasei DG ® CNCM 1-1572.
• compositions or mixtures M of the present invention In order to assess the number of live bacterial strains in the compositions or mixtures M of the present invention, these compositions or mixtures M can be analysed by plate count method to determine the CFU value.
• the strain(s) of bacteria, the prebiotics and/or further (optional) active components included in the mixture (M) of the invention may also be administered separately (preferably in a time interval from 30 minutes to 60 minutes) and in any order but, preferably, are administered to a subject at the same time, even more preferably in a single composition to achieve a faster effect and for ease of administration.
• said strains and (optional) active components are administered in a single composition, said single composition corresponds to the composition of the present invention.
• composition or mixture or other comprising a component in an amount "within a range from x to y" means that said component may be present in the composition or other in all amounts present in said range, even if not explicitly stated, extremes of the range included.
• a statement that a composition 'comprises' one or more components or substances means that other components or substances may be present in addition to that, or those, specifically indicated.
• Method of treatment in the context of the present invention means an intervention on a subject in need thereof, comprising the administration of the strain of bacteria or a composition of the invention to the subject in a therapeutically effective amount, having as its purpose the elimination, reduction/decrease or prevention of a pathology or disease and related symptoms or disorders.
• subject(s) in the context of the present invention refers to mammals (animals and humans), preferably human subjects of neonatal age (from 0 to 4 weeks of age) and/or from 1 month to ⁇ 12 months of life (from the beginning of the 2nd month until before the end of the 12th month).
• subjects treated or administered with the strains or mixtures or compositions of the invention may be newborn subjects or from 1 month to ⁇ 12 months of life born by caesarean section and/or born preterm.
• 'therapeutically effective amount refers to the amount of active compound and/or strain of bacteria that elicits the biological or medicinal response in a tissue, system, mammal or human being that is sought and defined by an individual, researcher, veterinarian, physician or other clinician or health care worker.
• Preferred embodiments FRn of the present invention are as follows.
• a mixture M comprising or, alternatively, consisting of at least one strain belonging to the species Lactobacillus paracaser, and
• strain for use or the composition for use according to FR1 wherein said strain of bacteria is selected from Lactobacillus paracasei DG® CNCM 1-1572, Lactobacillus paracasei LPC-S01 DSM 26760, and a mixture thereof; wherein said composition comprises:
• a mixture M comprising or, alternatively, consisting of: Lactobacillus paracasei DG® CNCM 1-1572 and/or Lactobacillus paracasei LPC-S01 DSM 26760; and
• FR4 The strain for use or the composition for use according to FR1 or FR2, wherein said strain or composition is for use in a newborn subject and/or a subject from 1 month to ⁇ 12 months in the treatment, preventive and/or curative, of gastro-intestinal infections by pathogenic micro-organisms or gastro intestinal infections by parasites.
• FR5. The strain for use or the composition for use according to FR1 or FR2, wherein said strain or composition is for use in a newborn subject and/or a subject from 1 month to ⁇ 12 months in the treatment, preventive and/or curative, of allergies.
• FR6 The strain for use or the composition for use according to FR1 or FR2, wherein said strain or composition is for use in a newborn subject and/or a subject from 1 month to ⁇ 12 months in the treatment, preventive and/or curative, of immune-mediated, autoimmune disorders, such as coeliac disease Graves' disease, rheumatoid arthritis, Flashimoto's thyroiditis, type 1 diabetes mellitus, systemic lupus erythematosus (lupus), vasculitis, Addison's disease, polymyositis, Sjogren's syndrome, progressive systemic sclerosis, glomerulonephritis (inflammation of the kidneys) and infertility.
• autoimmune disorders such as coeliac disease Graves' disease, rheumatoid arthritis, Flashimoto's thyroiditis, type 1 diabetes mellitus, systemic lupus erythematosus (lupus), vasculitis, Addison'
• FR7 The strain for use or the composition for use according to FR1 or FR2, wherein said strain or composition is for use in a newborn subject and/or a subject from 1 month to ⁇ 12 months in the treatment, preventive and/or curative, of supporting the body weight gain of the subject.
• antioxidant substances such as glutathione, polyphenols such as resveratrol and trans-resveratrol, coenzyme Q10, astaxanthin, lycopene;
• - minerals or salts thereof e.g. zinc, selenium, magnesium, potassium; - monounsaturated fatty acids, such as omega 9, and/or polyunsaturated fatty acids, such as omega 3 and omega 6;
• prebiotic preferably selected from group (II) consisting of: inulin, fructo-oligosaccharide (FOS), galacto-oligosaccharide (GOS), xylitol-oligosaccharide (XOS), guar gum, lactoferrin, and a mixture thereof; more preferably inulin; and and a mixture thereof
• FR10 The strain per use or the composition per use according to any one of FR1-9, wherein said strains are viable bacterial strains or derivatives of bacterial strains, wherein said derivatives of bacterial strains are selected from: tyndallized, sonicated, radiation-inactivated (preferably gamma radiation) bacterial strain, lysate or homogenised bacterial strain, extract or wall fraction of bacterial strain; preferably tyndallized bacterial strain.
• the Applicant carried out a clinical study in newborns in order to evaluate the effects on the gut microbiota of the host in the first 3 months of life following the administration of the Lactobacillus paracasei DG strain ® CNCM 1-1572 probiotic (viable cells).
• compositions according to the invention in drops form (oil-based) for oral administration comprising the L. paracasei strain CNCM 1-1572 (in short, composition under analysis) for 28 days changes the composition of the faecal gut microbiota in terms of the concentration of Lactobacillus paracasei CNCM 1-1572 in the study population.
• Sunflower seed oil Heliantus annuus L., DL-alpha-tocopherol, Lactobacillus paracasei CNCM 1-1572 (not less than 14 billion of live cells per 8 ml).
• the 60 subjects enrolled were randomised in a 1 :1 ratio into each of the three groups in two arms:
• Test composition in drops form, 9 drops twice a day, equal to 2 billion CFU (colony-forming units) of L. paracasei DG ® (CNCM 1-1572) for 4 weeks (28 days).
• a 12-week period allows to evaluate the changes in the gut microbiota based on the persistence of the L. casei DG strain ® ( Lactobacillus paracasei CNCM 1-1572) determined by the intake of the composition under analysis.
• the 12-week period is divided into 4 weeks of treatment and 8 weeks of follow-up. Including an enrolment period of approximately 24 weeks, the total duration of the study will be 36 weeks.
• ELBW Extremely low birth weight
• the newborn will be considered eligible for the Per Protocol population if he/she has taken at least 80% and no more than 120% of the prescribed treatment.
• V1 day 1 (0-48 hours of life); V2 day 10 (+/- 2 days); V3 day 28 (+/- 3 days); V4 day 56 (+/- 3 days); V5 day 84 (+/- 3 days) ( Figure 1).
• microbiota was analysed through nucleotide sequence analysis of portions of the gene coding for the bacterial ribosomal subunit 16S rRNA. Specifically, a metagenomic strategy consisting of the following steps will be adopted:
• VOCs volatile organic compounds
• GC-MS/SPME Gas Chromatography coupled to Mass Spectrometry by Solid Phase Micro Extraction
• CAR-PDMS carboxy-polydimethylsiloxane coated fibre (85 pm) was used in the SPME process. From each sample, analysed in triplicate, an average of 100-500 mg was placed in 10 ml glass vials with the addition of 4-methyl-2-pentanol as an internal standard (IS). Subsequently, faecal samples were equilibrated for 10 minutes at 45 °C.
• volatilome i.e. volatile faecal metabolites (chemical categories: short chain fatty acids (SCFA), alcohols, ketones, aldehydes, thiols, acids, esters, pyrazines, pyridines, phenols, furans, terpenes, alkanes, alkenes, etc.), by assessing the change in the overall metabolic profile of the microbiota at day 28, 56, 84;
• SCFA short chain fatty acids
• Categorical data are represented in terms of counts and percentages, while continuous data as averages and standard deviations or medians and ranges.
• Comparisons of categorical data are made by the Chi-square test or Fisher's exact test, as appropriate. Continuous data are compared by Student's t-test (for independent or dependent data), Anova and Anova for repeated measures, where the appropriate statistical tests determined the normality of the data; in the case of data not normally distributed, non-parametric tests such as sum rank test (Mann Whitney Rank sum test) or sum signed rank test (Wilkoxon Signed Rank), Kruskal Wallis test or Friedman's test are used. Multivariate regression techniques are used if indicated to assess the simultaneous effect of several independent variables on a response variable, or to identify possible confounders and/or analyses for temporal data. Results are considered statistically significant with a P-value ⁇ 0.05.
• Such diagrams show the concentrations of L. casei DG ® in the placebo group and in the 'Tc' group to which the composition under analysis was administered, for each follow-up point. For each group, the median, 25th percentile, 75th percentile, minimum and maximum value of the L casei DG ® concentration (CFU/ml) are shown.
• the probiotic L. casei DG ® detected in the intestines of subjects in the treatment group starting 10 days after the first administration, was persistent up to 84 days after intake, with a slight decrease starting at point V3 of the follow-up (56th day of treatment).
• Figures 3A to 3D show histograms of the relative abundance of Lactobacillus spp. at the time points of control visits V2-V5.
• Tx group causes a statistically significant increase in the relative abundance of the genus Lactobacillus spp. up to ten days (point V2) after the start of administration (pFDR ⁇ 0.05) and that this increase continues to have a trend with p ⁇ 0.05 at time points V4 and V5.
• Figure 4 shows a diagram of the results of the PLS-DA metabolomic analysis: the vertical axis lists the identified metabolites, and the horizontal axis shows the variable importance in projection (VIP).
• PLS-DA analysis identified butanoic acid and propanoic acid metabolites (SCFAs) as the main players in the clustering between the placebo group and the Tx group with an increase in the concentration of said metabolites in the Tx samples.
• SCFAs butanoic acid and propanoic acid metabolites
• SCFA short chain fatty acids
• the number of evacuations observed at each study visit was substantially higher in subjects treated with the composition under analysis than in subjects treated with placebo.
• composition under analysis proved to be safe and well tolerated with a safety profile comparable to that of placebo.
• the aim of the present double-blind, placebo-controlled study was to confirm the ability of a paediatric formulation of LCDG to pass alive through the gastrointestinal tract of newborns (stratified into three groups according to birth weight: Normal birth weight, Low birth weight, Very low birth weight) during and after the administration period, by assessing its ability to positively influence the microbiota composition.
• the safety of the product also monitoring weight, length and head circumference parameters, defecation frequency, stool consistency and occurence of colic were also evaluated.
• Lactobacillus paracasei (L casei DG® - CNCM 1-1572; LCDG) was supplied by SOFAR S.p.A.
• short bowel syndrome; bowel obstruction; ductus arteriosus patency may be included if asymptomatic and does not require treatment), known severe primary or secondary maternal immunodeficiency, known maternal food allergies, maternal diabetes (including gestational diabetes), recent or suspected history of alcohol or drug abuse by the mother, any severe condition that the investigator believes may interfere with treatment, inadequate reliability or presence of conditions that could result in non-compliance or non-observance of the protocol by the patient.
• newborns were prohibited from receiving probiotics other than the trial product for the entire duration of the study, and breastfeeding mothers were prohibited from receiving any probiotic for the entire duration of the study.
• Each parent/guardian of the newborn signed an informed consent to participate in the study and received an explanatory sheet with the conditions of use of the trial product. Based on the inclusion/exclusion criteria, 60 preterm and term newborns were selected.
• Visit 1 Visit 1; 0-48 hours after birth
• Visit 2 Visit 2;10 days +/- 2 days
• Visit 3 Visit 3; 28 days +/- 3 days; end of product intake
• Visit 4 Visit 4; 56 days +/- 3 days
• Visit 5 Visit 5; 84 days +/- 3; end of study.
• birth weight >2500 g, NBW
• low birth weight (1500-2500 g LBW)
• very low birth weight 1000-1500 g, VLBW.
• Eligible patients thus stratified will be randomised at a ratio of 1 :1 into one of the following arms: Arm 1: Treatment group: 9 drops twice a day of LCDG corresponding to 2 billion CFU (colony-forming units) for 4 weeks (28 days) starting at 48 hours of life; Arm 2: Placebo group: 9 drops twice a day of placebo (product indistinguishable from the test product) for 4 weeks (28 days) starting at 48 hours of life.
• the primary outcome was the composition of the faecal gut microbiota in terms of bacterial genome counts for LCDG at day 28 th (V3) of treatment by Real Time PCR.
• the secondary outcomes assessed during the study were: modification of the functional activity of the faecal gut microbiota (metabolomics analysis - a- and b-diversity) with prediction of the microbiota ecology at 28, 56 and 84 days (V3,V4 and V5 respectively) after the start of administration; change in the composition (real-time PCR) of the faecal gut microbiota with prediction of the ecology of the microbiota at 56 and 84 days (V4 and V5 respectively) from the start of administration; change in stool quality and amount (frequency and consistency); occurrence of neonatal colic, defined according to Rome IV criteria [Castelluzzo]; safety and tolerability of the product; patient independence.
• Real-time PCR analyses were carried out to assess the bacterial load of the L. casei DG ® strain (according to an already validated and published method; Ferrario) and metagenomic and metabolomic analyses were performed, characterising the gut microbiota before and after administration of the probiotic.
• the microbiota was assessed through nucleotide sequence analysis of portions of the gene coding for the bacterial ribosomal subunit 16S rRNA. Metagenomic DNA was extracted from stool samples, quantified and normalised. The V3-V4 hypervariable regions of the bacterial gene coding for 16S rRNA were amplified by PCR and the PCR products were quantified and sequenced using the lllumina MiSeq technique.
• Bioinformatic sequence analysis was performed for microbial community characterisation, hierarchical clustering, taxonomic analysis and construction of phylogenetic dendrograms with heatmaps.
• VOCs volatile organic compounds
• metabolites of microbial origin produced as volatile organic compounds (VOCs - chemical categories: short-chain fatty acids, alcohols, ketones, aldehydes, thiols, acids, esters, pyrazine, pyridine, phenols, furans, terpenes, alkanes, alkenes, etc.) were identified and quantified.
• VOCs volatile organic compounds
• These compounds were extracted with a GC-MS/SPME (Gas Chromatography coupled to Mass Spectrometry by Solid Phase Micro-Extraction) system.
• a carboxyl-polydimethylsiloxane (CAR- PDMS) coated fibre 85 pm was used in the SPME process.
• the scale classifies the amount of stools into four classes: 1 : smear, 2: up to 25%, 3: 25-50% and 4:>50%.
• the stool consistency is classified as: A: watery, B: soft, C: formed and D: hard.
• the diagnosis of infant colic must be based on the presence of all the following criteria: The child is less than 5 months old at symptom onset and resolution; recurrent periods of prolonged crying, agitation or irritability that occur without apparent cause and that the parent is unable to prevent or resolve; no evidence of growth deficit, fever or illness.
• the term 'agitation' refers to intermittent vocalisation and is defined as '[behaviour] that is not exactly crying, but neither is that of someone who is awake and happy'. Children often alternate between crying and agitation, making it difficult to distinguish the two symptoms.
• the diagnosis of infant colic must include the previous criteria, together with both of the following: During a telephone call or one-on-one interview with a researcher or physician, parents report a period of crying or agitation lasting at least 3 hours per day for at least 3 days per week; the duration of crying and agitation, reported as continuous for 24 hours in a selected group of children, confirmed as lasting at least 3 hours, is prospectively quantified through the completion of a daily diary.
• L casei DG ® Lactobacillus paracasei CNCMI-1572; LCDG
• LCDG Lactobacillus paracasei CNCMI-1572
• L. salivaris which, in an identical study, showed a difference after 28 days [Putignani et al, in submission ] of 2.36 Log10/200mg stool copies (from 0.04 with SD 0.4 to 2.5 with SD 2.5).
• NBW, LBW, VLBW recognising the intake in the treatment group of 2 billion CFU/day, a sample of 9 patients in each treatment group would give an 80% power to the study, with a two-tailed t-test and 95% significance level in each stratum.
• Categorical data were represented as counts and percentages, while continuous data were expressed in terms of averages and standard deviations or medians and ranges.
• Comparisons of categorical data were performed using the Chi-square test or Fisher's exact test, as appropriate. Continuous data were compared using Student's t-test (for independent or dependent data), Anova's test and Anova for repeated measures, where the appropriate statistical tests determined the normality of the data; in the case of non-normal distribution, non-parametric tests such as Mann Whitney Rank sum test or Wilkoxon Signed Rank test, Kruskal Wallis or Friedman tests were applied. Multivariate regression methods were used if indicated to assess the simultaneous effect of a set of independent variables on a response variable, or to identify potential confounders and/or analyses for temporal data. The results obtained were considered statistically significant with a P-value ⁇ 0.05. All analyses were performed with Stata 15 software (StatCorp).
• L case/ DG L case/ DG was detected in the gut from 10 days after the first administration and remained persistent until the last visit (84 days).
• a multivariate analysis was conducted using a supervised approach, that is partial least squares discriminant analysis (PLS-DA), for each time point.
• PLS-DA partial least squares discriminant analysis
• LCDG had an impact on both the ecology and function of the microbiota, modifying the ecological niches of bacteria residing in the gut and the corresponding functional profile.
• Lactobacillus paracasei (CFU/mL) correlated positively (p ⁇ 0.05) with the metabolites cyclohexanone, n-decanoic acid and 3,4-dimethyl heptane and with the OTUs Lactobacillus, Granulicatella.
• SCFAs including butanoic acid, which shows significant correlations (p ⁇ 0.05) with Ruminococcus, Prevotella, Collinsella, Faecalibactarium and Oscillospira.
• Pentanoic acid shows significant correlations (p ⁇ 0.05) with Streptococcus, Granulicatella and Enterococcus, while propionic acid has significant correlations (p ⁇ 0.05) with Eggerthella, Bacteroides and Bifidobacterium.
• Lactobacillus paracasei (CFU/mL) was positively correlated (p ⁇ 0.05) with 2,3-dimethyl pentanal, 1-hexanol, decanal and dodecanoic acid (Figure 10A), whereas no significant correlations were found between Lactobacillus paracasei (CFU/mL) and the various OTUs ( Figure 10B).
• Figure 10A Lactobacillus paracasei
• Figure 10B Lactobacillus paracasei
• Figure 10B several statistically significant positive correlations (p ⁇ 0.05) between OTUs and metabolites can be noted, in particular, butanoic and propanoic acid show significant correlations (p ⁇ 0.05) with Collinsella, while pentanoic acid has a positive correlation with Propionibacterium, Faecalibacterium, Prevotella and Coprococcus (Figure 10C).
• the investigator recorded an episode of colic if the crying was more than 3 hours for at least 3 days a week together with a state of agitation of at least 3 hours a day.
• the mothers had to fill out a diary in which they had to keep track of the infant's crying if the crying was longer than 3 hours.
• no statistically significant differences were found between the two study groups (placebo and Enterolactis Baby).
• the number of patients who cried more than 3 hours per day and the average duration of crying in each study group were almost similar at each study visit, with a total number of 8 colics occurring during the study (4 in the placebo group and 4 in the LCDG group).
• the 143 and 221 total evacuations recorded for PTs belonging to the placebo and treatment group were classified with colour II
• 7 (4.9%) evacuations for the placebo and no evacuations for the treatment group were classified with colour IV.
• AEs adverse events
• probiotic administration has been correlated with a lower occurrence of necrotising enterocolitis and a reduced mortality rate in preterm newborns [Lundelin, Strunk, Shane, Costeloe], According to these findings, probiotics are characterised by an excellent safety profile. Cases of bacteremia caused by bacterial strains contained in probiotics have been recorded, although they are extremely rare and are determined by the presence of severe immunodeficiency [Lundelin, Salminen, Thomas],
• test product showed to be safe and well-tolerated as placebo in this study.
• Figure 5 Flow chart of the study design. 60 participants were enrolled for the clinical trial and randomised into one of the administration groups in a 1:1 ratio. 20 and 26 completed the study in the placebo and treatment group, respectively. 20 and 26 subjects completed the study in the placebo and treatment group, respectively.
• FIG. 8 LCDG treatment was able to preserve the heterogeneity of the gut microbiome.
• A a diversity analysis between the placebo group and the treatment group at days 10, 28, 56 and 84 (V2, V3, V4 and V5 respectively).
• B /3-diversity analysis. The diversity matrix was obtained using the Unweighted UniFrac algorithm, represented as PCoA at the various time points between the placebo group and the treatment group.
• D Histograms showing the relative abundance of Lactobacillus spp. at all time-points.
• FIG. 9 The administration of LCDG increased the concentration of short-chain fatty acids (SCFAs).
• Metabolite analysis is shown as Partial Least Squares Discriminant Analysis (PLS-DA; left: score graph; right: VIP scores) comparing placebo with treatment at each time point: A) V1; B) V2; C) V3; D) V4; E) V5.
• PLS-DA Partial Least Squares Discriminant Analysis
• FIG. 10 The administration of LCDG had an impact on both the ecology and function of the microbiota, modifying the ecological niches of bacteria residing in the gut and the corresponding functional profile.

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