ES2929638T3 - Use of lactic acid bacteria to treat or prevent gestational diabetes mellitus - Google Patents

Abstract

The present invention provides methods for treating or preventing gestational diabetes by administering Lactobacillus rhamnosus HN001 or derivatives thereof, as well as uses, compositions, and medicaments comprising Lactobacillus rhamnosus HN001 or derivatives thereof for treating or preventing gestational diabetes mellitus. (Automatic translation with Google Translate, without legal value)

Description

DESCRIPTION

Use of lactic acid bacteria to treat or prevent gestational diabetes mellitus

technical field

This invention relates to the use of probiotic bacteria and, in particular, to the use of a strain of lactic acid bacteria for treating or preventing gestational diabetes mellitus (g Dm ).

Background

Lifestyle factors, such as changes in food consumption patterns with economic development, have given rise to the well-recognized and increasing problems of obesity and associated diseases, including gestational diabetes mellitus (GDM). , both in New Zealand (NZ)(1) and in other developed countries(2).

Overweight and obesity before pregnancy have been shown to account for 46% of GDM(3) with excessive weight gain during pregnancy, prior GDM or a family history of diabetes, polycystic ovary syndrome (PCOS), advanced age, and higher parity also identified as risk factors(4). GDM itself increases the risk of preeclampsia, miscarriage, preterm labor, macrosomia, induction of labor, and cesarean section(2, 3). GDM also increases the risk of subsequent maternal and child obesity and subsequent type 2 diabetes mellitus(5).

There is currently no international consensus on the diagnostic criteria for GDM. A diagnostic criterion of the International Association of Diabetes and Pregnancy Study Group (IADPSG) is an oral glucose tolerance test threshold of >5.1 mmol/L (fasting plasma glucose), or a glucose level below at 75 g in 1 hour of >10 mmol/L or at 2 hours >8.5mmol/L. The New Zealand Ministry of Health guideline definitions for GDM specify a higher baseline and 2-hour glucose test threshold (fasting plasma glucose >5.5 mmol/L or 2-hour glucose level after 75 g >9 mmol/L)(1). US 2011/177044 A1 describes a symbiotic composition comprising the probiotic Lactobacillus rhamnosus HN001 and a carbohydrate-based prebiotic such as fructooligosaccharides, galactooligosaccharides, human milk oligosaccharides or combinations thereof and the use of the composition for the prevention and / or the treatment of allergic diseases. WO 2010/064930 A1 describes the use of Lactobacillus rhamnosus HN001 to treat or prevent eczema. WO 99/10476 A1 discusses the immune enhancing effects of ingested Lactobacillus rhamnosus HN001. Bartow et al., BMC Pregnancy Childbirth 2016; 16(1):133 evaluates the effects of Lactobacillus rhamnosus HN001 supplementation in women from early pregnancy and during lactation on childhood eczema and atopic sensitization. Barrett et al, Cochrane Database of Systematic Reviews 2014:1-13 discusses the treatment of gestational diabetes mellitus with Lactobacillus rhamnosus GG. WO 2016/198528 A1 describes oral compositions comprising Lactobacillus probiotic bacteria and their use to normalize plasma glucose concentrations and increase insulin sensitivity in pregnant and/or lactating women.

The current treatment or prevention of GDM is generally based on maintaining a healthy lifestyle in the subject. Prevention of GDM may include maintaining a healthy weight and blood sugar during pregnancy through dietary restrictions and regular exercise. Treatment may also include daily blood glucose tests and insulin injections. Dietary counseling is another method currently used to prevent GDM.

There remains a need for methods and compositions useful for treating or preventing obesity and related diseases, particularly GDM, and particularly methods and compositions using or comprising other lactobacilli. In addition, there is a need for methods and compositions for treating or preventing GDM that do not rely on changes in dietary modification and advice. Methods and compositions for the prevention or amelioration of the risks associated with GDM or the sequelae of GDM are also desirable.

It is an object of this invention to go some way toward fulfilling one or more of these desires or at least to offer the public a useful choice.

Summary of the invention

In a first aspect, the invention provides Lactobacillus rhamnosus HN001, AGAL deposit number NM97/09514 dated August 18, 1997 for use in the treatment or prevention of gestational diabetes mellitus (GDM) or one or more risks associated with GDM or one or more sequelae of GDM in an adult subject, or for use in the treatment or prevention of one or more risks associated with GDM or one or more sequelae of GDM in a fetal, neonatal, infant subject or child, wherein the use for treatment or prevention comprises administration of L. rhamnosus HN001 to the adult subject or to the mother of the fetal, neonate, infant or child subject; and the mean fasting blood glucose level is reduced in the adult subject, or in the mother of a fetal subject, neonate, infant or child to less than approximately 4.35 mmol/l. The invention further provides a composition comprising L. rhamnosus HN001, AGAL deposit number NM97/09514 dated August 18, 1997, for use in the treatment or prevention of GDM or one or more risks associated with the GDM or one or more sequelae of GDM in an adult subject, or for use in the treatment or prevention of one or more risks associated with GDM or one or more sequelae of GDM in a fetal, neonatal, infant or child, wherein the use for treatment or prevention comprises administration of L. rhamnosus HN001 to the adult subject or to the mother of the fetal, neonate, infant or child subject; and the mean fasting blood glucose level is reduced in the adult subject, or in the mother of a fetal, neonate, infant, or child subject to less than about 4.35 mmol/L.

In one embodiment, the L. rhamnosus HN001 is administered in the form of a composition with a physiologically acceptable diluent, adjuvant, carrier, or excipient.

In the present disclosure it is anticipated that HN001 may be the only probiotic bacterium administered.

It is further envisioned herein that HN001 may be administered with one or more prebiotics.

In one embodiment, said physiologically acceptable diluent, adjuvant, carrier or excipient is a food. In one embodiment, the food is fermented milk, yogurt, cheese, dairy drink, or powdered milk.

Alternatively, the composition is a pharmaceutical composition and said excipient or diluent is a pharmaceutically acceptable diluent, adjuvant, carrier or excipient.

In embodiments where the subject is a fetal subject, treating or preventing one or more risks associated with GDM or one or more sequelae of GDM may comprise administering L. rhamnosus HN001 or a composition comprising L. rhamnosus HN001 to the subject's mother fetal. It will be appreciated that in such embodiments, administration to the subject may be considered indirect administration. In one embodiment, the composition is a maternal formula or a maternal supplement.

In certain embodiments where the subject is a neonate, infant, or child subject, treating or preventing one or more risks associated with GDM or one or more sequelae of GDM may comprise administering a composition comprising L. rhamnosus HN001 to the subject. It will be appreciated that in such embodiments, administration to the subject may be considered direct administration.

In other embodiments, such as when the subject is a neonatal, infant or child subject, treatment or prevention of one or more risks associated with GDM or one or more sequelae of GDM may comprise administration of L. rhamnosus HN001 or a composition comprising L. rhamnosus HN001 to the subject's mother. It will be appreciated that in such embodiments, administration to the subject may be considered indirect administration.

The composition may be a formula, eg, a maternal formula, follow-up formula, growth formula, or dietary product, including hypoallergenic forms of such compositions. In preferred embodiments where the subject is an adult subject, the method comprises administering a composition comprising L. rhamnosus HN001 to the adult subject. Preferably, the composition is a supplement, formula, dietary product, or food.

In certain embodiments, L. rhamnosus HN001 is in a reproductively viable form, preferably in a reproductively viable form and quantity. In other embodiments, L. rhamnosus HN001 is killed, lysed, cleaved, or attenuated.

The composition may be suitable for oral administration or for parenteral administration. In embodiments related to the prevention of one or more risks associated with GDM or one or more sequelae of GDM in a fetal subject, the composition is suitable for oral administration to a pregnant mother during gestation. In various embodiments, the adult subject or the mother of a fetal, neonate, infant or child subject is a subject who is at increased risk for GDM. In one example, the adult subject or the mother has previously suffered from GDM. In one example, the adult subject or mother is over the age of 35, eg, a subject who is over the age of 35 at the time of conception. In another example, the adult subject or mother has previously been pregnant.

In various examples, the subject may have a BMI of less than 30 kg/m2.

Preventing the recurrence of GDM in a subject who has previously suffered from GDM, may comprise administering an effective amount of HN001 to a subject in need thereof.

In one embodiment, the composition is administered after the first trimester of pregnancy. In one embodiment, the composition is administered between 14-16 weeks of gestation.

The invention reduces mean fasting blood glucose levels in the adult subject or in the mother of the fetal, neonate, infant or child subject below about 4.35 mmol/L.

The term "comprising" as used in this description means "consisting at least in part of". In construing each statement in this description that includes the term "comprising", features other than the foregoing may also be present. Related terms such as "comprising" and "comprises" are to be interpreted in the same way.

In this description where reference is made to patent descriptions, other external documents or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless otherwise specifically stated, reference to such external documents should not be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art or part of common general knowledge in the technique.

Brief description of the drawings

Figure 1 is a diagram of the status of study participants during the trial.

Figure 2 shows the associations of HN001 with GDM stratified by A) Age >35 years vs <35 years B) BMI >30 kg/m2 vs <30 kg/m2 C) History of GDM D) Use of systemic antibiotics since enrollment .

Figure 3 shows the graphs of the prevalence of GDM by subgroups. The graph on the left shows the effect of HN001 administration in subjects who had previously been pregnant versus those who had not been (parity, left) and the graph on the right shows the effect of HN001 administration in subjects older than 35 years versus those under 35 (maternal age, right).

Detailed description of the invention

The present invention recognizes for the first time the beneficial effects of administration of the lactic acid bacterium L. rhamnosus HN001 on the incidence and severity of GDM.

Accordingly, in a first aspect, the invention provides Lactobacillus rhamnosus HN001, AGAL deposit number NM97/09514 dated August 18, 1997 for use in the treatment or prevention of gestational diabetes mellitus (GDM) or one or more risks associated with GDM or one or more sequelae of GDM in an adult subject, or for use in the treatment or prevention of one or more risks associated with GDM or one or more sequelae of GDM in a fetal subject, neonate, infant or child, wherein the use for treatment or prevention comprises administration of L. rhamnosus HN001 to the adult subject or to the mother of the fetal, neonate, infant or child subject; and the mean fasting blood glucose level is reduced in the adult subject, or in the mother of the fetal, neonate, infant, or child subject to less than about 4.35 mmol/L. The risks associated with GDM in a pregnant woman are, for example, but not limited to, high blood pressure, urinary tract infections, cesarean delivery, and increased risk of type 2 diabetes. The risks associated with GDM or the sequelae of GDM in a fetal, neonate, infant, child, or adult subject (in particular, subjects whose biological mother suffered from GDM during her period in utero) are, for example, but not limited to preterm labor, shoulder dystocia, macrosomia, defects congenital and neonatal complications such as hypoglycemia, jaundice and respiratory distress, type 2 diabetes, cardiovascular disease, obesity and metabolic problems.

While various routes and methods of administration are contemplated, oral administration of L. rhamnosus HN001 is currently preferred, such as in a composition suitable for oral administration. Of course, it will be appreciated that other routes and methods of administration may be used or preferred in certain circumstances. For example, a parenteral route can be used with a composition comprising killed or attenuated L. rhamnosus HN001.

The term "oral administration" includes oral, buccal, enteral and intragastric administration.

The term "parenteral administration" includes but is not limited to topical (including administration to any dermal, epidermal, or mucosal surface), subcutaneous, intravenous, intraperitoneal, and intramuscular administration.

A "subject" is an animal, preferably a mammal, more preferably a mammalian companion animal or human. Preferred companion animals include cats, dogs, and horses. In one embodiment, the human is an adult, child, infant, neonate, or fetus. In various embodiments, the human child, infant, or neonate is a nursing child, infant, or neonate.

The term "treat" and its derivatives should be interpreted in its broadest possible context. The term is not to be understood as implying that a subject is treated until full recovery. Accordingly, "treat" includes, in general terms, the amelioration and/or prevention of the onset of symptoms or the severity of a particular condition.

It will be appreciated that treatment includes prophylactic treatment, such as for example, the prophylactic treatment of a subject, such as a subject who is at an expected or established increased risk of GDM and/or a subject who is attempting or recently pregnant, or the prophylactic treatment of one or more risks associated with GDM or one or more sequelae of GDM in a fetal subject by indirect administration of a composition of the invention by administration of the composition to the mother of the fetal subject.

In another example, the prophylactic treatment of one or more risks associated with GDM or one or more sequelae of GDM is of a neonatal, infant, or child subject by indirect administration of a composition of the invention by administration of the composition to the mother of the lactating subject.

It will further be appreciated that treatment includes therapeutic treatment, such as, for example, treatment of GDM or one or more symptoms or risks associated with GDM, including, for example, treatment of a neonatal, infant, or child subject by administration indirect control of a composition of the invention by administration of the composition to the mother of the subject.

Accordingly, the invention provides L. rhamnosus HN001 or a composition comprising L. rhamnosus HN001 for use in the treatment or prevention of GDM or one or more risks associated with GDM or one or more sequelae of GDM in a adult subject, or for use in the treatment or prevention of one or more risks associated with GDM or one or more sequelae of GDM in a fetal, neonate, infant, or child subject, wherein use to treat or prevent includes administration of L. rhamnosus HN001 to the adult subject or to the mother of the fetal, neonate, infant or child subject; and the mean fasting blood glucose level is reduced in the adult subject, or in the mother of a fetal, neonate, infant, or child subject to less than about 4.35 mmol/L.

In certain modalities, the pregnant woman is older, for example, 35 years or older.

The pregnant woman may have a history of GDM.

In certain embodiments, L. rhamnosus HN001 or a composition comprising L. rhamnosus HN001 is administered from 14 to 16 weeks of gestation until delivery.

In other examples, L. rhamnosus HN001 or a composition comprising L. rhamnosus HN001 can be administered from 14 to 16 weeks of gestation to 6 months postpartum.

Accordingly, in some aspects, the invention provides L. rhamnosus HN001 or a composition comprising L. rhamnosus HN001 for use in a method of preventing one or more risks associated with GDM or one or more sequelae of GDM in a subject. fetal, the method comprises administering L. rhamnosus HN001 or a composition comprising L. rhamnosus HN001 to the subject's mother.

Furthermore, L. rhamnosus HN001 or a composition comprising L. rhamnosus HN001 is provided for use in a method of treating or preventing one or more risks associated with GDM or one or more sequelae of GDM in a neonatal, infant, or child, the method preferably comprises administering L. rhamnosus HN001 or a composition comprising L. rhamnosus HN001 to the subject's mother.

Furthermore, a composition comprising L. rhamnosus HN001 is contemplated for use in a method of treating one or more risks associated with GDM or one or more sequelae of GDM in an infant or child comprising administering said composition consisting of or consisting essentially of L. rhamnosus HN001.

The infant or child may be one or more years of age.

The infant or child may be a food sensitized infant or child.

The infant or child may be considered to be at risk of one or more risks associated with GDM or one or more sequelae of GDM due to the prior incidence of GDM in the mother of the infant or child.

1 Lactobacillus rhamnosus HN001

As described in applicant's PCT international application PCT/NZ98/00122 (published as WO 99/10476), a freeze-dried culture of Lactobacillus rhamnosus HN001 was deposited with the Australian Government Analytical Laboratories (AGAL), The New South Wales Regional Laboratory, 1 Suakin Street, Pymble, NSW 2073, Australia, on August 18, 1997 and was granted deposit number NM97/09514. This repository recognized by the Budapest Treaty is now no longer known as AGAL, but as the National Measurement Institute of Australia (NMIA). The genome sequence of L. rhamnosus HN001 is available from Genebank under accession number: NZ_ABWJ00000000.

1.1 Morphological properties

The morphological properties of L. rhamnosus HN001 are described below.

Short to medium-sized rods with square ends in chains, usually 0.7 x 1.1 x 2.0 - 4.0 pm, when grown in MRS broth.

Immobile, non-spore forming, Gram positive, catalase negative, facultative anaerobic rods with an optimum growth temperature of 37±1°C and an optimum pH of 6.0 - 6.5. These are facultatively heterofermentative bacteria and gas is not produced from glucose.

1.2 Fermentative properties

An API 50 CH Sugar Fermentation Kit was used to determine the carbohydrate fermentation pattern of L. rhamnosus HN001, yielding a score of 5757177 (based on scores for 22 prominent sugars - see PCT/NZ98/00122). .

1.3 Additional characterization

L. rhamnosus strain HN001 can be further characterized by the functional attributes described in PCT/NZ98/00122, including its ability to adhere to human intestinal epithelial cells, and by improvements in phagocyte function, on antibody responses, on natural killer cell activity, and on lymphocyte proliferation elicited by dietary intake or in vitro model systems. It will be appreciated that there are a wide variety of methods known and available to the person skilled in the art that can be used to confirm the identity of L. rhamnosus HN001, wherein illustrative methods include DNA fingerprinting, genomic analysis, sequencing, and genomic techniques and related proteomics.

As described herein, certain embodiments of the present invention utilize live L. rhamnosus HN001.

As used herein, the term "derivative" and its grammatical equivalents when used with reference to bacteria (including use with reference to a specific strain of bacteria such as L. rhamnosus HN001) contemplates mutants and homologues or derivatives. of bacteria, killed or attenuated bacteria such as, but not limited to, heat-killed, lysed, fractionated, pressure-killed, irradiated, and UV- or light-treated bacteria, and material derived from bacteria, including, but not limited to to compositions of bacterial cell walls, bacterial cell lysates, lyophilized bacteria, probiotic factors of bacteria, and the like, wherein the derivative retains probiotic activity. Methods for producing such derivatives, such as but not limited to one or more L. rhamnosus HN001 mutants or one or more probiotic factors, are well known in the art, and particularly those derivatives suitable for administration to a subject (for example, in a composition).

It will be appreciated that methods suitable for identifying L. rhamnosus HN001, such as those described above, are equally suitable for identifying derivatives of L. rhamnosus HN001, including, for example, mutants or homologues of L. rhamnosus HN001, or for example, probiotic factors of L. rhamnosus HN001.

The term "probiotic factor" refers to a bacterial molecule responsible for mediating probiotic activity, including but not limited to bacterial DNA motifs, surface proteins, small organic acids, polysaccharides, or cell wall components such as lipoteichoic acids. and peptidoglycan, or a mixture of any two or more of these. While, as noted above, these molecules have not been clearly identified, and without wishing to be bound by theory, such molecules will be present if a probiotic organism is present.

The term "probiotic activity" refers to the ability of certain microorganisms to stimulate the immune system. Measurement of the type and level of activity of a probiotic microorganism is known to those skilled in the art; see, for example, Mercenier et al., (2004), Leyer et al., (2004) or Cummings et al., (2004). For example, probiotic activity can be assessed by a PBMC cytokine secretion assay.

Reference to retention of probiotic activity is intended to mean that a derivative of a probiotic microorganism, such as a mutant or homologue of a probiotic microorganism or an attenuated or killed probiotic microorganism, still has useful probiotic activity, or that a composition comprising a probiotic microorganism or a derivative thereof is capable of supporting the maintenance of useful probiotic activity. Although the bacterial molecules responsible for mediating probiotic activity have not been clearly identified, molecules that have been proposed as potential candidates include bacterial DNA motifs, surface proteins, small organic acids, polysaccharides, and cell wall components such as acids lipoteichoic and peptidoglycan. It has been postulated that these interact with components of the host's immune system to produce an immunomodulatory effect. Preferably, the retained activity is at least about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% of the activity of an untreated control (ie , vivid, or unattenuated) and useful ranges may be selected between any of these values (for example, from about 35 to about 100%, from about 50 to about 100%, from about 60 to about 100%, from about 70 to about 100 %, from about 80 to about 100% and from about 90 to about 100%).

Using conventional solid substrate and liquid fermentation technologies well known in the art, L. rhamnosus HN001 can be grown in quantities sufficient to permit use as contemplated herein. For example, L. rhamnosus HN001 can be produced in bulk for formulation using submerged culture or nutrient film growth techniques, eg, under conditions as described in WO99/10476. Briefly, growth is effected under aerobic conditions at any temperature satisfactory for the growth of the organism. For example, for L. rhamnosus HN001 a temperature range of 30 to 40°C is preferred, preferably 37°C. The pH of the growth medium is slightly acidic, preferably about 6.0 to 6.5. The incubation time is sufficient for the isolate to reach a stationary growth phase.

L. rhamnosus HN001 cells can be harvested by methods well known in the art, eg, by conventional sedimentation or filtration methodologies (eg, centrifugation) or dry-harvested using a cyclone system. L. rhamnosus HN001 cells can be used immediately or stored, preferably lyophilized or refrigerated at -20°C to 6°C, preferably -4°C, for as long as needed using standard techniques.

2 Compositions

A composition useful herein may be formulated as a food, beverage, food additive, beverage additive, dietary supplement, nutritional product, medicinal food, enteral or parenteral feeding product, meal replacement, cosmeceutical, nutraceutical or pharmaceutical product. Appropriate formulations can be prepared by one skilled in the art with respect to that skill and the teaching of this disclosure.

Compositions useful herein include any edible consumer product that is capable of harboring bacteria. Examples of suitable edible consumer products include powders, liquids, confectionery products including chocolate, gels, ice creams, reconstituted fruit products, snack bars, food bars, muesli bars, spreads, sauces, dressings, products dairy including yogurts and cheeses, beverages including dairy and non-dairy beverages (such as milk drinks and yogurt drinks), powdered milk, sports supplements including dairy and non-dairy sports supplements, food additives such as protein granules, dietary supplement products including daily supplement tablets, weaning foods and yoghurts, and formulas such as powdered or liquid maternal formula, including hypoallergenic versions of such compositions. Within this embodiment, a preferred composition useful in the present disclosure may be a maternal formula, in powder or liquid form. Suitable nutraceutical compositions useful in the present disclosure may be provided in similar forms.

Examples of formulas such as maternal formula, in powder or liquid form, include the following. It is to be understood that the following formulations are indicative only and variations may be made in accordance with known principles for formulating such products. For example, non-dairy protein sources can be supplemented with the listed dairy proteins. Likewise, hypoallergenic embodiments of these products can be provided where the protein source is fully or partially hydrolyzed. Such hydrolyzates are known in the art. An example of a maternal formula, useful in the present description comprises (w/w)

30 - 60% lactose

15 - 35% vegetable oils

0 - 40% skimmed milk powder

0-40% whey protein, such as a WPC or WPI, preferably 80% WPC (WPC80) 0.001-50% L. rhamnosus HN001.

Another example of a maternal formula, useful in the present description, comprises (w/w)

40 - 60% lactose

20 - 30% vegetable oils

10 - 15% skimmed milk powder

6 - 8% whey protein, preferably WPC80

0.001 - 10% L. rhamnosus HN001.

Another example of a maternal formula, in the present description comprises (w/w)

40 - 60% lactose

20 - 30% vegetable oils

10 - 15% skimmed milk powder

6 - 8% whey protein, preferably WPC80

0.001 - 5% L. rhamnosus HN001.

Another example of a maternal formula, useful in the present description, comprises (w/w)

40 - 60% lactose

20 - 30% vegetable oils

10 - 15% skimmed milk powder

6 - 8% whey protein, preferably WPC80

0.001 - 2% L. rhamnosus HN001.

Any of these formulas may further comprise 0.1 to 4% w/w, preferably 2 to 4% w/w of one or more of a vitamin premix, a mineral premix, lecithin, one or more antioxidants, one or more stabilizers, or one or more nucleotides, or a combination of two or more of these. In some embodiments, these infant formulas can be formulated to provide between 2700 and 3000 kJ/L.

Examples of edible consumer products of the invention, such as dairy beverages (such as dairy beverages and yogurt beverages) will typically comprise and may consist of a protein source (such as a dairy protein source), a of lipids, a carbohydrate source, in addition to L. rhamnosus HN001. In addition, flavorants, colorants, and other additives, carriers, or excipients well known to those skilled in the art may be included.

A further example of an edible consumer product usable in the present invention is the Unistraw™ delivery system (Unistraw International Limited, Australia) as described in PCT international application PCT/AU2007/000265 (published as WO 2007 /098564) and PCT international application PCT/AU2007/001698 (published as WO 2008/055296). Those skilled in the art will appreciate that L. rhamnosus HN001, optionally together with one or more probiotic factors or additional probiotic agents, can be coated onto a substrate (eg, a water-soluble bead) for use in such delivery systems.

Alternatively, the compositions useful herein can be formulated to allow administration to a subject by any chosen route, including, but not limited to, oral or parenteral (including topical, subcutaneous, intramuscular, and intravenous) administration.

For example, a nutraceutical composition for use in accordance with the invention may be a dietary supplement (for example, a capsule, minipouch, or tablet) or a food product (for example, milk, juice, soft drink, tea bag). herbs or confectionery). The composition can further include other nutrients, such as a protein, a carbohydrate, vitamins, minerals or amino acids. The composition may be in a form suitable for oral use, such as a tablet, hard or soft capsule, aqueous or oily suspension, or syrup; or in a form suitable for parenteral use, such as an aqueous propylene glycol solution or a buffered aqueous solution. The amount of the active ingredient in the nutraceutical composition largely depends on the specific need of the subject. The amount also varies, as recognized by those skilled in the art, depending on the route of administration and the possible concomitant use of other probiotic factors or probiotic agents.

It will be appreciated that in certain embodiments, the compositions of the invention may be formulated to have a suitable caloric content, for example, to supply a suitable amount of energy or a suitable percentage of the recommended daily energy intake. For example, an edible consumer product can be formulated to provide from about 200 to about 2,000 kJ per serving, or from about 500 to about 2,000 kJ per serving, or from about 1,000 to about 2,000 kJ per serving.

Thus, a useful pharmaceutical composition according to the invention may be formulated with an appropriate pharmaceutically acceptable carrier (including excipients, diluents, auxiliaries, and combinations thereof) selected with respect to the intended route of administration and standard pharmaceutical practice. For example, a composition useful in accordance with the invention may be administered orally as a powder, liquid, tablet or capsule, or topically as an ointment, cream or lotion. Suitable formulations may contain additional agents as needed, including emulsifying, antioxidant, flavoring, or coloring agents, and may be adapted for immediate, delayed, modified, sustained, pulsed, or controlled release.

The term "pharmaceutically acceptable carrier" refers to a carrier that includes but is not limited to an excipient, diluent, or auxiliary, pharmaceutically acceptable carrier includes a solvent, a dispersion medium, a coating, an antibacterial and antifungal agent, and an antifungal agent. delayed absorption and isotonic or combination thereof that can be administered to a subject as a component of a composition described herein that does not reduce the activity of the composition and is non-toxic when administered in doses sufficient to deliver an effective amount of a useful compound or composition of the present disclosure. The formulations can be administered orally, nasally, or parenterally (including topical, intramuscular, intraperitoneal, subcutaneous, and intravenous).

Certain compositions of the invention (such as, for example, a nutraceutical or pharmaceutical composition of the invention) may be provided as a capsule. Capsules may contain any standard pharmaceutically acceptable material, such as gelatin or cellulose. Tablets can be formulated according to conventional procedures by compressing mixtures of the active ingredients with a solid carrier and a lubricant. Examples of solid carriers include starch and sugar bentonite. The active ingredients may further be administered in the form of hard-shell tablets or capsules containing a binder, eg lactose or mannitol, a conventional filling agent and a tableting agent. The pharmaceutical compositions can also be administered parenterally. Examples of parenteral dosage forms include aqueous solutions, isotonic saline or 5% glucose of the active agent, or other well known pharmaceutically acceptable excipients. Cyclodextrins or other solubilizing agents well known to those skilled in the art can be used as excipients for delivery of the therapeutic agent.

Certain compositions of the invention may comprise live L. rhamnosus HN001. Methods for producing such compositions are well known in the art and one such method is exemplified herein in the examples.

It will be appreciated that a wide range of additives or carriers may be included in such compositions, for example, to improve or preserve bacterial viability or to increase the therapeutic efficacy of L. rhamnosus HN001. For example, additives such as surfactants, moisturizers, humectants, adhesives, dispersing agents, stabilizers, penetrants, and so-called stress additives can be included to enhance bacterial cell vigour, growth, replication, and survivability (such as chloride potassium, glycerol, sodium chloride and glucose), as well as cryoprotectants such as maltodextrin. Additives can also include compositions that help maintain the viability of microorganisms in long-term storage, for example, unrefined corn oil or "invert" emulsions containing a mixture of oils and waxes on the outside and water, alginate sodium and bacteria inside.

In certain embodiments, L. rhamnosus HN001 is in a reproductively viable form and quantity.

The composition may comprise a carbohydrate source, such as a disaccharide including, for example, sucrose, fructose, glucose, or dextrose. Preferably, the carbohydrate source is one that can be used aerobically or anaerobically by L. rhamnosus HN001.

In such cases, the composition is preferably capable of supporting the reproductive viability of L. rhamnosus HN001 for a period greater than about two weeks, preferably greater than about one month, about two months, about three months, about four months, about five months, more preferably greater than about six months, most preferably at least about 2 years to about 3 years or more.

The composition for use in a method of treating or preventing GDM or the risks associated with GDM or the sequelae of GDM may comprise a probiotic comprising L. rhamnosus HN001 and a prebiotic, for example fructooligosaccharides, galactooligosaccharides, oligosaccharides of human milk and combinations thereof.

Use in a method of treating and preventing GDM or the risks associated with GDM or the sequelae of GDM in a subject as described herein may comprise administering to an individual an effective amount of a composition comprising L. rhamnosus HN001 and a prebiotic, eg, fructooligosaccharides, galactooligosaccharides, human milk oligosaccharides, and combinations thereof. In certain instances, an oral composition is formulated to allow administration of a sufficient amount of L. rhamnosus HN001 to establish a population in the subject's gastrointestinal tract when ingested. The established population may be a transient or permanent population.

In theory, one colony-forming unit (cfu) should be sufficient to establish a population of L. rhamnosus HN001 in a subject, but in real situations a minimum number of units is required to do so. Therefore, for therapeutic mechanisms that rely on a viable living population of probiotic bacteria, the number of units administered to a subject will affect therapeutic efficacy.

As presented herein in the examples, Applicants have determined that a dosage rate of 6 * 109 cfu L. rhamnosus HN001 per day is sufficient (but may not be necessary) to establish a population in the gastrointestinal tract of subjects. humans. Accordingly, in one example, a composition formulated for administration will be sufficient to provide at least about 6*10 9 cfu of L. rhamnosus HN001 per day.

Methods for determining the presence of a population of intestinal flora, such as L. rhamnosus HN001, in the gastrointestinal tract of a subject are well known in the art and examples of such methods are presented herein. In certain instances, the presence of a population of L. rhamnosus HN001 can be determined directly, for example, by analyzing one or more samples that are obtained from a subject and determining the presence or quantity of L. rhamnosus HN001 in said sample. In other cases, the presence of a population of L. rhamnosus HN001 can be determined indirectly, for example, by observing a reduction in GDM symptoms or a decrease in the number of other intestinal flora in a sample obtained from a subject. Furthermore, combinations of such methods are envisioned.

The efficacy of a composition useful according to the invention can be assessed both in vitro and in vivo. See, for example, the examples below. Briefly, the composition can be evaluated for its ability to prevent or treat GDM, or to modulate glucose tolerance. For in vivo studies, the composition can be fed or injected into an animal model (eg, a mouse) or administered to human subjects (including pregnant women) and then evaluated for its effects on the incidence and severity of GDM or disease. glucose tolerance and associated conditions. Based on the results, an appropriate dosage range and route of administration can be determined.

Methods for calculating the appropriate dose may depend on the nature of the active agent in the composition. For example, when the composition comprises live L. rhamnosus HN001, the dose can be calculated with reference to the number of live bacteria present. For example, as described in the examples herein, the dose may be established with reference to the number of colony-forming units (cfu) to be administered per day. In examples where the composition comprises one or more L. rhamnosus HN001 derivatives, the dose may be calculated with reference to the amount or concentration of the L. rhamnosus HN001 derivative present. For example, for a composition comprising L. rhamnosus HN001 cell lysate, the dose can be calculated with reference to the concentration of L. rhamnosus HN001 cell lysate present in the composition.

By way of general example, administration of about 1*106 cfu to about 1*1012 cfu of L. rhamnosus HN001 per kg body weight per day is contemplated, preferably about 1*106 cfu to about 1*1011 cfu/kg/ day, about 1 * 106 cfu to about 1 * 1010 cfu/kg/day, about 1 * 106 cfu to about 1 * 109 cfu/kg/day, about 1 * 106 cfu to about 1 * 108 cfu/kg/day, about 1*106 cfu to about 5*107 cfu/kg/day or about 1*106 cfu to about 1*107 cfu/kg/day. Preferably, administration of about 5*106 cfu to about 5*108 cfu per kg body weight of L. rhamnosus HN001 per day is contemplated, preferably about 5*106 cfu to about 4*108 cfu/kg/day, about 5 * 106 cfu at about 3 * 108 cfu/kg/day, about 5 * 106 cfu at about 2 * 108 cfu/kg/day, about 5 * 106 cfu at about 1 * 108 cfu/kg/day, about 5 * 106 cfu at about 9 * 107 cfu/kg/day, about 5 * 106 cfu at about 8 * 107 cfu/kg/day, about 5 * 106 cfu at about 7 * 107 cfu/kg/day, about 5 * 106 cfu at about 6 * 107 cfu/kg/day, about 5 * 106 cfu at about 5 * 107 cfu/kg/day, about 5 * 106 cfu at about 4 * 107 cfu/kg/day, about 5 * 106 cfu at about 3 * 107 cfu/kg/day, approximately 5 * 106 cfu to approximately 2 * 107 cfu/kg/day or approximately 5 * 106 cf u at about 1 * 107 cfu/kg/day.

Periodic doses need not vary with body weight or other characteristics of the subject. In such examples, administration of from about 1*106 cfu to about 1*1013 cfu of L. rhamnosus HN001 per day is contemplated, preferably about 1*106 cfu to about 1*1012 cfu/day, about 1*106 cfu at about 1 x 1011 cfu/day, about 1 x 106 cfu at about 1 x 1010 cfu/day, about 1 x 106 cfu at about 1 * 109 cfu/day, about 1 * 106 cfu at about 1 * 108 cfu/day , about 1*106 cfu to about 5*107 cfu/day or about 1*106 cfu to about 1*107 cfu/day. Preferably, administration of about 5*107 cfu to about 5*1010 cfu per kg body weight of L. rhamnosus HN001 per day is contemplated, preferably about 5*107 cfu to about 4*1010 cfu/day, about 5* 107 cfu at about 3 * 1010 cfu/day, about 5 * 107 cfu at about 2 * 1010 cfu/day, about 5 * 107 cfu at about 1 * 1010 cfu/day, about 5 * 107 cfu at about 9 * 109 cfu /day, about 5 * 107 cfu to about 8 * 109 cfu/day, about 5 * 107 cfu to about 7 * 109 cfu/day, about 5 * 107 cfu to about 6 * 109 cfu/day, about 5 * 107 cfu at about 5 * 109 cfu/day, about 5 * 107 cfu at about 4 * 109 cfu/day, about 5 * 107 cfu at about 3 * 109 cfu/day, about 5*107 cfu to about 2*109 cfu/day or about 5*107 cfu to about 1*109 cfu/day.

For example, as presented herein in the examples, an effective dose of lyophilized L. rhamnosus HN001 was determined to be 6*10 9 cfu per day.

It will be appreciated that the composition is preferably formulated to allow administration of an effective dose of L. rhamnosus HN001. The dose of the composition administered, the period of administration, and the general administration regimen may differ between subjects depending on variables such as the severity of a subject's symptoms, the type of disorder to be treated, the mode of administration chosen, and the nature of the administration. age, sex and/or general health of a subject. Furthermore, as described above, the appropriate dose may depend on the nature of the active principle in the composition and on the form of formulation. For example, when the composition comprises live L. rhamnosus HN001, the dose can be calculated with reference to the number of live bacteria present. For example, as described in the examples herein, the dose may be established with reference to the number of colony-forming units (cfu) to be administered per day. In examples where the composition comprises one or more L. rhamnosus HN001 derivatives, the dose may be calculated with reference to the amount or concentration of L. rhamnosus HN001 derivative to be administered per day. For example, for a composition comprising L. rhamnosus HN001 cell lysate, the dose can be calculated with reference to the concentration of L. rhamnosus HN001 cell lysate present in the composition.

It will be appreciated that preferred compositions are formulated to provide an effective dose in a convenient form and amount. In certain cases, such as but not limited to those where the periodic dose need not vary with body weight or other characteristics of the subject, the composition may be formulated for unit dose. It is to be appreciated that administration may include a single daily dose or the administration of several discrete divided doses, as appropriate. For example, as presented herein in the examples, an effective dose of L. rhamnosus HN001 can be formulated into a capsule for oral administration.

However, by way of general example, the inventors contemplate administration of about 1 mg to about 1000 mg per kg of body weight of a composition useful herein per day, preferably about 50 to about 500 mg per kg per day. , alternatively about 150 to about 410 mg/kg/day or about 110 to about 310 mg/kg/day. For example, the inventors contemplate the administration of about 0.05 mg to about 250 mg per kg of body weight of a composition useful in the present disclosure.

Examples of infant formula, follow-up formula or growth formula are presented in the present description. Compositions such as these can be formulated so that the concentration of L. rhamnosus HN001 present in the composition is such that an effective dose can be prepared by using a readily measurable amount of the composition. For example, in certain embodiments, such as when the composition is an infant formula, the L. rhamnosus HN001 is provided in a concentration sufficient to deliver an effective dose in an amount of formula readily measurable by a parent or caregiver when preparing formula for administration, such as, for example, with a measuring spoon or the like, as is commonly provided with infant formulas. Illustrative non-limiting concentrations of L. rhamnosus HN001 for use in such compositions include from about 5 * 105 cfu per gram of formula up to about 109 cfu per gram of formula or from about 106 cfu per gram of formula up to about 108 cfu per gram of formula

A composition useful herein may comprise, consist essentially of, or consist of at least about 0.1, 0.2, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 99.5, 99.8 or 99.9% by weight of L. rhamnosus HN001 and useful ranges may be selected from any of those above values (for example, from about 0.1 to about 50%, from about 0.2 to about 50%, from about 0.5 to about 50%, from about 1 to about 50%, from about 5 to about 50%, about 10 to about 50%, about 15 to about 50%, about 20 to about 50%, about 25 to about 50%, about 30 to about 50%, about 35 to about 50 %, from about 40 to about 50%, from about 45 to about 50%, from about 0.1 to about 60%, from about 0.2 to about 60%, from about 0.5 to about 60%, from about 1 to about 60%, from about 5 to about 60%, from about 10 to about 60%, from about 15 to about 60%, about 20 to about 60%, about 25 to about 60%, about 30 to about 60%, about 35 to about 60%, about 40 to about 60%, about 45 to about 60%, from about 0.1 to about 70%, from about 0.2 to about 70%, from about 0.5 to about 70%, from about 1 to about 70%, from about 5 to about 70% , from about 10 to about 70%, from about 15 to about 70%, from about 20 to about 70%, about 25 to about 70%, about 30 to about 70%, about 35 to about 70%, about 40 to about 70%, about 45 to about 70%, about 0.1 to about about 80%, about 0.2 to about 80%, about 0.5 to about 80%, about 1 to about 80%, about 5 to about 80%, about 10 to about 80%, about 15 to about 80%, from about 20 to about 80%, from about 25 to about 80%, from about 30 to about 80%, from about 35 to about 80%, from about 40 to about 80%, from about 45 to about 80%, about 0.1 to about 90%, about 0.2 to about 90%, about 0.5 to about 90%, about approximately 1 to approximately 90%, approximately 5 to approximately 90%, approximately 10 to approximately 90%, approximately 15 to approximately 90%, approximately 20 to approximately 90%, approximately 25 to approximately 90%, approximately 30 to about 90%, from about 35 to about 90%, from about 40 to about 90%, from about 45 to about 90%, from about 0.1 to about 99%, from about 0.2 to about 99%, from from about 0.5 to about 99%, from about 1 to about 99%, from about 5 to about 99%, from about 10 to about 99%, from about 15 to about 99%, from about 20 to about 99%, from about 25 to about 99%, about 30 to about 99%, about 35 to about 99%, about 40 to about 99 % and from about 45 to about 99%).

For example, a composition useful herein may comprise, consist essentially of, or consist of at least about 0.001, 0.01, 0.05, 0.1, 0.15, 0.2, 0.3, 0 .4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 grams of L. rhamnosus HN001 and useful ranges may be selected from any of those above values (for example, from about 0.01 to about 1 grams, from about 0.01 to about 10 grams, from about 0.01 to about 19 grams, from about 0.1 to about 1 grams, about 0.1 to about 10 grams, about 0.1 to about 19 grams, about 1 to about 5 grams, about 1 to about 10 grams, about 1 to about 19 grams, about 5 to about 10 grams, and about 5 to about 19 grams).

A composition useful herein comprising L. rhamnosus HN001 may further comprise about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 , 65, 70, 75, 80, 85, 90, 95, 97, 99 or 99.9% by weight of fresh whole milk or a milk derivative and useful ranges between any of these above values (for example, from about 0.1 to about 50%, about 0.2 to about 50%, about 0.5 to about 50%, about 1 to about 50%, about 5 to about 50%, about 10 to about 50% , from about 15 to about 50%, from about 20 to about 50%, from about 25 to about 50%, from about 30 to about 50%, from about 35 to about 50%, from about 40 to about 50% and from about 45 to about 50%). The milk derivative is preferably selected from recombined, powdered or fresh skimmed milk, reconstituted or recombined whole or skimmed powdered milk, skimmed milk concentrate, skimmed milk retainer, concentrated milk, ultrafiltered milk retainer, protein concentrate Milk Protein Isolate (MPC), Milk Protein Isolate (MPI), Calcium Free Milk Protein Concentrate (MPC), Low Fat Milk, Low Fat Milk Protein Concentrate (MPC), Casein, Caseinate , milk fat, cream, butter, shortening, anhydrous milk fat (AMF), whey, buttermilk, beta whey, hard milk fat fractions, soft milk fat fractions, sphingolipid fractions, milk fat globular membrane, phospholipid fractions, complex lipid fractions, colostrum, a colostrum fraction, colostrum protein concentrate (CPC), colostrum whey, a colostrum immunoglobulin fraction colostrum, whey (including sweet whey, lactic acid whey, mineral acid whey, or reconstituted powdered whey), whey protein isolate (WPI), whey protein concentrate (WPC), a composition derived from any flow from milk or colostrum processing, a composition derived from the retentate or permeate obtained by ultracentrifugation or microfiltration of any milk or colostrum processing stream, a composition derived from the advance or adsorbed fraction obtained by chromatographic separation (including, but not limited to not limited to gel and ion permeation chromatography) of any milk or colostrum processing stream, extracts of any of these milk derivatives, including extracts prepared by multi-step fractionation, differential crystallization, solvent fractionation, supercritical fractionation , near supercritical fractionation, distillation, centrifugal fractionation or fractionation with a modifier (for example, soaps or emulsifiers), hydrolysates of any of these derivatives, fractions of the hydrolysates and combinations of these derivatives, fractions of the hydrolysates and any combination of any two or more of these derivatives, including the combinations of the hydrolyzed and/or non-hydrolyzed fractions. It should be understood that the source of these derivatives can be milk or colostrum or a combination of these.

It will be apparent that the concentration of L. rhamnosus HN001 in a composition formulated for administration may be less than that in a composition formulated, for example, for distribution or storage, and that the concentration of a composition formulated for storage and subsequent formulation in a composition suitable for administration must be suitable to allow said composition for administration to also be sufficiently concentrated to be able to be administered in a dose with therapeutic efficacy.

The compositions useful in the present disclosure may be used alone or in combination with one or more other therapeutic agents. The therapeutic agent can be a food, beverage, food additive, beverage additive, food component, beverage component, dietary supplement, nutritional product, medicinal food, nutraceutical, medicament, or pharmaceutical. The therapeutic agent may be a probiotic agent or a probiotic factor, and is preferably effective in treating, preventing, or attenuating GDM or one or more of the symptoms of GDM, or one or more risks associated with GDM, or one or more sequelae. of the GDM.

When used in combination with another therapeutic agent, administration of a composition useful herein and the other therapeutic agent can be simultaneous or sequential. Simultaneous administration includes the administration of a single dosage form comprising all of the components or the administration of separate dosage forms, substantially at the same time. Sequential administration includes administration according to different schedules, preferably such that there is an overlap in the periods during which the composition useful herein and another therapeutic agent are provided.

Suitable agents with which the compositions useful in the present disclosure may be administered separately, simultaneously or sequentially include one or more probiotic agents, one or more prebiotic agents, one or more phospholipids, one or more gangliosides, other suitable agents known in the art technique and combinations of these. Useful prebiotics include galactooligosaccharides (GOS), short-chain GOS, long-chain GOS, fructooligosaccharides (FOS), human milk oligosaccharides (HMO), short-chain FOS, long-chain FOS, inulin, galactans, fructans, lactulose. and any mixture of any two or more of these. Some prebiotics are reviewed by Boehm G and Moro G (Structural and Functional Aspects of Prebiotics Used in Infant Nutrition, J. Nutr. (2008) 138(9):1818S-1828S). Other useful agents may include dietary fiber such as a wholly or partially insoluble or indigestible dietary fiber. Accordingly, L. rhamnosus HN001 can be administered separately, simultaneously or sequentially with one or more agents selected from one or more probiotics, one or more prebiotics, one or more sources of dietary fiber, one or more galactooligosaccharides, one or more galactooligosaccharides of short-chain, one or more long-chain galactooligosaccharides, one or more fructooligosaccharides, one or more short-chain galactooligosaccharides, one or more long-chain galactooligosaccharides, one or more human milk oligosaccharides, inulin, one or more galactans, one or more plus fructans, lactulose, or any mixture of two or more of these.

A composition useful herein may include or be administered simultaneously or sequentially with milk components such as whey protein, whey protein fractions (including acidic or basic whey protein fractions or a combination thereof), glycomacropeptide, lactoferrin, iron-lactoferrin, a functional variant of lactoferrin, a functional fragment of lactoferrin, a vitamin D or calcium, or combinations of these. Compositions containing useful dairy components include compositions such as a food, beverage, food additive, beverage additive, dietary supplement, nutritional product, medicinal food, or nutraceutical. Milk fractions enriched for these components can also be used. Useful lactoferrins, fragments and compositions are described in international patent applications WO 03/082921 and WO 2007/043900.

It is to be understood that the additional therapeutic agents listed above (both food-based and pharmaceutical agents) may also be employed in a method according to the invention where they are administered separately, simultaneously or sequentially with a composition useful in the present disclosure.

A composition useful herein may further comprise a pharmaceutically acceptable carrier. Other compositions may be or be formulated as a food, beverage, food additive, beverage additive, dietary supplement, nutritional product, medicated food, enteral feeding product, parenteral feeding product, meal replacement, cosmeceutical, nutraceutical, medicament or pharmaceutical product. . A composition may be in the form of a tablet, a capsule, a pill, a hard or soft capsule, or a lozenge. The composition may be in the form of a cachet, a powder, a dispensable powder, granules, a suspension, an elixir, a liquid, or any other form that can be added to food or beverages, including, for example, water, milk, or juice. of fruit. The composition may further comprise one or more constituents (such as antioxidants) that prevent or reduce degradation of the composition during storage or after administration. These compositions can include any edible consumer product that is capable of carrying bacteria or bacterial derivatives, including heat-killed, pressure-killed, lysed, light- or UV-treated, irradiated, fractionated, or otherwise killed or attenuated bacteria. Examples of suitable edible consumer products include aqueous products, baked goods, confectionery products including chocolate, gels, ice creams, reconstituted fruit products, snack bars, food bars, muesli bars, spreads, sauces, dressings , Dairy products including yogurts and cheeses, beverages including dairy and non-dairy beverages, milk, powdered milk, sports supplements including dairy and non-dairy sports supplements, fruit juices, food additives such as protein granules, and dietary supplement products , including daily supplement tablets, weaning foods and yoghurts and formulas such as infant formula, monitoring formula or growth formula, in powder or liquid form. Suitable nutraceutical compositions useful in the present disclosure may be provided in similar forms.

It will be appreciated that different compositions for use of the invention may be formulated with a view to administration to a particular group of subjects. For example, the formulation of a composition suitable for administration to a pregnant mother (for example, for indirect administration to a fetal subject or to a neonatal, infant, or nursing child subject) may differ from that of a composition for administration directly to the subject. It should further be appreciated that the formulation of a composition to be administered prophylactically may differ from that of a composition formulated for administration once GDM or one or more symptoms of GDM is present.

The composition for prophylactic use may further comprise L. rhamnosus HN001 or may be used in combination with a probiotic agent such as Lactobacillus rhamnosus GG, Lactobacillus acidophilus (eg Lactobacillus acidophilus (LAVRI-A1), Lactobacillus reuteri (eg Lactobacillus reuteri ATCC 55730) or Bifidobacterium lactis (eg, Bifidobacterium lactis strain HN019 or Bifidobacterium lactis Bb12) or a combination of two or more of these.

Compositions for prophylactic administration, and in particular prophylactic indirect administration, may further comprise or L. rhamnosus HN001 may be used in combination with a probiotic agent such as Lactobacillus rhamnosus GG, Lactobacillus acidophilus (for example, Lactobacillus acidophilus (LAVRI -A1), Lactobacillus reuteri (eg Lactobacillus reuteri ATCC 55730) or Bifidobacteria lactis (eg Bifidobacteria lactis strain HN019) or a combination of two or more of these.

It will be appreciated that the term "prophylactic" and grammatical equivalents as used herein contemplate treatment, use, administration, and the like before GDM or symptoms of GDM become evident.

In embodiments for use in treating a subject having GDM or one or more symptoms of GDM, the composition may further comprise or L. rhamnosus HN001 may be combined with a probiotic agent such as Lactobacillus rhamnosus GG, Lactobacillus acidophilus (for example , Lactobacillus acidophilus (LAVRI-A1), Lactobacillus reuteri (eg Lactobacillus reuteri ATCc 55730) or Bifidobacteria lactis (eg Bifidobacteria lactis strain HN019) or a combination of two or more of these.

As used herein, the term "therapeutic" and grammatical equivalents contemplate treatment, uses, or administration in which GDM or symptoms of GDM are present.

Reference to a range of numbers described herein (for example, 1 to 10) is intended to also incorporate reference to all rational numbers within that range (for example, 1, 1,1, 2, 3 , 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5, and 3.1 to 4.7) and therefore all subranges of all ranges expressly described herein are expressly described herein. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value listed are to be deemed expressly indicated in this application in a similar manner.

3 Gestational Diabetes Mellitus

Along with global obesity trends, the rate of gestational diabetes mellitus (GDM) is also increasing in both the developed and developing world*10*. GDM typically affects between 9% and 26% of all pregnancies depending on country and ethnic group. In certain cases, the prevalence of GDM is as high as 36%. Using the IADPSG diagnostic criteria(11), 18% of pregnant women in the United States develop GDM during pregnancy(10). There is a strong upward trend in prevalence in the last 20 years.

GDM is associated with adverse short- and long-term outcomes for both women and infants, including maternal gestational hypertension, polyhydramnios, preeclampsia, delivery of infants large for gestation, instrumental or cesarean delivery, and maternal death (10, 12)

Adverse outcomes in infants include preterm delivery, shoulder dystocia, macrosomia, birth defects, and neonatal complications such as hypoglycemia, jaundice, and respiratory distress(10). Furthermore, in the longer term, women with GDM are at increased risk of metabolic syndrome(13), type 2 diabetes, and cardiovascular disease. Children of women with GDM are at increased risk of diabetes, obesity, and metabolic problems with evidence of altered insulin secretion and lipid profiles regardless of the infant's weight(14).

Lifestyle interventions to prevent GDM related to diet, weight loss, and exercise are often unsuccessful(10, 15); therefore, primary prevention of GDM could provide significant multigenerational health and economic benefits.

Various aspects of the invention will now be illustrated with reference to the following examples.

examples

To determine whether the probiotic Lactobacillus rhamnosus HN001 (HN001) taken by pregnant mothers early in pregnancy could reduce the prevalence of GDM between 26-28 weeks of gestation, a double-blind, randomized, placebo-controlled parallel trial was conducted.

Materials and methods

Pregnant women in Auckland and Wellington, NZ, were recruited for the study through health professionals and study information was placed in pregnancy packs. Women were considered eligible if they were less than 16 weeks' gestation, spoke English, intended to breastfeed, and if they or the biological father of the unborn child had a history of asthma, hay fever, or eczema that required medication. Women were excluded from the study if they were less than 16 years of age, planned to move outside the study centers for the duration of the study, had a history of immunological disorders or medication, heart valve disease, required in vitro fertilization, had significant fetal abnormalities , were using probiotic beverages or supplements, were participating in another randomized controlled trial (RCT), refused to notify their clinical caregivers, were taking epinephrine for cow's milk allergy, had a history of transplantation or HIV, had used antibiotic therapy continuous for at least 3 months, aborted between screening and enrollment, or were deemed unsuitable. Eligible women were enrolled in the study at 14-16 weeks' gestation, where gestation was based on the earliest ultrasound in the first trimester and, when not available, the date of the last menstrual period.

Study design

Participating women were randomized to receive capsules containing HN001 (6*109 colony-forming units (cfu)) or placebo (corn-derived maltodextrin, identical in appearance and odor to the probiotic) to be taken daily from enrollment throughout the pregnancy and up to six months after delivery if you are still breastfeeding. Powder HN001 was manufactured by Fonterra Co-operative Group Ltd using aseptic fermentation, concentration and freeze drying, as previously described®. The placebo powder, corn-derived maltodextrin, was manufactured by Grain Processing Corp. Oregon, USA. Women were instructed to keep the capsules in the refrigerator and to avoid taking them within 10 minutes of consuming food or hot liquids.

Fonterra retained capsule samples at 4°C which were tested monthly to ensure content viability over time. The viability of the contents of a selection of unused capsules returned from the field was evaluated three times per month. The loss of viability was less than 0.1 log and within the uncertainty limit of the enumeration method.

Capsule randomization was performed by a Fonterra statistician who had no contact with the investigators or study participants. Randomization was stratified by study center and was performed in blocks of 20 according to a computer-generated randomization schedule and an allocation ratio of 1:1. Research staff screened and enrolled participants, providing eligible participants with the next available sequentially numbered container of capsules. All investigators, laboratory personnel, and participants were blinded to study assignment.

Baseline information collected included age, ethnicity, parity, prior polycystic ovary syndrome (PCOS), body mass index (BMI) (weight (kg)/height (m)2), waist circumference, antibiotic use during the pregnancy but prior to enrollment and type 2 diabetes mellitus in the participant or a first-degree relative. Among women with previous pregnancies of more than 20 weeks, we also collected a history of previous GDM and birth weights of previous babies.

outcome measures

The outcome of the GDM was defined a priori mainly as the diagnosis of GDM according to the recommendations of the IADPSG(6): a fasting plasma glucose >5.1 mmol/l, or a glucose load after 75 g of >10 mmol/l in 1 hour or >8.5 mmol/l in 2 hours. A secondary analysis was performed using NZ thresholds of >5.5 mmol/L fasting or >9 mmol/L 2 hours to define GDM(1).

GDM assessment was performed at 24-30 weeks' gestation after a 12-h overnight fast, using a glucose tolerance test (GTT) performed in a community laboratory. Only women without diabetes before pregnancy were invited to undergo a GTT study. Women who received a GTT-based diagnosis of GDM prior to the GTT study were included in the study outcome only if there was evidence of previous negative tests confirming that their diabetes was gestational. When repeat GTTs were performed later in pregnancy (for clinical purposes), testing completed at 24-30 weeks' gestation determined study GDM status.

Women with GDM were asked to have a postpartum HbA1c at least 3 months after delivery. A postpartum HbAlc level >6.5 % (48 mmol/mol) or a postpartum fasting glucose >7 mmol/l and/or 2-hour glucose >11.1 mmol/l was used to indicate the presence of type diabetes. 2 coexisting. If women met these criteria, they were excluded from the GDM analysis.

Other outcomes were collected at 4-7 days postpartum, including maternal weight (kg) and waist circumference (cm), gestation (in weeks), and prematurity (<37 weeks). The infant's five-minute Apgar score and birth weight were collected from medical records. Infant length (cm), Weight Index (PI) (birth weight (kg)/length (m)3), head circumference (cm), type of delivery (vaginal or cesarean section) and admission to the Neonatal Intensive Care Unit (NICU) were assessed by the investigator at 47 days postpartum.

Protocol adherence

A supply of capsules for more than three months (n=105) was placed in each bottle. The bottles were replaced at 26-28 weeks of gestation and delivery, where 2 bottles were given to the mother to cover the 6-month period after delivery. Returned vials were counted by personnel not involved in study assessments as a measure of adherence to the protocol.

Power

Assuming a 15% prevalence of GDM, a 63% reduction due to the probiotic as found in a Finnish study(8) and a sample size of 195 in each group, the study would have a power of 87% with a level of significance of 5%.

Statistic analysis

Analysis was performed using SAS 9.3 and 9.4 (SAS Institute, Cary, NC). All analyzes were by intention to treat. Differences between treatment groups in the prevalence of GDM and dichotomous birth outcomes were estimated using relative rates (RRs) and 95% CIs. Although not prespecified, we performed an analysis of GDM stratified by factors that were significantly associated with GDM (maternal age, BMI, history of GDM) and by pre-GTT study antibiotic use using a model generalized linear with a logarithmic linkage and a binomial distribution. For continuous variables, differences between treatment groups are reported as differences in means (95% CI) compared using t-tests or ratios of geometric means (95% CI) compared using analysis of covariance. of the recorded values, adjusted for the initial recorded measurements; other differences were compared using Wilcoxon rank sum tests. Apgar scores were compared between groups using ordinal logistic regression. The tests were two-sided and P<0.05 was considered statistically significant.

Missing GTT measurements were estimated with 1,000 multiple imputations using treatment, fasting, one- and two-hour measurements, ethnicity, age, BMI at enrollment, family history of diabetes in first-degree relatives, ovarian syndrome prior polycystic and the combination of prior GDM and number of prior pregnancies >20 weeks (grouped as no prior pregnancy, one prior pregnancy with GDM, or one prior pregnancy without GDM, two or more prior pregnancies with GDM, or two or more prior pregnancies without GDM ). A participant who did not have GTT because she had been diagnosed with GDM and prescribed insulin since early pregnancy was assumed to have GDM on all imputations.

This study was conducted in accordance with the guidelines set forth in the Declaration of Helsinki and all procedures involving humans were approved by the Multiregional Health and Disability Ethics Committee. Written informed consent was obtained from all subjects. The trial was registered with the Australian NZ Clinical Trials Registry: ACTRN12612000196842, https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=362049&isReview=true

Results

Participants (n=423) were randomized to HN001 (n=212) or placebo (n=211) between December 2012 and November 2014 at an average rate of 4.2 per week. Gestational diabetes screenings are they completed in February 2015, and the last infant was born in May 2015. Rates of loss to follow-up were similar between study groups, but more participants in the HN001 group had discontinued the intervention before GTT (Figure 1). In contrast, the majority of participants lost to follow up between GTT and delivery visits were in the placebo group and a small number discontinued the intervention in both study groups. There was one maternal death in the placebo group, due to subarachnoid haemorrhage.

Among randomized participants, GTT outcomes from 24 to 30 weeks included all three time points (fasting, 1-hour and 2-hour values, as required by IADPSG guideline definitions) in 184 (87%) participants in the HN001 group and 189 (90%) in the placebo group, with a mean of 27.7 (SD 4.6) and 28.0 (SD=8.6) weeks of gestation, respectively. An additional 10 HN001 participants and 11 placebo participants only had fasting and 2-hour GTT results available, which was sufficient for diagnosis of GDM according to standard NZ guidelines. In the HN001 group, 194 (92%) and in the placebo group, 200 (95%) participated in any of the GTT assessments, all of whom were able to contribute data to the analysis according to NZ guidelines.

There were no substantial differences between the study groups in any maternal characteristics at enrollment, including age, ethnicity, parity, weight, waist, BMI, use of antibiotics during pregnancy prior to enrollment, family history of diabetes, family income, and among those with previous births, largest infant weight, and history of GDM (Table 1).

Table 1 Characteristics of the study population at the time of enrollment

HN001 (N=212) Placebo (N=211) _p

Previous pregnancy ^67.5%

_(143/212) 73.8 (155/210) 0.15*

Diabetes in first degree relative ^17.9%

_(38/212) 18.5% (39/211) 0.88*

Previous polycystic ovary syndrome ^8.1%

_(17/209) 10.6% (22/208) 0.39*

Age (years), median (range 34 (30-36)

interquartile) _(n=212) 34 (31-37) (n=210) 0.65*

Weight (kg), median (interquartile range) 69 (63-80) (n=211) 71 (63-82) (n=210) 0.45t Waist circumference (cm), median (range

_{interquartile)} 87 (80-94) (n=211) 87 (81-99) (n=210) 0.30t BMI, median (interquartile range) 25 (23-29) (n=211) 26 (23-30 ) (n=210) 0.28t Ethnicity N=212 N=211

Maori 10.9% (23) 16.6% (35)

Pacific 3.8% (8) 1.9% (4)

Asian 7.7% (15) 7.6% (16) 0.27* European 78.3% (166) 73.5% (155)

Other 0.0% (0) 0.5% (1)

Systemic antibiotic use during ^{previous pregnancy}

at enrollment 12.4% (25/201) 13.9% (28/202) 0.67* Household income (NZ$) N=207 N=204

0-49k 8.2% (17) 7.8% (16)

^{50-99k 30.4% (63) 32.4% (66)}

_{100-149k 35.8 (74) 34.8% (71)} 0.98* 150+k 25.6% (53) 25.0% (51)

Among women with previous pregnancies > 20 weeks (n=248)

Previous gestational diabetes 5.0% (6/121) 7.1% (9/127) 0.48* ^{Weight (g) of previous largest baby, median (range 3520 (3260-3900) 3547 (3232-3856)} _{interquartile) (n=119) (n=124)} 0.62t Macrosomia (previous larger baby >4000g) 20.2% (24/119) 14.5% (18/124) 0.25* •Based on chi-square t distribution Based on ranks

The prevalence of GDM (defined using the IADPSG criteria) in the HN001 group was lower than in the placebo group, but this difference was not statistically significant (Table 2). However, by using the more specific definition of NZ, the prevalence of GDM was significantly lower in the HN001 group. These analyzes were repeated with imputed results for missing values, but there was little change in the RR estimates. Using IADPSG guidelines, imputed RR = 0.64 (95% CI: 0.36, 1.12) and using NZ guidelines, imputed RR = 0.39 (95% CI: 0.36, 1.12). : 0.14, 1.07).

Mean blood glucose levels at baseline and after one and two hours were slightly lower in the HN001 group compared to placebo, but were only significant at baseline (Table 2).

Table 2 Effects of treatment on the prevalence (95% CI) of gestational diabetes mellitus defined according to international* and NZf standards, and mean (95% CI) levels of blood glucose.

HN001 Placebo RR p-value (95% CI) _p -value multiple imputation

IADPSG* 8.2% (15/184) 13.8% (26/189) (4.6

(n=373) 13.1%) (n=184) %, (9.2%, 19.5%) 0.59 (0.32,1.08) 0.08 0.12

(n=189)

NZf 2.1% (4/194) (0.6 6.5% (13/200)

(n=394) %, 5.2%) (3.5%, 10.9%) 0.32 (0.11, 0.96) 0.03 0.07

(n=194) (n=200)

(mmol/l) (mmol/l) ^{Difference in mean}

_{(mmol/L) (95% CI)}

Fasting ^{4.32 (4.27, 4.37)} 4.40 (4.34, 4.46)

_{(n=195) (n=202)} -0.08 (-0.15, 0.00) 0.048 0.06

1 hour ^{6.71 (6.46, 6.96) 6.89 (6.63, 7.15)}

_{(n=185) (n=189)} -0.18 (-0.55, 0.18) 0.31 0.42

2 hours ^{5.65 (5.47, 5.83) 5.78 (5.57, 5.99)}

_{(n=194) (n=200)} -0.13 (-0.41, 0.15) 0.36 0.39 IADPSG, International Association of Diabetes and Pregnancy Study Groups

*Fasting >5.1 mmol/l, 1 hour >10 mmol/l, 2 hours >8.5 mmol/l

fFasting >5.5 mmol/l, 2 hours >9 mmol/l__________________________________

Among the 44 participants diagnosed with GDM according to the NZ or IADPSG guidelines, 40 participants had their HbA1c measured between one and fifteen months postpartum, with values between 5.0% (31 mmol/mol) and 6. 4% (46 mmol/mol). One had a GTT after delivery, with values within normal limits. Three participants did not have a postpartum HbA1c because one declined, one withdrew from the study, and one died.

Table 3 shows that maternal age, BMI, and history of GDM were significantly associated with GDM in this study. These factors were then used to stratify the analysis (Figure 2), using only the IADPSG definition of GDM. There was a significant interaction of treatment by age (as a continuous variable) (p=0.005) and a non-significant interaction with age dichotomized as >35 years versus <35 years (p=0.06).

In the older age group, HN001 was associated with a three-fold reduction in the prevalence of GDM compared with the prevalence among women in the placebo group (RR = 0.31 (95% CI: 0.12, 0.81), p=0.009). In women under 35 years of age, the prevalence in each study group was similar (RR=1.04 (95% CI 0.45, 2.39)). There was also no significant difference in effect depending on whether the BMI was 30 kg/m2 or more (RR = 0.86 (95% CI 0.37-1.96)) or less than 30 kg/m2 (RR = 0.51 (95% CI 0.22-1.17)). An interaction effect could not be assessed because the GDM was not repeated in any of the HN001 participants who had a history of GDM.

Among those with a history of GDM, HN001 protected against a recurrence of GDM, RR=0.00 (95% CI: 0.00 to 0.66) and for those without prior GDM, RR=0.50 (95% CI: 0.00 to 0.66). %: 0.20 to 1.27). Three women (20%) with a history of GDM did not have full GTT according to IADSPG guidelines. In all imputations these were positive for GDM giving an imputed RR = 0.38 ((95% CI 0.05-1.00) p=0.043, Barnard's exact test). The effect of HN001 on GDM was significantly protective among participants who had not used antibiotics between study enrollment and the GTT, but there was no significant effect of HN001 for those who had used antibiotics during this period.

The prevalence plots depicted in Figure 3 show the significant interaction for HN001 administration and maternal age (Figure 3, right plot), where those over 35 benefited more from HN001 than younger mothers and also the interaction for HN001 administration and previous pregnancy (Figure 3, parity, left diagram), where the effect of HN001 on gestational diabetes was significantly different depending on whether or not the woman had had a previous pregnancy of more of 20 weeks. In women who had had a previous pregnancy, those taking HN001 had approximately one-third the rate of GD compared to those taking placebo.

Table 3 The association of maternal risk factors with gestational diabetes mellitus, after adjustment for treatment group.

N RR (95% CI) _p Maternal age at enrollment* 372 1.11 (1.03,1.20) 0.004 Maternal BMI at enrollmentt 371 1.11 (1.08, 1.15) <0.0001 Family history of diabetes 67/373 1.13 (0.55, 2.33) 0.74 Ethnicity 373

European 293 1.00

Maori 46 1.49 (0.69, 3.22) 0.31 Pacific 7 3.05 (0.92, 10.07) 0.07 Asian 26 1.62 (0.62, 4.22) 0, 32 Polycystic Ovarian Syndrome 37/367 1.19 (0.50, 2.83) 0.70 Use of systemic antibiotics since enrollment 49/372 1.78 (0.91, 3.49) 0.09 Previous pregnancy (of any duration) 259/372 1.30 (0.66, 2.56) 0.45 Among women with a previous pregnancy (259/372)

Number of spontaneous abortions* 259 1.12 (0.78, 1.62) 0.54 Previous pregnancy >20 weeks 216/372 1.24 (0.68, 2.26) 0.47 Among women with previous pregnancies> 20 weeks (216/372)

Previous gestational diabetes 12/216 6.59 (3.95, 11.01) <0.0001 Macrosomia§ in previous child 35/213 0.66 (0.19, 2.35) 0.53 * for each additional year old

ffor each additional BMI unit; two values>45 truncated by 45 to ensure model fit.

* for each additional miscarriage

§ birth weight >4000 grams.

Similarly, among those not using antibiotics, mean fasting blood glucose levels were significantly (p=0.001) lower in the HN001 group (4.28, 95% CI 4.23-4.33). compared to the placebo group (4.42, 95% CI 4.35-4.48). Differences in mean glucose levels one and two hours after glucose loading were also smaller but not significant. At one hour, blood glucose levels were 6.63 (% CI 6.37-6.89) in the HN001 group and 6.88 (95% CI 6.60-7.16) in the HN001 group. placebo, p=0.20, and at two hours, levels were 5.56 (95% CI 5.38-5.73) in HN001 and 5.77 (95% CI 5.53-6, 01) in the placebo group, p=0.15.

HN001 was not significantly associated with any maternal anthropometric measures (after adjustment for baseline measures), or infant birth weight, gestation, cesarean delivery, or NICU admission, or, at 4-7 days postpartum, infant length, PI or head circumference. Infants whose mothers were in the HN001 group had significantly higher five-minute Apgar scores than infants in the placebo group (Table 4).

Table 4 Effects of treatment on birth outcomes

HN001 Placebo Geometric ^{Mean Ratios} _{(95% CI) p}

Maternal weight (kg) postpartum 76.7 (76.1, 76.8 (76.2, 1.00 (0.99, 1.01) 0.79 (geometric mean, 95% CI) 77, 2) (n=197) 77.4) (n=194)

Maternal waist (cm) after 97.7 (96.8, 97.3 (96.4, 1.00 (0.99, 1.02) 0.53 parta (geometric mean, 95% CI) 98, 7) (n=195) 98.2) (n=195)

Maternal postpartum BMI ^ (geometric mean 28.0 (27.8, 28.1 (27.9, 1.00 (0.99, 1.01) 0.69, 95% CI 28.2) (n=197) 28.3) (n=194)

Number of weeks of gestation 39.7 (38.7, 39.6 (38.7, -0.1 (-0.4, 0.1) 0.3* (median, interquartile range) 40.7) (n= 205) 40.4) (n=201)

Mean Differences (95% CI)

Child birth weight (kg) (mean, CI 3.6 (3.5, 3.7) 3.5 (3.4, 3.6) 0.1(-0.1, 0.2) 0 .36 of 95%) (n=205) (n=202)

Infant length at birth (cm) (mean, 51.3 (51.0, 51.2 (50.8, 0.2 (-0.3, 0.7) 0.48 95% CI) 51, 7) (n=205) 51.5) (n=199)

Child weight index (mean, CI 25.9 (25.5, 25.7 (25.4, 0.1 (-0.4, 0.7) 0.59 95%) 26.2) (n =204) 26.1) (n=199)

Child head circumference (cm) 35.3 (35.1, 35.4 (35.2, -0.1 (-0.40.2) 0.67 (mean, 95% CI 35.6) (n=205) 35.6) (n=201)

RR (95% CI) Macrosomia (>4000 grams) 22.4% 15.8% 1.41 (0.94, 2.12) 0.10

(46/205) (32/202)

Preterm (<37 weeks of 7.8% (16/205) 4.0% (8/201) 1.96 (0.86, 4.48) 0.10 gestation)

Cesarean delivery 27.7% 25.4% 1.09 (0.79, 1.51) 0.60

(57/206) (51/201)

NICU admission 11.3% 11.0% 1.04 (0.60, 1.80) 0.90

(23/203) (22/201)

Apgar score >7 at 5 minutes 98.5% 98.0% *1.51 (1.01, 2.27) 0.04

(200/203) (198/202)

NICU, neonatal intensive care unit

•Analysis of covariance on recorded values, adjusted for measurements recorded at baseline, geometric means adjusted for baseline geometric mean

"•"Wilcoxon rank sum test

*Odds ratio of having a higher Apgar score, pooled Apgar scores 0-3, 4-6, 7, 8, 9, 10. Ordinal logistic regression._______________________________________________________________

Maternal GDM, defined according to IADPSG recommendations, was associated with higher maternal weight (p=0.0002), waist circumference (p<0.0001), and BMI (p<0.0001). after delivery, but was not associated with any infant anthropometric measurements, gestation, cesarean delivery, NICU admission, or 5-minute Apgar score (data not shown).

Discussion

To the applicant's knowledge, this is the first study to report a role for probiotics in the prevention of GDM among women not selected on the basis of gDm risk. The applicant's data suggest that the probiotic HN001 at a dose of 6 * 109 cfu/day can reduce the rate of GDM from 13.8% to 8.2%, a reduction of 40% using the guidelines of the ⁱ A ^d PSG(6) or a 68% reduction from 6.5% to 2.1% using NZ guidelines.

This study showed that the HN001 effect was stronger by using higher NZ glucose thresholds than IADPSG thresholds to define GDM, suggesting that the effect is greater in preventing more severe GDM.

The gut microbiota is profoundly altered during the three trimesters of pregnancy towards a less diverse state, with the lowest microbial richness found in women with GDM(9). In contrast to obesity-related gut microbiota, late trimester gut microbiota has been associated with a greater amount of energy lost in feces compared to first trimester, indicating that the impact of gut microbiota alterations during pregnancy in the adiposity of the host and in the host glucose metabolism are not necessarily identical(9). Without wishing to be bound by theory, Applicants believe that HN001 supplementation altered the composition and function of the gut microbiota in favor of improved insulin sensitivity and host inflammation, thereby reducing the propensity for GDM.

The absence of any deleterious effect on birth outcomes supports HN001 as a safe intervention from early pregnancy (14-16 weeks' gestation), which may also be beneficial to the infant, as reflected by the Apgar score. These findings are important given the small amount of data available on the effects of probiotic interventions in early pregnancy.

The promotion of good health in pregnancy through diet or weight control programs has been largely ineffective, partly due to poor adherence to the interventions. This study provides evidence that L. rhamnosus HN001 is an effective intervention to reduce the prevalence of GDM, particularly in older mothers and those with a history of GDM.

industry application

This invention relates to the use of probiotic bacteria, particularly Lactobacillus rhamnosus HN001, in particular in the treatment or prevention of GDM.

References

1. Ministry of Health (2014) Screening, diagnosis and management of gestational diabetes in New Zealand: A clinical practice guideline. Ministry of Health, Wellington.

2. American Committee of Obstetricians and Gynecologists Committee Opinion number 549 (January 2013) Obesity in pregnancy. Obstet Gynecol 121: 213-217.

3. Kim SY, England L, Wilson HG, Bish C, Satten GA, Dietz P (2010) Percentage of gestational diabetes mellitus attributable to overweight and obesity. Am J Pub Health 100: 1047-1052.

4. Ben-Haroush A, Yogev Y, Hod M (2004) Epidemiology of gestational diabetes mellitus and its association with Type 2 diabetes. Diabet Med 21: 103-113.

5. Kim C, Newton KM, Knopp RH (2002) Gestational diabetes and the incidence of type 2 diabetes. Diabetes Care 25: 1862-1868.

6. International Association of Diabetes and Pregnancy Study Groups Consensus Panel (2010) International Association of Diabetes and Pregnancy Study Groups Recommendations on the diagnosis and classification of hyperglycemia in pregnancy. Diabetes Care 33: 676-682.

7. Luoto R, Laitinen K, Nermes M, Isolauri E (2010) Impact of maternal probiotic-supplemented dietary counseling on pregnancy outcome and prenatal and postnatal growth: a double-blind, placebo-controlled study. Br JNutr 103: 1792-1799.

8. Wickens K, Black PN, Stanley TV, et al., (2008) A differential effect of 2 probiotics in the prevention of eczema and atopy: A double-blind, randomized, placebo-controlled trial. J Allergy Clin Immunol 122: 788-794.

9. Koren O, Goodrich JK, Tyler CC, et al., (2012) Host remodeling of the gut microbiome and metabolic changes during pregnancy. Cell 150: 470-480.

10. Barrett HL, Dekker Nitert M, Conwell LS, Callaway LK. Probiotics for preventing gestational diabetes. Cochrane Database Syst Rev. 2014; Art. No.: CD009951. DOI: 10.1002/14651858.CD009951.

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Claims (14)

1. L. rhamnosus HN001, AGAL deposit number NM97/09514 dated August 18, 1997, for use in the treatment or prevention of gestational diabetes mellitus (GDM) or one or more risks associated with GDM or a or more sequelae of GDM in an adult subject, or for use in the treatment or prevention of one or more risks associated with GDM or one or more sequelae of GDM in a fetal, neonatal, infant, or child subject, in where a. use in treatment or prevention comprises administration of L. rhamnosus HN001 to the adult subject or to the mother of the fetal, neonate, infant or child subject; Y b. the mean fasting blood glucose level is reduced in the adult subject, or in the mother of the fetal, neonate, infant, or child subject to less than about 4.35 mmol/L. 2. A composition comprising L. rhamnosus HN001, AGAL deposit number NM97/09514 dated August 18, 1997, for use in the treatment or prevention of GDM or one or more risks associated with GDM or one or more sequelae of GDM in an adult subject, or for use in the treatment or prevention of one or more risks associated with GDM or one or more sequelae of GDM in a fetal, neonate, infant, or child subject, where a. use in treatment or prevention comprises administration of L. rhamnosus HN001 to the adult subject or to the mother of the fetal, neonate, infant or child subject; Y b. the mean fasting blood glucose level is reduced in the adult subject, or in the mother of a fetal, neonate, infant, or child subject to less than about 4.35 mmol/L. The composition for use of claim 2, wherein the composition comprises a physiologically acceptable diluent, adjuvant, carrier or excipient. The composition for use of claim 2 or 3, wherein the composition is or comprises a food, preferably a food selected from fermented milk, yogurt, cheese, milk drink and powdered milk. The composition for use of claim 2 or 3, wherein the composition is (1) a pharmaceutical composition comprising a pharmaceutically acceptable diluent, adjuvant, carrier, or excipient, or (2) a maternal formula or a maternal supplement. The composition for use of any one of claims 2 to 5, wherein L. rhamnosus HN001 is in a reproductively viable form. The composition for use of any one of claims 2 to 6, wherein the L. rhamnosus HN001 is killed, lysed, cleaved, or attenuated. The composition for use of any one of claims 2 to 7, wherein the subject is a mother or a fetal, neonate, infant or child subject. The composition for use of any one of claims 2 to 8, wherein the adult subject, or the mother of a fetal, neonate, infant or child subject, is at increased risk of GDM. The composition for use of claim 8 or 9, wherein the adult subject, or the mother, has previously suffered from GDM. The composition for use of any one of claims 8 to 0, wherein the adult subject, or the mother, is over 35 years of age. The composition for use of any of claims 2 to 11, wherein the adult subject, or the mother, has previously been pregnant. The composition for use of any one of claims 2 to 12, wherein the composition is administered after the first trimester of pregnancy. The composition for use of any one of claims 2 to 11, wherein the composition is administered between 14-16 weeks of gestation.