U.S. patent application number 17/239889 was filed with the patent office on 2021-12-23 for synthetic composition for preventing or treating cvd.
The applicant listed for this patent is GLYCOM A/S. Invention is credited to Emma Elison, Bruce McConnell, Louise Kristine Vigsn.ae butted.s.
Application Number | 20210393657 17/239889 |
Document ID | / |
Family ID | 1000005810939 |
Filed Date | 2021-12-23 |
United States Patent
Application |
20210393657 |
Kind Code |
A1 |
Vigsn.ae butted.s; Louise Kristine
; et al. |
December 23, 2021 |
SYNTHETIC COMPOSITION FOR PREVENTING OR TREATING CVD
Abstract
A method for reducing the likelihood of a non-infant human
experiencing a cardiovascular disease (CVD) associated with
hypercholesterolemia, hypertension, or a metabolic disorder such as
type II diabetes and/or insulin resistance. Various examples of the
method include selecting an amount of one to five human milk
oligosaccharides (HMOs) selected from the fucosylated HMOs
2'-fucosyllatcose (2'-FL), difucosyllactose (DFL), 3-fucosyllactose
(3-FL), and lacto-N-fucopentaose I (LNFP-I) and the non-fucosylated
neutral HMOs lacto-N-tetraose and lacto-N-neotetraose that is
effective for increasing the relative abundance of Bifidobacterium
adolescentis in the gastrointestinal microbiota of the non-infant
human; increasing the relative abundance of Bifidobacterium
adolescentis in the gastrointestinal microbiota of the non-infant
human and reducing serum levels of low-density lipoprotein (LDL)
cholesterol and/or increasing GLP-1 or reducing hypertension in the
non-infant human by administering the selected amount of the
selected HMOs during an initial treatment phase.
Inventors: |
Vigsn.ae butted.s; Louise
Kristine; (Kobenhavn, DK) ; McConnell; Bruce;
(La Tour de Peilz, CH) ; Elison; Emma; (Hjarup,
SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GLYCOM A/S |
Horsholm |
|
DK |
|
|
Family ID: |
1000005810939 |
Appl. No.: |
17/239889 |
Filed: |
April 26, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15183431 |
Jun 15, 2016 |
10987368 |
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17239889 |
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15104794 |
Jun 15, 2016 |
10828313 |
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PCT/DK2015/050385 |
Dec 8, 2015 |
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15183431 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/702 20130101;
A61K 9/2054 20130101; A61K 9/4841 20130101; A61K 2035/115 20130101;
A61K 35/745 20130101; A23L 33/21 20160801; A23L 33/135 20160801;
A23V 2002/00 20130101 |
International
Class: |
A61K 31/702 20060101
A61K031/702; A61K 35/745 20060101 A61K035/745; A61K 9/20 20060101
A61K009/20; A61K 9/48 20060101 A61K009/48; A23L 33/21 20060101
A23L033/21; A23L 33/135 20060101 A23L033/135 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 8, 2014 |
DK |
PA 2014 70768 |
Claims
1. A method for reducing the likelihood of a non-infant human
experiencing a cardiovascular disease (CVD) associated with
hypercholesterolemia, the method comprising: selecting an amount of
one to five human milk oligosaccharides (HMOs) selected from the
fucosylated HMOs 2'-fucosyllatcose (2'-FL), difucosyllactose (DFL),
3-fucosyllactose (3-FL), and lacto-N-fucopentaose I (LNFP-I) and
the non-fucosylated neutral HMOs lacto-N-tetraose and
lacto-N-neotetraose that is effective for increasing the relative
abundance of Bifidobacterium adolescentis in the gastrointestinal
microbiota of the non-infant human; and increasing the relative
abundance of Bifidobacterium adolescentis in the gastrointestinal
microbiota of the non-infant human and reducing serum levels of
low-density lipoprotein (LDL) cholesterol in the non-infant human
by administering the selected amount of the selected HMOs during an
initial treatment phase.
2. The method of claim 1, further comprising increasing serum
levels of high-density lipoprotein (HDL) cholesterol in the
non-infant human by administering the selected amount of the
selected HMOs.
3. The method of claim 1, wherein at least one of the selected HMOs
is 2'-FL.
4. The method of claim 1, wherein at least one of the selected HMOs
is LNnT.
5. The method of claim 1, wherein the initial treatment phase is at
least 14 days.
6. The method of claim 1, wherein the effective amount of the
selected HMOs during the initial treatment phase is from about 3.5
g to about 7.5 g per day.
7. The method of claim 6, further comprising administering a dosage
of the selected HMOs during a maintenance period that is reduced
relative to the effective amount administered during the initial
treatment phase.
8. A method for reducing the likelihood of a non-infant human
experiencing a cardiovascular disease (CVD) associated with
hypertension, the method comprising: selecting an amount of one to
five human milk oligosaccharides (HMOs) selected from the
fucosylated HMOs 2'-fucosyllatcose (2'-FL), difucosyllactose (DFL),
3-fucosyllactose (3-FL), and lacto-N-fucopentaose I (LNFP-I) and
the non-fucosylated neutral HMOs lacto-N-tetraose and
lacto-N-neotetraose that is effective for increasing the relative
abundance of Bifidobacterium adolescentis in the gastrointestinal
microbiota of the non-infant human; and increasing the relative
abundance of Bifidobacterium adolescentis in the gastrointestinal
microbiota of the non-infant human and reducing the risk of the
non-infant human experiencing hypertension by administering the
selected amount of the selected HMOs during an initial treatment
phase.
9. The method of claim 8, further comprising increasing serum
levels glucagon-like peptide 1 (GLP-1) in the non-infant human by
administering the selected amount of the selected HMOs.
10. The method of claim 8, wherein at least one of the selected
HMOs is 2'-FL.
11. The method of claim 8, wherein at least one of the selected
HMOs is LNnT.
12. The method of claim 8, wherein the initial treatment phase is
at least 14 days.
13. The method of claim 8, wherein the effective amount of the
selected HMOs during the initial treatment phase is from about 3.5
g to about 7.5 g per day.
14. The method of claim 13, further comprising administering a
dosage of the selected HMOs during a maintenance period that is
reduced relative to the effective amount administered during the
initial treatment phase.
15. A method for reducing the likelihood of a non-infant human
having a metabolic disorder experiencing a cardiovascular disease
(CVD) associated with the metabolic disorder, the method
comprising: selecting an amount of one to five human milk
oligosaccharides (HMOs) selected from the fucosylated HMOs
2'-fucosyllatcose (2'-FL), difucosyllactose (DFL), 3-fucosyllactose
(3-FL), and lacto-N-fucopentaose I (LNFP-I) and the non-fucosylated
neutral HMOs lacto-N-tetraose and lacto-N-neotetraose that is
effective for increasing the relative abundance of Bifidobacterium
adolescentis in the gastrointestinal microbiota of the non-infant
human; and increasing the relative abundance of Bifidobacterium
adolescentis in the gastrointestinal microbiota of the non-infant
human and reducing the risk of the non-infant human experiencing
hypertension by administering the selected amount of the selected
HMOs during an initial treatment phase.
16. The method of claim 15, further comprising increasing serum
levels of glucagon-like peptide 1 (GLP-1) in the non-infant human
by administering the selected amount of the selected HMOs.
17. The method of claim 15, wherein the metabolic disorder
comprises one or more of type II diabetes and insulin
resistance.
18. The method of claim 15, wherein at least one of the selected
HMOs is 2'-FL.
19. The method of claim 15, wherein at least one of the selected
HMOs is LNnT.
20. The method of claim 15, wherein: the initial treatment phase is
at least 14 days; and the effective amount of the selected HMOs
during the initial treatment phase is from about 3.5 g to about 7.5
g per day.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. patent application Ser. No.
15/183,431, filed Jun. 15, 2016, which is a continuation-in-part of
U.S. patent application Ser. No. 15/104,794, filed Jun. 15, 2016,
now U.S. Pat. No. 10,828,313, which is a national stage filing in
accordance with 35 U.S.C. .sctn. 371 of PCT/DK2015/050385, filed
Dec. 8, 2015, which claims the benefit of the priority of Denmark
Patent Application No. PA 2014 70768, filed Dec. 8, 2014, the
contents of each of which are incorporated herein by reference for
all purposes permissible by law.
FIELD
[0002] This disclosure relates to a method and composition for
reducing the risk of, preventing, or treating CVD and associated
co-morbidities in overweight or obese humans.
BACKGROUND
[0003] The increasing trend of obese individuals has become a major
health issue over the past several decades and World Health
Organization (WHO) has declared obesity as a global epidemic.
According to WHO, it was estimated that more than 1.9 billion
adults were overweight in 2014, and among them, at least 600
million were obese. This means that worldwide, obesity has more
than doubled since 1980 (WHO, fact sheet from January 2015). The
rapid increase in obesity over such a short time frame makes a
novel genetic cause per se unlikely and strongly favours modified
environmental factors over the past 30 years. Such environmental
factors include dietary habits, exercise or energy expenditure, and
lifestyle. Indeed, there appears to be a strong correlation between
Westernization in terms of diet and lifestyle and obesity. A shift
from more traditional diets, rich in whole-plant foods like
whole-grain cereals, fruits, and vegetables, to modern
Western-style diets rich in refined carbohydrates, fat, and
red/processed meats and low in fibre and whole-plant foods, is
strongly correlated with increased body weight, obesity, and the
diseases of obesity (Conterno et al. Genes Nutr. 6, 241). Some
cardiovascular conditions which are often found in obese humans may
also have an association with gut dysbiosis.
SUMMARY
[0004] A method for reducing the likelihood of a non-infant human
experiencing a cardiovascular disease (CVD) associated with
hypercholesterolemia. In some examples, the method includes
selecting an amount of one to five human milk oligosaccharides
(HMOs) selected from the fucosylated HMOs 2'-fucosyllatcose
(2'-FL), difucosyllactose (DFL), 3-fucosyllactose (3-FL), and
lacto-N-fucopentaose I (LNFP-I) and the non-fucosylated neutral
HMOs lacto-N-tetraose and lacto-N-neotetraose that is effective for
increasing the relative abundance of Bifidobacterium adolescentis
in the gastrointestinal microbiota of the non-infant human;
increasing the relative abundance of Bifidobacterium adolescentis
in the gastrointestinal microbiota of the non-infant human and
reducing serum levels of low-density lipoprotein (LDL) cholesterol
in the non-infant human by administering the selected amount of the
selected HMOs during an initial treatment phase.
[0005] A method is also disclosed for reducing the likelihood of a
non-infant human experiencing a cardiovascular disease (CVD)
associated with hypertension. In some examples, the method includes
selecting an amount of one to five human milk oligosaccharides
(HMOs) selected from the fucosylated HMOs 2'-fucosyllatcose
(2'-FL), difucosyllactose (DFL), 3-fucosyllactose (3-FL), and
lacto-N-fucopentaose I (LNFP-I) and the non-fucosylated neutral
HMOs lacto-N-tetraose and lacto-N-neotetraose that is effective for
increasing the relative abundance of Bifidobacterium adolescentis
in the gastrointestinal microbiota of the non-infant human and
increasing the relative abundance of Bifidobacterium adolescentis
in the gastrointestinal microbiota of the non-infant human and
reducing the risk of the non-infant human experiencing hypertension
by administering the selected amount of the selected HMOs during an
initial treatment phase.
[0006] A further method is disclosed for reducing the likelihood of
a non-infant human having a metabolic disorder experiencing a
cardiovascular disease (CVD) associated with the metabolic
disorder. In certain examples, the method includes selecting an
amount of one to five human milk oligosaccharides (HMOs) selected
from the fucosylated HMOs 2'-fucosyllatcose (2'-FL),
difucosyllactose (DFL), 3-fucosyllactose (3-FL), and
lacto-N-fucopentaose I (LNFP-I) and the non-fucosylated neutral
HMOs lacto-N-tetraose and lacto-N-neotetraose that is effective for
increasing the relative abundance of Bifidobacterium adolescentis
in the gastrointestinal microbiota of the non-infant human and
increasing the relative abundance of Bifidobacterium adolescentis
in the gastrointestinal microbiota of the non-infant human and
reducing the risk of the non-infant human experiencing hypertension
by administering the selected amount of the selected HMOs during an
initial treatment phase.
[0007] In some embodiments, a method is disclosed that includes
selecting a non-infant patient having an obesity-related metabolic
disorder and being diagnosable with one or more of obesity,
obesity-induced pre-diabetes, and obesity-induced type 2
diabetes.
[0008] The present disclosure provides synthetic compositions
comprising one or more HMOs that can be advantageously used to
reduce the risk of, prevent or treat CVD or CVD-associated
pathologic condition or disease in a human, preferably, in an
overweight or obese human individual.
[0009] Accordingly, in addition to the above-described
examples:
[0010] a first aspect of this disclosure relates to a human milk
oligosaccharide or a mixture of two to five human milk
oligosaccharides for reducing the propensity of a cardiovascular
disease (CVD) and/or a CVD-associated pathological condition or
disease in a human, preferably, in an overweight or obese human
individual;
[0011] a second aspect of this disclosure relates to a human milk
oligosaccharide or a mixture of two to five human milk
oligosaccharides for preventing development of a cardiovascular
disease (CVD) and/or a CVD-associated pathological condition or
disease in a human, preferably, in an overweight or obese human
individual;
[0012] a third aspect of this disclosure relates to a human milk
oligosaccharide or a mixture of two to five human milk
oligosaccharides for treating a cardiovascular disease (CVD) and/or
a CVD-associated pathological condition or disease in a human,
preferably, in an overweight or obese human individual;
[0013] a fourth aspect of this disclosure provides a method for
reducing the propensity of a cardiovascular disease (CVD) and/or a
CVD-associated pathological condition or disease in a human,
preferably, in an overweight or obese human individual, the method
comprising administering to the human an effective amount of a
human milk oligosaccharide or an effective amount of a mixture of
two to five human milk oligosaccharides, or a composition
comprising an effective amount of a human milk oligosaccharide or
an effective amount of mixture of said two to five human milk
oligosaccharides;
[0014] a fifth aspect of this disclosure provides a method for
preventing development of a cardiovascular disease (CVD) and/or a
CVD-associated pathological condition or disease in a human,
preferably, in an overweight or obese human individual, the method
comprising administering to the human an effective amount of a
human milk oligosaccharide or a mixture of two to five human milk
oligosaccharides, or a composition comprising an effective amount
of said human milk oligosaccharide, or an effective amount of
mixture of said two to five human milk oligosaccharides;
[0015] a sixth aspect of this disclosure provides a method for
treating a cardiovascular disease (CVD) and/or a CVD-associated
pathological condition or disease in a human, preferably, in an
overweight or obese human individual, the method comprising
administering to the human an effective amount of a human milk
oligosaccharide or a mixture of two to five human milk
oligosaccharides, or a composition comprising an effective amount
of said human milk oligosaccharide or an effective amount of
mixture of said two to five human milk oligosaccharides;
[0016] a seventh aspect of this disclosure provides a method for
increasing the abundance of bifidobacteria in a human, preferably,
in an overweight or obese human having a propensity of, or
diagnosed with a cardiovascular disease (CVD), the method
comprising administering to the patient one or more HMOs selected
from the group consisting of fucosylated HMOs and core HMOs,
preferably of a mixture of one or more fucosylated HMOs and one or
more core HMOs.
DETAILED DESCRIPTION
Introduction
[0017] Overweight and obesity may be associated with accumulated
abdominal visceral fat and can be related to psycho-sociological
behavioural disorders. It is often associated with the development
of several chronic complications, which may increase the risk of
developing metabolic diseases such as type 2 diabetes and
cardiovascular diseases (CVD).
[0018] High levels of low-density lipoprotein cholesterol (LDL-C)
and triglyceride concentrations and low levels of high-density
lipoprotein cholesterol (HDL-C) in the blood is a precursor to
hypertension, hyperlipidaemia, and causes the formation and
build-up of atherosclerotic plaque in the arteries leading to
higher risk of CVD. Cardiovascular risk factors are not only
observed in adults, but also obese children and young adults suffer
from dyslipidaemia, hypertension, hyperinsulinemia, or insulin
resistance.
[0019] Cholesterol concentrations within the circulatory pool are
products of input from gut absorption and endogenous synthesis
relative to clearance through hepatic and extrahepatic tissue
pathways. A disruption in any of these mechanisms can alter this
balance, which is reflected in plasma cholesterol concentrations
and subsequent CVD progression. Complex interplay between the gut
intestinal microbiota and the diverse human physiological systems
are taking plays in the human body, and it has been implicated that
an imbalance in this host-microbiota interaction can disrupt the
energy homeostasis and lipid metabolism (Zhang et al. EBioMedicine
2, 966 (2015); Conterno et al. Genes Nutr. 6, 241 (2011)).
[0020] Gut microbiota is a specific entity within the body which
has its own genome whose gene pool is much more abundant than the
one of its host. It has been estimated that the human intestine
harbours 10.sup.13 to 10.sup.14 bacterial cells and the number of
bacteria outnumbers the total number of cells in the body by a
factor of 10 (Gill et al. Science 312, 1355 (2006)).
[0021] In diet-induced obesity, over-nutrition can alter
composition of the gut microbiota, with dietary nutrients
influencing the growth of certain species. Diets rich in
cholesterol, saturated fats, and simple carbohydrates are
associated with a gut microbiota rich in particular organisms
belonging to the Firmicutes phylum. In line with this, it has been
shown that there are marked differences in the gut microbiota
between healthy, obese, and type 2 diabetic patients (Backhed et
al. PNAS 101, 15718 (2004), Qin et al. Nature 490, 55 (2012)) with
fewer Bacteroidetes and more Firmicutes in obese than lean people.
However, this proportion has shown to change with weight loss
leading to increase in the abundance of Bacteroidetes and decrease
in the abundance of Firmicutes (Ley et al. Nature 444, 1022
(2006)). Additionally, specific changes at genus level have been
observed with lower number of bifidobacteria in obese versus lean
and diabetic versus non-diabetic individuals (Schwiertz et al.
Obesity 18, 190 (2009)).
[0022] It would be advantageous to be able to prevent or reduce the
damaging consequences of a dysbiotic microbiota in overweight and
obesity. Modulation of the microbiota increasing the abundance of
beneficial bacteria could be a way to interrupt the processers
involved in CVD and hence improve cardiovascular health. Beneficial
bacteria such as bifidobacteria have shown to ameliorate both
metabolic and immunological dysfunctions related to obesity. As an
example, Bifidobacterium pseudocatenulatum has shown to reduce
serum cholesterol, triglyceride and glucose levels and decrease
insulin resistance and improve glucose tolerance in obese mice.
Additionally, the species can reduce liver steatosis and the number
of larger adipocytes in enterocytes of obese mice (Cano et al.,
Obesity, 21, 2310 (2013)).
[0023] One mode of action for lowering cholesterol by
bifidobacteria is the processing of bile salts. Metabolism of
cholesterol, a precursor of bile acids, is mediated through the
bacteria expressing the enzyme bile salt hydrolase (BSH). Some
bifidobacteria have high BSH activity promoting deconjugation of
bile acids in the gut to secondary amino acid conjugates. When
these secondary conjugates are excreted, cholesterol is broken down
to replace the processed bile salts. Overall, this process promotes
the catabolism of cholesterol, leading to reduced serum levels
(Ettinger et al., Gut Microbes 5, 719 (2014)). Another mechanism is
through bacterial metabolites like short chain fatty acids (SCFA),
including acetate, propionate, and butyrate. Acetate has shown to
be negatively associated with visceral adipose tissue and insulin
levels in obese individuals and propionate has shown to reduce
lipogenesis and cholesterol synthesis inhibition (Verbeke et al.,
Nutrition Research Reviews 28, 42 (2015)).
[0024] Probiotic supplementation could be an approach, however, the
addition of a small number of different probiotics to the intestine
is unlikely to fully promote a beneficial intestinal microbiota
composition with sufficient production of metabolites.
[0025] WO 2013/154725 describes that some sialylated and
fucosylated HMOs has a positive effect on the growth of certain
strains of bifidobacteria that are typically found in both infant
and adult microbiota.
[0026] EP-A-1332759 discloses that oral doses of 2'-FL, 3'-SL,
6'-SL, LNnT and sialic acid promote insulin secretion in type 2
diabetes-model mice.
[0027] EP-A-2143341 discloses that a mixture of GOS, sialylated
oligosaccharides and N-acylated oligosaccharides reduces
triglyceride concentration in liver in model mice.
[0028] EP-A-2332552 discloses that 3'-SL and 6'-SL reduce/prevent
fat accumulation in the liver and other organs in high-fat diet
mice and rats.
[0029] WO 2013/057061 discloses a composition for increasing
insulin sensitivity and/or reducing insulin resistance. The
composition contains long chain polyunsaturated fatty acids,
probiotics and a mixture of oligosaccharides containing at least
one of lacto-N-neotetraose (LNnT) and lacto-N-tetraose (LNT), at
least one N-acetylated oligosaccharide different from LNnT and LNT,
at least one sialylated oligosaccharide and at least one neutral
oligosaccharide, for use in increasing insulin sensitivity and/or
reducing insulin resistance. This composition can also contain
2'-O-fucosyllactose (2'-FL). The composition is particularly
adapted for use in infants who were born preterm and/or who
experienced IUGR, and in pregnant women suffering from gestational
diabetes. It is also stated that the composition can be given to
children, adolescents, and adults suffering from insulin resistance
and/or type II diabetes. It is stated that the efficacy of the
composition can be the result of the synergistic combination of
immunity modulator effects triggered by the probiotics and the
LC-PUFA through their stimulation with the specific oligosaccharide
mixture.
[0030] WO 2014/187464 discloses a synthetic mixture of
oligosaccharides comprising at least 6 oligosaccharides selected
from fucosylated, sialylated, sulfated, GlcNAc-, GalNAc- and
mannose-containing oligosaccharides, for treating a microbiota of a
human, to reduce or eliminate the activity and/or the proportion of
a microbe in the microbiota that is associated with the development
or maintenance of a cardiovascular disease.
[0031] However, there remains a need for effective interventions
which are able to prevent or reduce CVD and long-term effects of
CVD in CVD patients, especially where the patients are overweight
or obese, which are safe, well tolerated and well accepted.
[0032] It has now been surprisingly found that administration of
human milk oligosaccharides (HMOs) to an obese patient,
preferentially increases the abundance of bifidobacteria in the
gastrointestinal tract, reducing cholesterol and/or hypertension,
and through this reduces the risk of CVD and associated
co-morbidities. Further, the abundance of members of the
Bifidobacterium adolescentis phylogenetic group is increased, in
particular B. adolescentis and/or B. pseudocatenulatum.
[0033] The increased abundance of bifidobacteria leads to
production of SCFAs though the fermentation of HMOs and increased
activity of BSH. Thus, it has been discovered that HMOs can, by
oral or enteral ingestion, increase the production of SCFA and
activity of BSH, possibly through modulation of the intestinal
microbiota in human. As an outcome, a more beneficial intestinal
microbial community can be shaped and maintained, which contributes
to attenuation of hypercholesterolemia and hypertension. This can
result in reduced risk of, prevention of and/or treatment of, CVD
and associated co-morbidities.
Terms and Definitions
[0034] The terms "human", "non-infant human" and "non-infant" all
mean in the present context a human individual of at least 3 years
old. A human can be a child, a teenager, an adult, or an elderly
human, preferably, the human is an individual of at least 3 years
old that has an excess of body fat, more preferably, an individual
whose excess body fat has accumulated to the extent that it may
have a negative effect on health, i.e. an overweight or obese human
individual.
[0035] Body fat percentage preferably means total mass of body fat
divided by total mass of the body.
[0036] The term "obese human individual" means that a human
individual that has a body mass index (BMI), a measurement obtained
by dividing the individual's weight by the square of the
individual's height, over 30 kg/m.sup.2, with the range 25-30
kg/m.sup.2 defined as overweight.
[0037] Overweight and obesity for children and teens (human
individuals aged 3-19 years old) is defined as the following:
overweight is defined as a BMI at or above the 85th percentile and
below the 95th percentile for children and teens of the same age
and sex. Obesity is defined as a BMI at or above the 95th
percentile for children and teens of the same age and sex (see
Rolland-Cachera, Int. J. Pediatr. Obesity 6, 325 (2011)).
[0038] The term "cardiovascular disease (CVD)" refers broadly to
any disease of the heart and circulatory system (arteries and
veins). Cardiovascular disease generally refers to conditions that
involve narrowed or blocked blood vessels that can lead to a heart
attack, chest pain (angina) or stroke. Other heart conditions, such
as those that affect the heart muscle, valves, or rhythm, also are
also contemplated as forms of heart disease. Examples of CVD
include, but not limited to, coronary artery disease (blockage of
blood vessels that serve the heart), acute coronary syndrome
(symptoms such as pain, weakness, and tiredness caused by coronary
artery disease), angina pectoris (pain resulting from coronary
artery disease or other causes), myocardial infarction (heart
attack, with damage to heart muscle caused by coronary artery
disease), and left ventricular thrombus (blood clot in the left
side of the heart that pumps blood into your body).
[0039] CVD may be accompanied with health complications (that are
interchangeably referred herein as pathologic conditions) or
associated diseases, which are also contemplated herein. Some
non-limiting examples of relevant contemplated health complications
and CVD-associated diseases/pathologic conditions include heart
failure (occurs when the heart cannot adequately pump blood
throughout the body; this can cause shortness of breath, dizziness,
confusion, and the build-up of fluid in the body, causing
swelling), heart attack (occurs when the coronary arteries narrow
so much that they cut off blood supply to the heart; the heart
cells begin to die as they are deprived of oxygen and symptoms
include shortness of breath and severe chest pain that may radiate
to the back, jaw, or left arm), stroke (occurs due formation and
lodging of blood clots in a blood vessel in the brain and cutting
thus off blood flow; stroke symptoms include: numbness on one side
of the body, confusion, trouble, speaking, loss of balance or
coordination), pulmonary embolism (is similar to a stroke, but the
blocked blood vessel is in the lungs instead of the brain; symptoms
include shortness of breath, chest pain on breathing, and bluish
skin), cardiac arrest (occurs when the heart suddenly stops
beating; this will lead to death if not treated immediately),
Peripheral Artery Disease (PAD) (occurs due to narrowing in the
arteries that supply blood to the arms and legs; the main symptom
of PAD is severe leg pain when walking).
[0040] The term "patient" means a human who has been diagnosed by a
medical practitioner as having a disease or a pathological
condition. Both paediatric or adult patients are contemplated.
Embodiments of the disease and pathological condition are discussed
above. Preferably, the patient is an overweight or obese individual
that is having a CVD or a CVD-associated pathological condition or
disease.
[0041] The term "propensity "in the present context means natural
tendency of a human individual to develop later in life a medical
condition, such as a disease, in particular a CVD or a
CVD-associated pathological condition or disease.
[0042] The term "preventing CVD and/or CVD associated pathological
condition or disease" in the present context means eliminating or
minimising a chance of development of a CVD disease or a
pathological condition or disease associated with an CVD. Both
primary and secondary prevention are thus contemplated. The primary
prevention means preventing a CVD or associated disease or
condition before it occurs, and the secondary prevention means
preventing additional attacks of a CVD or development of associated
condition or disease after the first attack has occurred.
[0043] The term "enteral administration" means any conventional
form for delivery of a composition to a human that causes the
deposition of the composition in the gastrointestinal tract
(including the stomach). Methods of enteral administration include
feeding through a nasogastric tube or jejunum tube, oral,
sublingual, and rectal.
[0044] The term "oral administration" means any conventional form
for the delivery of a composition to a human through the mouth.
Accordingly, oral administration is a form of enteral
administration. The term "effective amount" preferably means an
amount of a human milk oligosaccharide sufficient to render a
desired treatment outcome in a patient. An effective amount can be
administered in one or more doses to the patient to achieve the
desired treatment outcome.
[0045] "Microbiota", "microflora" and "microbiome" preferably mean
a community of living microorganisms that typically inhabits a
bodily organ or part, particularly the gastrointestinal organs of
non-infant humans. The most dominant members of the
gastrointestinal microbiota include microorganisms of the phyla of
Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria,
Synergistetes, Verrucomicrobia, Fusobacteria, and Euryarchaeota; at
genus level Bacteroides, Faecalibacterium, Bifidobacterium,
Roseburia, Alistipes, Collinsella, Blautia, Coprococcus,
Ruminococcus, Eubacterium and Dorea; at species level Bacteroides
uniformis, Alistipes putredinis, Parabacteroides merdae,
Ruminococcus bromii, Dorea longicatena, Bacteroides caccae,
Bacteroides thetaiotaomicron, Eubacterium hallii, Ruminococcus
torques, Faecalibacterium prausnitzii, Ruminococcus lactaris,
Collinsella aerofaciens, Dorea formicigenerans, Bacteroides
vulgatus and Roseburia intestinalis. The gastrointestinal
microbiota includes the mucosa-associated microbiota, which is
located in or attached to the mucus layer covering the epithelium
of the gastrointestinal tract, and luminal-associated microbiota,
which is found in the lumen of the gastrointestinal tract.
[0046] The term "bifidobacteria" means a member of the
Bifidobacterium genus commonly found in the human gastrointestinal
tract. Examples of bifidobacteria are Bifidobacterium longum,
Bifidobacterium bifidum, and the members of the phylogenetic
Bifidobacterium adolescentis group. In non-infant humans,
bifidobacteria preferably include members of the phylogenetic
Bifidobacterium adolescentis group.
[0047] The term "Bifidobacterium of the Bifidobacterium
adolescentis phylogenetic group" means a bacterium selected from a
group consisting of Bifidobacterium adolescentis, Bifidobacterium
angulatum, Bifidobacterium catenulatum, Bifidobacterium
pseudocatenulatum, Bifidobacterium kashiwanohense, Bifidobacterium
dentum and Bifidobacterium stercoris (Duranti et al. Appl. Environ.
Microbiol. 79, 336 (2013), Bottacini et al. Microbial Cell Fact.
13:S4 (2014)).
[0048] The term "relative abundance of bifidobacteria" means the
abundance of bifidobacteria relative to other genera in the
microbiota of the gastrointestinal tract.
[0049] The term "human milk oligosaccharide" or "HMO" preferably
means a complex carbohydrate consisting of a small number,
typically 3-10, of monosaccharide units attached to each other by
an interglycosidic linkage that can be found in human breast milk
and that can be in acidic or neutral form. More than about 200
different HMO structures are known to exist in human breast milk
(Urashima et al.: Milk Oligosaccharides, Nova Biomedical Books, New
York, 2011). HMOs can be core, fucosylated and sialylated
oligosaccharides. Core HMOs are non-fucosylated neutral (that is
non-charged) HMOs and consist of Glu, Gal and GlcNAc (thus devoid
of Fuc and sialic acid). Examples of core HMOs include
lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT),
lacto-N-neohexaose (LNnH), lacto-N-hexaose (LNH) and
p-lacto-N-neohexaose (pLNnH). Fucosyl HMOs are fucosylated lactoses
or fucosylated core HMOs such as 2'-fucosyllactose (2'-FL),
lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I),
3-fucosyllactose (3-FL), difucosyllactose (DFL),
lacto-N-fucopentaose III (LNFP-III),
fucosyl-para-lacto-N-neohexaose (F-pLNnH), lacto-N-difucohexaose I
(LNDFH-I), fucosyl-lacto-N-hexaose II (FLNH-II),
lacto-N-fucopentaose V (LNFP-V), lacto-N-difucohexaose II
(LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I),
fucosyl-lacto-N-hexaose III (FLNH-III) and
fucosyl-para-lacto-N-neohexaose (F-pLNnH). Sialyl HMOs are
sialylated lactoses or sialylated core HMOs such as
3',6-disialyllacto-N-tetraose (DSLNT), 6'-sialyllactose (6'-SL),
3'-sialyllactose (3'-SL), 6'-sialyllacto-N-neotetraose (LST c),
3'-sialyllacto-N-tetraose (LST a) and 6-sialyllacto-N-tetraose (LST
b). Examples for sialylated and fucosylated HMOs include
disialyl-fucosyl-lacto-N-hexaose II (DSFLNH-II),
fucosyl-sialyl-lacto-N-neohexaose I (FSLNnH-I),
fucosyl-sialyl-lacto-N-hexaose I (FSLNH-I) and
3-fucosyl-3'-sialyllactose (FSL).
[0050] The HMOs can be isolated or enriched by well-known processes
from milk(s) secreted by mammals including, but not limited to
human, bovine, ovine, porcine, or caprine species. The HMOs can
also be produced by well-known processes using microbial
fermentation, enzymatic processes, chemical synthesis, or
combinations of these technologies. As examples, using chemistry
LNnT can be made as described in WO 2011/100980 and WO 2013/044928,
LNT can be synthesized as described in WO 2012/155916 and WO
2013/044928, a mixture of LNT and LNnT can be made as described in
WO 2013/091660, 2'-FL can be made as described in WO 2010/115934
and WO 2010/115935, 3-FL can be made as described in WO
2013/139344, 6'-SL and salts thereof can be made as described in WO
2011/100979, sialylated oligosaccharides can be made as described
in WO 2012/113404 and mixtures of human milk oligosaccharides can
be made as described in WO 2012/113405. As examples of enzymatic
production, sialylated oligosaccharides can be made as described in
WO 2012/007588, fucosylated oligosaccharides can be made as
described in WO 2012/127410, and advantageously diversified blends
of human milk oligosaccharides can be made as described in WO
2012/156897 and WO 2012/156898. With regard to biotechnological
methods, WO 2001/04341 and WO 2007/101862 describe how to make core
human milk oligosaccharides optionally substituted by fucose or
sialic acid using genetically modified E. coli.
[0051] The term "synthetic composition" means a composition which
is artificially prepared and preferably means a composition
containing at least one compound that is produced ex vivo
chemically and/or biologically, e.g., by means of chemical
reaction, enzymatic reaction or recombinantly. In some embodiments
a synthetic composition of the disclosure may be, but preferably is
not, identical with a naturally occurring composition. The
synthetic composition of the disclosure typically comprises one or
more compounds, advantageously HMOs, that are capable of
preferentially increasing the abundance of bifidobacteria, in
particular Bifidobacterium of the following species:
Bifidobacterium longum, Bifidobacterium bifidum, and/or members of
the phylogenetic Bifidobacterium adolescentis group. In some
embodiments, the synthetic composition may comprise one or more
compounds or components other than HMOs that may have an effect on
bifidobacteria of a human subject microbiota in vivo, e.g.,
non-digestible oligosaccharides or prebiotics. Also in some
embodiments, the synthetic compositions may comprise one or more
nutritionally or pharmaceutically active components which do not
affect adversely the efficacy of the above-mentioned compounds.
Some non-limiting embodiments of a synthetic composition of the
disclosure are also described below.
Exemplary Embodiments
[0052] One or more embodiments to single HMOs as substantially pure
single compounds, i.e., an HMO which grade of purity satisfies the
demand of a medical or food authority for marketing, or mixtures of
2 to 5 such substantially pure HMOs, or artificial compositions
comprising one to five HMOs. Embodiments of HMOs and compositions
comprising thereof are described below.
[0053] In particular, different embodiments of the disclosure
relate to HMOs for
[0054] reducing the propensity of a cardiovascular disease (CVD) in
a human individual, preferably in an overweight or obese human
individual,
[0055] preventing development of a cardiovascular disease (CVD) in
in a human individual, preferably in an overweight or obese human
individual, and/or
[0056] treating a cardiovascular disease (CVD) in in a human
individual, preferably in an overweight or obese human
individual,
[0057] where the HMOs may be a single HMO or a mixture of two to
five of any HMOs suitable for the purpose of the disclosure.
Preferably, the HMO is a fucosylated or a non-fucosylated neutral
HMO. More preferably, the disclosure relates to a mixture of HMOs,
the mixture comprising at least a first HMO and at least a second
HMO, wherein the first HMO is a fucosylated neutral HMO and the
second HMO is a non-fucosylated neutral HMO. In other embodiments
the mixture may comprise further a third, a fourth, and a fifth
HMO. Particularly, the mixture of HMOs may contain a fucosylated
HMO selected from the list consisting of 2'-FL, 3-FL, DFL, LNFP-I,
LNFP-II, LNFP-III, LNFP-V, LNDFH-I, LNDFH-II, LNDFH-III, FLNH-I,
FLNH-II, FLNnH, FpLNH-I and F-pLNnH II, and a non-fucosylated HMO
selected from the list consisting of LNT, LNnT, LNH, LNnH, pLNH and
pLNnH. Preferably, the mixture of HMOs contains a fucosylated HMO
selected from the list consisting of 2'-FL, 3-FL and DFL, and a
non-fucosylated HMO selected from the list consisting of LNT and
LNnT; advantageously the mixture comprises 2'-FL and LNnT and/or
LNT. In some embodiments, the mixture of HMOs essentially consists
of two neutral HMOs, e.g., a fucosylated HMO selected from the list
consisting of 2'-FL, 3-FL, DFL, LNFP-I, LNFP-II, LNFP-III, LNFP-V,
LNDFH-I, LNDFH-II, LNDFH-III, FLNH-I, FLNH-II, FLNnH, FpLNH-I and
F-pLNnH II, and a non-fucosylated HMO selected from the list
consisting of LNT, LNnT, LNH, LNnH, pLNH and pLNnH. Preferably, the
mixture essentially consists of a fucosylated HMO selected from the
list consisting of 2'-FL, 3-FL and DFL, and a non-fucosylated HMO
selected from the list consisting of LNT and LNnT; in one preferred
embodiment the mixture essentially consists of 2'-FL and LNnT, in
another preferred embodiment the mixture essentially consists of
2'-FL and LNT.
[0058] In a preferred embodiment, a mixture of 2'-FL and LNnT may
contain the amount of 2'-FL: LNnT from about 1.5:1 to about
4:1.
[0059] In other embodiments, the disclosure relates to a synthetic
composition for
[0060] reducing the propensity of a cardiovascular disease (CVD) in
a human individual, preferably in an overweight or obese human
individual,
[0061] preventing development of a cardiovascular disease (CVD) in
a human individual, preferably in an overweight or obese human
individual, and/or
[0062] treating a cardiovascular disease (CVD) in a human
individual, preferably in an overweight or obese human
individual,
[0063] which may comprise a single HMO or a mixture of two to five
of any HMOs suitable for the purpose of the disclosure as disclosed
above.
[0064] The synthetic composition can take any suitable form. For
example, the composition can be in the form of a nutritional
composition which contains other macronutrients such as proteins,
lipids, or other carbohydrates. The synthetic composition can also
be a pharmaceutical composition.
[0065] In other embodiments, the disclosure relates to a method
for
[0066] reducing the propensity of a cardiovascular disease (CVD),
or an CVD-associated pathologic condition or disease, in a human,
preferably, wherein said human is overweight or obese;
[0067] preventing development of a cardiovascular disease (CVD), or
an CVD-associated pathologic condition or disease, in a human,
preferably wherein said human is overweight or obese;
[0068] treating a cardiovascular disease (CVD), or an
CVD-associated pathologic condition or disease, in a human,
preferably, wherein said human is overweight or obese; and/or
[0069] increasing the abundance of bifidobacteria in a human having
an CVD disease, or an CVD-associated pathologic condition or
disease, preferably, wherein said human is overweight or obese,
said method comprising administering to the patient, preferably
daily at least 2 g of, a human milk oligosaccharide (HMO) selected
from the group consisting of fucosylated HMOs and core HMOs. The
HMOs suitable for the purpose of the method are disclosed
above.
Nutritional Compositions
[0070] A nutritional composition can contain sources of protein,
lipids and/or digestible carbohydrates and can be in solid,
powdered, or liquid forms. The composition can be designed to be
the sole source of nutrition or a nutritional supplement.
[0071] Suitable protein sources include intact, hydrolysed, and
partially hydrolysed protein, which can be derived from any
suitable source such as milk (e.g., casein, whey), animal (e.g.,
meat, fish), cereal (e.g., rice, corn), and vegetable (e.g., soy,
potato, pea), insect (e.g., locust) and combinations of these
sources. Examples of the source of protein include whey protein
concentrates, whey protein isolates, whey protein hydrolysates,
acid caseins, sodium casemates, calcium casemates, potassium
casemates, casein hydrolysates, milk protein concentrates, milk
protein isolates, milk protein hydrolysates, non-fat dry milk,
condensed skim milk, soy protein concentrates, soy protein
isolates, soy protein hydrolysates, pea protein concentrates, pea
protein isolates, pea protein hydrolysates, collagen proteins, and
combinations of these sources.
[0072] The amount of protein is preferably sufficient to provide
about 5 to about 30% of the energy of the nutritional composition;
for example, about 10% to about 25% of the energy. Within these
ranges, the amount of protein can vary depending upon the
nutritional needs of the intended individual.
[0073] The nutritional compositions can also include free amino
acids such as tryptophan, glutamine, tyrosine, methionine,
cysteine, taurine, arginine, carnitine, threonine, serine and
proline and combinations of these amino acids. Threonine, serine
and proline are important amino acids for the production of mucin
which aids gut barrier function.
[0074] Any suitable source of other carbohydrates can be used.
Examples include maltodextrin, hydrolysed or modified starch or
corn starch, glucose polymers, corn syrup, corn syrup solids,
rice-derived carbohydrates, sucrose, glucose, fructose, lactose,
high fructose corn syrup, honey, sugar alcohols (e.g., maltitol,
erythritol, sorbitol, etc.), isomaltulose, sucromalt, pullulan,
potato starch, slowly-digested carbohydrates, dietary fibres such
as oat fibre, soy fibre, gum arabic, sodium carboxymethylcellulose,
methylcellulose, guar gum, gellan gum, locust bean gum, konjac
flour, hydroxypropyl methylcellulose, tragacanth gum, karaya gum,
gum acacia, chitosan, arabinogalactans, glucomannan, xanthan gum,
alginate, pectin, low and high methoxy pectin, cereal beta-glucans
(i.e., oat beta-glucan, barley beta- glucan), carrageenan and
psyllium, Fibersol.TM., other resistant starches, and combinations
of these carbohydrate.
[0075] Preferably the carbohydrate source includes low glycemic
index carbohydrates having a GI score of 55 or below. Examples of
low glycemic index carbohydrates include sucromalt, Fibersol.TM.
(inulin), maltodextrins having a dextrose equivalence (DE) of less
than 15, rice syrup having a dextrose equivalence of less than 15,
fructooligosaccharides, resistant starches, starches, fruit sourced
fibres, vegetable sourced fibres, whole grains, beta-glucans, soy
fibres, oat fibres, locust bean gum, konjac flour, hydroxypropyl
methylcellulose, gum acacia, chitosan, arabinogalactans, xanthan
gum, alginate, low and high methoxy pectin, carrageenan, psyllium,
isomaltulose, glycerine and sugar alcohols.
[0076] The nutritional compositions can include carbohydrates in an
amount sufficient to provide about 30 to about 70% of the energy of
the composition, for example about 35 to about 65% of the energy.
Within these parameters, the amount of carbohydrate can vary
widely.
[0077] Suitable lipid sources include coconut oil, fractionated
coconut oil, soy oil, corn oil, olive oil, safflower oil, high
oleic safflower oil, medium chain triglycerides, sunflower oil,
high oleic sunflower oil, palm and palm kernel oils, palm olein,
canola oil, marine oils, cottonseed oils and combinations of these
oils. Fractionated coconut oils are a suitable source of medium
chain triglycerides. The lipids can contain polyunsaturated fatty
acids such as n-3 LC-PUFA. The n-3 LC-PUFA can be a C20 or a C22
n-3 fatty acid. Preferably the n-3 LC-PUFA is docosahexaenoic acid
(DHA, C22:6, n-3). The source of LC-PUFA can be, for example, egg
lipids, fungal oil, low EPA fish oil or algal oil.
[0078] The nutritional compositions can include lipids in an amount
sufficient to provide about 10 to about 50% of energy of the
nutritional composition, for example about 15 to about 40% of the
energy.
[0079] The nutritional composition preferably also includes
vitamins and minerals. If the nutritional composition is intended
to be a sole source of nutrition, it preferably includes a complete
vitamin and mineral profile. Examples of vitamins include vitamins
A, B-complex (such as B1, B2, B6 and B12), C, D, E and K, niacin,
and acid vitamins such as pantothenic acid, folic acid, and biotin.
Examples of minerals include calcium, iron, zinc, magnesium,
iodine, copper, phosphorus, manganese, potassium, chromium,
molybdenum, selenium, nickel, tin, silicon, vanadium, and
boron.
[0080] The nutritional composition can also include a carotenoid
such as lutein, lycopene, zeaxanthin, and beta-carotene. The total
amount of carotenoid included can vary from about 0.001 .mu.g/ml to
about 10 .mu.g/ml. Lutein can be included in an amount of from
about 0.001 .mu.g/ml to about 10 .mu.g/ml, preferably from about
0.044 .mu.g/ml to about 5 .mu.g/ml of lutein. Lycopene can be
included in an amount from about 0.001 .mu.g/ml to about 10
.mu.g/ml, preferably about 0.0185 .mu.g/ml to about 5 .mu.g/ml of
lycopene. Beta-carotene can comprise from about 0.001 .mu.g/ml to
about 10 .mu.g/ml, for example about 0.034 .mu.g/ml to about 5
.mu.g/ml of beta-carotene. The nutritional composition can also
include a source of anthocyanins. This can be in the form of a
fruit or a fruit extract. Particularly useful fruits and fruit
extracts include plum/prune, apple, pear, strawberry, blueberry,
raspberry, cherry, and their combinations.
[0081] The nutritional composition can also contain various other
conventional ingredients such as preservatives, emulsifying agents,
thickening agents, buffers, fibres and prebiotics (e.g.,
fructooligosaccharides, galactooligosaccharides), probiotics (e.g.,
B. animalis subsp. lactis BB-12, B. lactis HN019, B. lactis Bi07,
B. infantis ATCC 15697, L. rhamnosus GG, L. rhamnosus HN001, L.
acidophilus LA-5, L. acidophilus NCFM, L. fermentum CECT5716, B.
longum BB536, B. longum AH1205, B. longum AH1206, B. breve M-16V,
L. reuteri ATCC 55730, L. reuteri ATCC PTA-6485, L. reuteri DSM
17938), antioxidant/anti-inflammatory compounds including
tocopherols, carotenoids, ascorbate/vitamin C, ascorbyl palmitate,
polyphenols, glutathione, and superoxide dismutase (melon), other
bioactive factors (e.g., growth hormones, cytokines, TFG-.beta.),
colorants, flavours, and stabilisers, lubricants, and so forth.
[0082] The nutritional composition can be in the form of a food,
soluble powder, a liquid concentrate, or a ready-to-use
formulation. The composition can be eaten, drunk or can be fed via
a nasogastric. Various flavours, fibres and other additives can
also be present.
[0083] The nutritional compositions can be prepared by any commonly
used manufacturing techniques for preparing nutritional
compositions in solid or liquid form. For example, the composition
can be prepared by combining various feed solutions. A
protein-in-fat feed solution can be prepared by heating and mixing
the lipid source and then adding an emulsifier (e.g., lecithin),
fat soluble vitamins, and at least a portion of the protein source
while heating and stirring. A carbohydrate feed solution is then
prepared by adding minerals, trace, and ultra-trace minerals,
thickening, or suspending agents to water while heating and
stirring. The resulting solution is held for 10 minutes with
continued heat and agitation before adding carbohydrates (e.g., the
HMOs and digestible carbohydrate sources). The resulting feed
solutions are then blended together while heating and agitating and
the pH adjusted to 6.6-7.0, after which the composition is
subjected to high-temperature short-time processing during which
the composition is heat treated, emulsified, and homogenized, and
then allowed to cool. Water soluble vitamins and ascorbic acid are
added, the pH is adjusted to the desired range if necessary,
flavours are added, and water is added to achieve the desired total
solid level.
[0084] For a liquid product, the resulting solution can then be
aseptically packed to form an aseptically packaged nutritional
composition. In this form, the nutritional composition can be in
ready-to-feed or concentrated liquid form. Alternatively, the
composition can be spray-dried and processed and packaged as a
reconstitutable powder.
[0085] The nutritional composition can also be in the form of a
food such as a nutritional bar, a yoghurt, etc. These forms can be
produced using standard technologies and processes.
[0086] When the nutritional product is a ready-to-feed nutritional
liquid, the total concentration of HMOs in the liquid, by weight of
the liquid, is from about 0.0001% to about 2.0%, including from
about 0.001% to about 1.5%, including from about 0.01% to about
1.0%. When the nutritional product is a concentrated nutritional
liquid, the total concentration of HMOs in the liquid, by weight of
the liquid, is from about 0.0002% to about 4.0%, including from
about 0.002% to about 3.0%, including from about 0.02% to about
2.0%.
Pharmaceutical Compositions
[0087] A pharmaceutical composition of the disclosure contains an
effective amount of HMO or an effective amount of mixture of two to
five HMOs, wherein the HMOs are selected from any of described
above. The term "effective amount" in the present content means an
amount of a single HMO, or a combination of different HMOs that is
capable of increasing the abundance of bifidobacteria in the
gastrointestinal tract of a human individual of the disclosure,
preferably, relative abundance of members of the Bifidobacterium
adolescentis phylogenetic group in particular B. adolescentis
and/or B. pseudocatenulatum.
[0088] The pharmaceutical composition can further contain a
pharmaceutically acceptable carrier, e.g., phosphate buffered
saline solution, mixtures of ethanol in water, water, and emulsions
such as an oil/water or water/oil emulsion, as well as various
wetting agents or excipients. The pharmaceutical composition can
also contain other materials that do not produce an adverse,
allergic, or otherwise unwanted reaction when administered to
humans. The carriers and other materials can include solvents,
dispersants, coatings, absorption promoting agents, controlled
release agents, and one or more inert excipients, such as starches,
polyols, granulating agents, microcrystalline cellulose, diluents,
lubricants, binders, and disintegrating agents. If desired, tablet
dosages of the anti-infective compositions can be coated by
standard aqueous or non-aqueous techniques.
[0089] The pharmaceutical compositions can be administered orally,
e.g., as a tablet, capsule, or pellet containing a predetermined
amount, or as a powder or granules containing a predetermined
concentration or a gel, paste, solution, suspension, emulsion,
syrup, bolus, electuary, or slurry, in an aqueous or non-aqueous
liquid, containing a predetermined concentration. Orally
administered compositions can include binders, lubricants, inert
diluents, flavouring agents, and humectants. Orally administered
compositions such as tablets can optionally be coated and can be
formulated so as to provide sustained, delayed, or controlled
release of the mixture therein.
[0090] The pharmaceutical compositions can also be administered by
rectal suppository, aerosol tube, nasogastric tube or direct
infusion into the GI tract or stomach.
[0091] The pharmaceutical compositions can also include therapeutic
agents most commonly prescribed for heart disease such as:
[0092] ACE Inhibitors: ACE inhibitors are a type of medication that
dilates (widens) arteries to lower blood pressure and make it
easier for the heart to pump blood. They also block some of the
harmful actions of the endocrine system that may occur with heart
failure;
[0093] Aldosterone Inhibitor: Eplerenone (Inspra.RTM.) and
spironolactone (Aldactone.RTM.) and eplerenone are
potassium-sparing diuretics. They can be prescribed to reduce the
swelling and water build-up caused by heart failure. Diuretics
cause the kidneys to send unneeded water and salt from the tissues
and blood into the urine;
[0094] They may improve heart failure symptoms that are still
present despite use of other treatments. These drugs protect the
heart by blocking a chemical (aldosterone) in the body that causes
salt and fluid build-up. This medication is used to treat patients
with certain types of severe heart failure;
[0095] Angiotensin II Receptor Blocker (ARBs): ARBs are used to
decrease blood pressure in people with heart failure. ARBs decrease
certain chemicals that narrow the blood vessels so blood can flow
more easily through your body. They also decrease certain chemicals
that cause salt and fluid build-up in the body;
[0096] Beta-Blockers: Beta-blockers block the effects of adrenaline
(epinephrine) and thereby improve the heart's ability to perform.
They also decrease the production of harmful substances produced by
the body in response to heart failure. They cause the heart to beat
more slowly and with less force, lowering blood pressure;
[0097] Calcium Channel Blockers: Calcium channel blockers are
prescribed to treat angina (chest pain) and high blood pressure.
Calcium channel blockers affect the movement of calcium in the
cells of the heart and blood vessels. As a result, the drugs relax
blood vessels and increase the supply of blood and oxygen to the
heart, while reducing its workload;
[0098] Cholesterol -Lowering Drugs: Cholesterol helps your body
build new cells, insulate nerves, and produce hormones. But
inflammation may lead to cholesterol build-up in the walls of
arteries, increasing the risk of heart attack and stroke;
[0099] Digoxin: Digoxin helps an injured or weakened heart to work
more efficiently and to send blood through the body. It strengthens
the force of the heart muscle's contractions and may improve blood
circulation;
[0100] Diuretics: Diuretics, commonly known as "water pills," cause
the kidneys to get rid of unneeded water and salt from the tissues
and bloodstream into the urine. Getting rid of excess fluid makes
it easier for your heart to pump. Diuretics are used to treat high
blood pressure and reduce the swelling and water build-up caused by
various medical problems, including heart failure;
[0101] Inotropic Therapy: Inotropic therapy is used to stimulate an
injured or weakened heart to pump harder to send blood through the
body. It helps the force of the heart muscle's contractions and
relaxes constricted blood vessels so blood can flow more smoothly.
Inotropic therapy may also speed up the heart's rhythm;
[0102] Potassium or Magnesium: Potassium and magnesium are minerals
that can be lost because of increased urination when taking
diuretics. Low levels in the body can be associated with abnormal
heart rhythms. Some patients take them as supplements as directed
by their doctor.
[0103] Vasodilators: Vasodilators are used to treat heart failure
and control high blood pressure by relaxing the blood vessels so
blood can flow more easily through the body. Vasodilators are
prescribed for patients who cannot take ACE inhibitors.
[0104] Warfarin: Warfarin is an anticoagulant medication. "Anti"
means "against," and "coagulant" means "causing blood clotting."
Therefore, warfarin helps prevent clots from forming in the
blood.
[0105] The pharmaceutical composition may also contain other
compounds such as antibiotics, probiotics, analgesics, and
anti-inflammatory agents.
[0106] The proper dosage of these compositions for a human can be
determined in a conventional manner, based upon factors such as
severity of conditions of the human individual, e.g., the
individual's blood pressure, immune status, body weight, age,
etc.
Administration Dosing
[0107] For increasing the levels of the gut hormones GLP-1 and
GLP-2 in a person, the amount of human milk oligosaccharide(s)
required to be administered to the person will vary depending upon
factors such as the risk and condition severity, the age of the
person, the form of the composition, and other medications being
administered to the person. However, the required amount can be
readily set by a medical practitioner and would generally be in the
range from about 10 mg to about 20 g per day, in certain
embodiments from about 10 mg to about 15 g per day, from about 100
mg to about 10 g per day, in certain embodiments from about 500 mg
to about 10 g per day, in certain embodiments from about 1 g to
about 7.5 g per day. An appropriate dose can be determined based on
several factors, including, for example, the body weight and/or
condition of the patient being treated, the severity of the
condition, being treated, other ailments and/or diseases of the
person, the incidence and/or severity of side effects and the
manner of administration. Appropriate dose ranges can be determined
by methods known to those skilled in the art. During an initial
treatment phase, the dosing can be higher (for example 200 mg to 20
g per day, preferably 500 mg to 15 g per day, more preferably 1 g
to 10 g per day, in certain embodiments 2.5 g to 7.5 g per day).
During a maintenance phase, the dosing can be reduced (for example,
10 mg to 10 g per day, preferably 100 mg to 7.5 g per day, more
preferably 500 mg to 5 g per day, in certain embodiments 1 g to 2.5
g per day).
[0108] HMOs of this disclosure can be co-administered to an
individual who is also receiving a standard-of-care medication for
obesity or diabetes.
Methods of Treatment
[0109] The disclosure contemplates both prophylactic and
therapeutic methods of treatment depending on different
embodiments. The term "therapeutic method" means a method
comprising treatment of disease or medical disorder by remedial
agents and/or, e.g., administering an HMO(s) or a composition of
the disclosure to a CVD patient of the disclosure to cure the CVD
or the associated pathological condition or disease. The term
"prophylactic method" means a method comprising a measure taken to
fend off a disease or another unwanted consequence of the disease,
e.g., administering an HMO or a composition of the disclosure to a
human of the disclosure to reduce the propensity of or prevent
development of CVD or the associated pathological condition or
disease in the human.
[0110] In particular, the disclosure relates to the following
methods:
[0111] a method for reducing the propensity of a cardiovascular
disease (CVD), an CVD-associated pathologic condition or disease,
in a human, preferably, wherein said human is overweight or
obese;
[0112] a method for preventing development of a cardiovascular
disease (CVD), an CVD-associated pathologic condition or disease,
in a human, preferably wherein said human is overweight or
obese;
[0113] a method for treating a cardiovascular disease (CVD), an
CVD-associated pathologic condition or disease, in a human,
preferably, wherein said human is overweight or obese; and/or
[0114] a method for increasing the abundance of bifidobacteria in a
human having an CVD disease, an CVD-associated pathologic condition
or disease, preferably, wherein said human is overweight or
obese.
[0115] Various methods of the disclosure comprise a step of
administering daily to the human at least 2 g of an HMO selected
from the group consisting of fucosylated HMOs and core HMOs,
preferably, at least 2 g of a mixture of two to five HMOs
consisting of one or more fucosylated HMOs and one or more core
HMOs.
[0116] Preferably, an HMO of the disclosure is administered to a
human in need enteral, e.g., orally.
[0117] Preferably, the disclosure relates to a method increasing
the abundance of a bacterium of the B. adolescentis phylogenetic
group, especially Bifidobacterium adolescentis and/or B.
pseudocatenulatum.
[0118] In any of the methods, one or more HMOs, preferably, one to
five HMOs, may be administered as substantially pure compounds
(i.e., neat or undiluted) or diluted, e.g., in form of a solution,
power or syrup, or in the form of a synthetic composition,
nutritional or pharmaceutical composition, as any of the described
above, in one or more unit dosage forms, preferably in a single
unit dosage form.
[0119] Preferably, the HMOs are, or the synthetic, nutritional, or
pharmaceutical, composition contains, 2'-FL and LNnT, preferably
the 2'-FL:LNnT ratio is about 1.5:1 to about 4:1.
[0120] The dosage of one or more fucosylated HMOs and one or more
core HMOs per administration may vary from about 2 g to about 10 g,
preferably from about 3.5 g to about 7.5 g. Typically, the HMOs are
administered in a single dosage unit containing from about 2 g to
about 10 g, preferably from about 3.5 g to about 7.5 g of one of
more fucosylated HMOs and one or more core HMOs. The patient may
also additionally receive a dose of one or more species of
probiotic bacteria, e.g., bifidobacteria.
[0121] Typically, the patient is administered a daily dose of at
least 2 g of the mixture of one or more fucosylated HMOs and one or
more core HMOs for at least 14 days, preferably, for more than 14
days.
EXAMPLES
[0122] Examples are now described to further illustrate the
disclosed method(s):
Example 1
Treating High Fat Diet Induced Obesity and Diabetes
[0123] 10-week-old C57BL/6J mice (100 mice) are housed in groups of
five mice per cage, with free access to food and water. The mice
are divided into 10 groups of 10 mice, one control group and 9
treatment groups. All of the mice are fed a high-fat (HF) diet (60%
fat and 20% carbohydrates [kcal/100 g], or an HF diet supplemented
with HMO (20 g/kg of diet) for 8 weeks. Food and water intake are
recorded twice a week. The 9 treatment groups are each administered
one of the following: a) 2'-FL, b) 3-FL, c) 3'-SL, d) 6'-SL, e)
LNT, f) LNnT, g) LNFP-I, h) DSLNT and i) a combination of these
saccharides. The control group is administered the HF diet only.
Fresh food is given daily.
[0124] Intraperitoneal or oral glucose tolerance tests are
performed as follows: 6-h-fasted mice are injected with glucose
into the peritoneal cavity (1 g/kg glucose, 20% glucose solution)
or by gavage (3 g/kg glucose, 66% glucose solution). Blood glucose
is determined with a glucose meter (Roche Diagnostics) on 3.5 .mu.l
blood collected from the tip of the tail vein. A total of 20 .mu.l
blood is sampled 30 min before and 15 or 30 min after the glucose
load to assess plasma insulin concentration.
[0125] Plasma triglyceride and cholesterol is measured from blood
taken during the treatment period.
[0126] To assess intestinal permeability in vivo, the intestinal
permeability of 4000 Da fluorescent dextran-FITC (DX-4000-FITC) is
measured. Mice are fasted for 6 h before given DX-44-FITC by gavage
(500 mg/kg body weight, 125 mg/ml). After 1 h and 4 h, 120 ml of
blood is collected from the tip of the tail vein. The blood is
centrifuged at 4.degree. C., 12 000 g for 3 min. Plasma is diluted
in an equal volume of PBS (pH 7.4) and analysed for DX-4000-FITC
concentration with a fluorescence spectrophotometer at an
excitation wavelength of 485 nm and emission wavelength of 535 nm.
Standard curves are obtained by diluting FITC-dextran in
non-treated plasma diluted with PBS (1:3 v/v).
[0127] Mice are anaesthetised (ketamine/xylazine, intraperineally,
100 and 10 mg/kg, respectively) after a 5 h period of fasting,
blood samples and tissues are harvested for further analysis. Mice
are killed by cervical dislocation. Liver, caecum (full and empty),
and adipose tissues (mesenteric and corresponding lymph nodes,
epididymal, subcutaneous and visceral) are precisely dissected,
weighed and stored at -80.degree. C., for further analysis.
[0128] Total and active GLP-1 are measured from blood with ELISA
(Millipore, Molsheim, France).
[0129] To assess the microbiota profile, the caecal contents
collected postmortem from mice are stored at -80.degree. C. DNA is
isolated from the caecal content samples using QIAamp DNA Stool
Mini Kit. The DNA concentration of extracts is measured using
NanoDrop. Aliquots of 100 ng of extracted DNA are subjected to PCR
using the 16S rDNA universal heteroduplex analysis (HDA) primers
HDA1-GC and HDA2 which are disclosed in Walter et al. Appl.
Environ. Microbiol. 66, 297 (2000) at 56.degree. C. for strand
annealing. Initial denaturation at 94.degree. C. for 4 min is
followed by thirty cycles of 30 s at 94.degree. C., 30 s at
56.degree. C. and 1 min at 72.degree. C. The quality of PCR
products is verified by agarose gel electrophoresis. Amplified 16S
rDNA fragments are separated by denaturing gradient gel
electrophoresis (DGGE) using an INGENYphorU system equipped with 6%
polyacrylamide gels with a denaturant in the range of 30-55%, where
100% denaturant is equivalent to 7M-urea and 40% formamide.
Electrophoresis is carried out at 130 V for 4-5 hours at 60.degree.
C. Polyacrylamide gels are stained with GelRede nucleic acid stain
for 45 min, destained in ultrapure water and viewed under UV light.
Bands of interest are excised from gels and lysed in ultrapure
water. Extracted DNA is re-amplified using the same primers and PCR
conditions. To purify the bacterial DNA, PCR products are reloaded
on a denaturant gradient gel followed by excision and lysis of
selected bands. DNA samples recovered from lysed bands of the
second DGGE are re-amplified by PCR before purification using the
QIAquick PCR Purification Kit and sequenced. Species identification
is done using the Ribosomal Microbiome Database Project Classifier
tool. Because of the limited sensitivity of DGGE to quantify
microbial diversity, the microbial composition of DNA samples is
also analysed using high-throughput sequencing. The V5-V6 region of
16S rRNA from caecal content DNA samples is amplified using a
forward primer and a reverse primer which are both disclosed in
Andersson et al. PloS ONE 3, e2836 (2008). Amplicons are
pyrosequenced using a Roche 454 GS-FLX system. Sequences of at
least 240 nucleotides and containing no more than two undetermined
bases are retained for taxonomic assignment. The QIIME software is
used for chimera check and the Greengenes database is used for
classification. Bacterial diversity is determined at the phylum,
family, and genus levels.
[0130] The results show that HMOs are able to change the intestinal
microbiota by increasing the abundance of bifidobacteria.
Additionally, HMO supplementation reduces cholesterol, body weight,
fat accumulation and glucose tolerance.
Example 2
Human Trial in Overweight and Obese Children
[0131] A total of 60 male and female patients, enrolled to a
childhood obesity treatment program, are recruited to participate
in the study. Patients are randomized into three groups, each of 20
patients, with 2 groups receiving different investigational
products and one group receiving a placebo product for 8 weeks. The
investigational products contain 4.5 grams of either 2'-FL alone or
a combination of 2'-FL and LNnT while the placebo product contains
4.5 grams glucose. All products are in powder form in a unit dosage
container.
[0132] The patients are eligible to participate if: they are
between 5 and 10 years of age, have a BMI SDS of .gtoreq.2.0 and
are enrolled in the childhood obesity treatment program at the
Children's Obesity Clinic. All recruited patients and their
representatives are able and willing to understand and comply with
the study procedures. Patients are excluded if: they have
participated in a clinical study one month prior to the screening
visit and throughout the study; have any gastrointestinal
disease(s) that may cause symptoms or may interfere with the trial
outcome; have other severe disease(s) such as malignancy, kidney
disease or neurological disease; have psychiatric disease; have
used highly dosed probiotic supplements (yoghurt allowed) 3 months
prior to screening and throughout the study; have consumed
antibiotic drugs 3 months prior to screening and throughout the
study; and consume on a regular basis medication that might
interfere with symptom evaluation 2 weeks prior to screening and
throughout the study.
[0133] At the initial visit (screening) patients and their
representatives are given both oral and written information about
the study; the children are asked for informed assent and their
representatives to sign an informed consent form.
[0134] Eligibility criteria are checked and for children who are
enrolled to the study, medical history and concomitant medication
are registered. A physical examination is done and pubertal staging
is determined. Blood pressure, pulse rate, height and bodyweight
are measured, and body composition is determined by a DXA (dual
energy x-ray absorptiometry)-scan and bioimpedance. BMI SDS is
calculated, waist and hip circumferences measured and food intake
registered. Fasting blood samples are collected for safety and
biomarker studies and for biobanking.
[0135] The serum from the blood samples is transferred to cryotubes
and stored at -80.degree. C. The following biomarkers are measured;
Lipopolysaccharides (LPS), hsCRP, free fatty acids, total
cholesterol, HDL, LDL, HbA1c, glucose, insulin, triglycerides,
TNF-.alpha., IL-1.beta., IL-6, IL-8, IL-10, GLP-1, GLP-2,
Adiponectin, and Zonulin.
[0136] Equipment for collecting faecal samples is distributed. The
faecal samples are stored at -80.degree. C. until analysis. SCFA
and Microbiological analysis is performed on the faecal
samples.
[0137] The Gastrointestinal Symptom Rating Scale (GSRS)
questionnaire is completed on site by the participating child's
representative(s), and the Bristol Stool Form Scales (BSFS) is
distributed to the participant's representative(s) with
instructions to assess the stool consistency during the study and
at each faecal sampling point using the BSFS.
[0138] At the second visit (randomization), patients and their
representatives are asked about adverse events, faecal samples are
collected and equipment for collection of new samples is
distributed. BSFS is collected and new BSFS is distributed. Study
products are distributed together with a compliance form (diary).
Patients and their representatives are reminded to follow the
healthy dietary habits.
[0139] The study runs for 8 weeks with the patients consuming
either a placebo or one of two investigational products daily.
Patients are instructed to consume the products in the morning with
breakfast. Compliance is monitored via a compliance form (diary) to
be filled in daily.
[0140] Four weeks after commencement there is an intermediate
check. Patients and their representatives are asked about adverse
events and any changes in the patient's usual medication. Faecal
samples are collected and equipment for collection of new samples
is distributed. Blood pressure, pulse rate, waist and hip
circumference, height and bodyweight are measured and BMI SDS
calculated. The GSRS is completed on site by the participating
child's representative. The BSFS is collected and new BSFS is
distributed to the participant's representative(s) with
instructions to assess the stool consistency at each faecal
sampling point using the BSFS. Patients and their representatives
are reminded to follow the healthy dietary habits.
[0141] At the end of intervention (8 weeks), each patient has a
visit with the medical team. Patients and their representatives are
asked about adverse events and any changes in the patient's usual
medication. Study products and compliance forms are collected to
check compliance. BSFS and faecal samples are collected and
equipment for collection of new samples is distributed. A physical
examination is done and pubertal staging is determined. Blood
pressure, pulse rate, height and bodyweight are measured, and body
composition is determined by a DXA (dual energy x-ray
absorptiometry)-scan and bioimpedance. BMI SDS is calculated, waist
and hip circumferences measured and food intake registered. Fasting
blood samples are collected for safety and biomarker studies and
for biobanking, and equipment for collecting faecal samples is
distributed. The GSRS questionnaire is completed on site by the
participating child's representative(s).
[0142] To examine potential long-term effects of the intervention,
an un-blinded follow-up period follows with a visit 8 weeks after
end of intervention. A physical examination is done and pubertal
staging is determined. Blood pressure, pulse rate, height and
bodyweight are measured, and body composition is determined by a
DXA (dual energy x-ray absorptiometry)-scan and bioimpedance. BMI
SDS is calculated, waist and hip circumferences measured and food
intake registered. Fasting blood samples are collected for safety
and biomarker studies and for biobanking. Faecal samples are
collected.
[0143] The results show that oral ingestion of HMOs modulate the
intestinal microbiota, and specifically stimulate the growth of
bifidobacteria, particular species belonging to the B. adolescentis
phylogenetic group, and change the SCFA profile. The blood
biomarker analysis indicated that the patients given the
investigational products have a lipid profile with lower
triglyceride levels and higher high-density lipoprotein
cholesterol. Additionally, the blood pressure and body composition
are decreased. The abundance of bifidobacteria correlates
negatively with the level of low-density lipoprotein cholesterol
and positively with the level of high-density lipoprotein
cholesterol. Collectively, HMOs are able to increase bifidobacteria
and change the intestinal environment, and by this, improve the
lipid profile, hypertension and body composition, all incidence
reducing the risk of CVD.
Example 3
Nutritional Composition
[0144] A ready to feed nutritional composition is prepared from
water, maltodextrin, milk protein concentrate, sucromalt,
glycerine, cocoa powder, soy protein isolate, fructose, high oleic
safflower oil, soy oil, canola oil, plant sterol esters, HMOs, soy
lecithin, magnesium chloride, calcium phosphate, carrageenan,
sodium ascorbate, potassium citrate, sodium phosphate, calcium
citrate, choline chloride, potassium chloride, sodium citrate,
magnesium oxide, taurine, L-carnitine, alpha-tocopheryl acetate,
zinc sulphate, ferrous sulphate, niacinamide, calcium pantothenate,
vitamin A palmitate, citric acid, manganese sulphate, pyridoxine
hydrochloride, vitamin D3, copper sulphate, thiamine mononitrate,
riboflavin, beta carotene, folic acid, biotin, potassium iodide,
chromium chloride, sodium selenate, sodium molybdate, phytonadione,
vitamin B12.
[0145] The composition has an energy density of 0.8 kcal/ml with an
energy distribution (% of kcal) as follows: protein: 20%,
carbohydrate: 48%, fat: 32%.
Example 5
Tablet Composition
[0146] A tablet is prepared from HMO, hydroxypropyl
methylcellulose, sodium alginate, gum, microcrystalline cellulose,
colloidal silicon dioxide, and magnesium stearate. All raw
materials except the magnesium stearate are placed into a high
shear granulator and premixed. Water is sprayed onto the premix
while continuing to mix at 300 rpm. The granulate is transferred to
a fluidised bed drier and dried at 75.degree. C. The dried powder
is sieved and sized using a mill. The resulting powder is then
lubricated with magnesium stearate and pressed into tablets. The
tablets each contain 325 mg of HMO. The tablets each have a weight
of 750 mg.
Example 6
Capsule Composition
[0147] A capsule is prepared by filling about 1 g of HMO into a 000
gelatine capsule using a filing machine. The capsules are then
closed. The HMO are in free flowing, powder form.
* * * * *