U.S. patent application number 17/577252 was filed with the patent office on 2022-05-05 for synthetic composition.
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 | 20220133759 17/577252 |
Document ID | / |
Family ID | 1000006082440 |
Filed Date | 2022-05-05 |
United States Patent
Application |
20220133759 |
Kind Code |
A1 |
Vigsn.ae butted.s; Louise Kristine
; et al. |
May 5, 2022 |
SYNTHETIC COMPOSITION
Abstract
A method of increasing the relative abundance of Bifidobacterium
adolescentis in the gastrointestinal tract and regulating levels of
one or both of tryptophan and serotonin in a non-infant human is
disclosed. In some examples, the method is effective for treating
migraines. The method includes administering for an initial
treatment period an amount of one or more human milk
oligosaccharides (HMOs) selected from 2'-fucosyllactose (2'-FL),
3-fucosyllactose (3-FL), lacto-N-fucopentaose I (LNFP-I),
difucosyllactose (DFL), lacto-N-tetraose (LNT), and
lacto-N-neotetraose (LNnT), where the amount is effective to
increase the relative abundance of Bifidobacterium adolescentis in
the gastrointestinal tract of the non-infant human.
Inventors: |
Vigsn.ae butted.s; Louise
Kristine; (Copenhagen, DK) ; McConnell; Bruce;
(La Tour de Peilz, CH) ; Elison; Emma; (Malmo,
SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Glycom A/S |
Horsholm |
|
DK |
|
|
Family ID: |
1000006082440 |
Appl. No.: |
17/577252 |
Filed: |
January 17, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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16303131 |
Nov 19, 2018 |
11224605 |
|
|
PCT/DK2017/050165 |
May 19, 2017 |
|
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17577252 |
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Current U.S.
Class: |
514/54 |
Current CPC
Class: |
A61K 31/702 20130101;
A61P 1/00 20180101; A61P 25/00 20180101 |
International
Class: |
A61K 31/702 20060101
A61K031/702; A61P 25/00 20060101 A61P025/00; A61P 1/00 20060101
A61P001/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 19, 2016 |
DK |
PA 2016 70333 |
Claims
1. A method of increasing the relative abundance of Bifidobacterium
adolescentis in the gastrointestinal tract and regulating levels of
one or both of tryptophan and serotonin in a non-infant human, the
method comprising administering for an initial treatment period an
amount of one or more human milk oligosaccharides (HMOs) selected
from 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL),
lacto-N-fucopentaose I (LNFP-I), difucosyllactose (DFL),
lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and
combinations thereof, wherein the amount is effective to increase
the relative abundance of Bifidobacterium adolescentis in the
gastrointestinal tract of the non-infant human.
2. The method of claim 1, wherein the non-infant human is an
irritable bowel syndrome (IBS) patient.
3. The method of claim 1, wherein during the initial treatment
period the selected total amount of the one or more HMOs that is
effective to increase the relative abundance of Bifidobacterium
adolescentis in the gastrointestinal tract of the non-infant human
is from 3 g to 10 g per day.
4. The method of claim 1, wherein the selected amount of the one or
more HMOs is effective to increase the level of serotonin in the
blood of the non-infant human.
5. The method of claim 1, wherein administering to the non-infant
human the selected amount of the one or more HMOs further
comprises: administering, during a maintenance treatment period of
one or more days following the initial treatment period, a reduced
amount, relative to the selected amount administered during the
initial treatment period, of the one or more HMOs that is effective
for increasing the relative abundance of one or both of
Bifidobacterium longum and Bifidobacterium bifidum in the
microbiota in the gastrointestinal tract of the non-infant
human.
6. The method of claim 5, wherein the maintenance treatment period
is at least one month.
7. The method of claim 1, wherein the selected amount of the one or
more HMOs is effective to decrease the concentration of one or both
of IFN-.gamma. and TNF-.alpha. in the blood of the non-infant
human.
8. A method of increasing the relative abundance of Bifidobacterium
adolescentis and regulating serotonin to improve gut motility in a
non-infant human with irritable bowel syndrome (IBS), the method
comprising: selecting an effective amount of one or more human milk
oligosaccharides (HMOs) chosen from the group consisting of
2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL),
lacto-N-fucopentaose I (LNFP-I), difucosyllactose (DFL),
lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and
combinations thereof; and increasing the relative abundance of
Bifidobacterium adolescentis in the gastrointestinal tract of the
non-infant human and regulating serotonin to improve gut motility
by administering to the non-infant human the selected amount of the
one or more HMOs during an initial treatment period.
9. The method of claim 8, wherein during the initial treatment
period the selected total amount of the one or more HMOs that is
effective to increase the relative abundance of Bifidobacterium
adolescentis in the gastrointestinal tract of the non-infant human
is from 3 g to 10 g per day.
10. The method of claim 8, wherein the selected amount of the one
or more HMOs is effective to increase the level of serotonin in the
blood of the non-infant human.
11. The method of claim 8, wherein administering to the non-infant
human the selected amount of the one or more HMOs further
comprises: administering, during a maintenance treatment period of
one or more days following the initial treatment period, a reduced
amount, relative to the selected amount administered during the
initial treatment period, of the one or more HMOs that is effective
for increasing the relative abundance of one or both of
Bifidobacterium longum and Bifidobacterium bifidum in the
microbiota in the gastrointestinal tract of the non-infant
human.
12. The method of claim 8, wherein the non-infant human is an IBS-D
patient and the method further comprises decreasing stool frequency
and improving stool consistency by administering to the non-infant
human the selected amount of the mixture.
13. The method of claim 8, wherein the non-infant human is an IBS-C
patient and the method further comprises increasing stool frequency
and softening stool consistency by administering to the non-infant
human the selected amount of the mixture.
14. The method of claim 8, wherein the non-infant human is an IBS-A
patient and the method further comprises normalizing stool
frequency and stool consistency by administering to the non-infant
human the selected amount of the mixture.
15. A method for treating migraines associated with serotonin
dysregulation in a non-infant human, the method comprising:
selecting an effective amount of one or more human milk
oligosaccharides (HMOs) chosen from the group consisting of
2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL),
lacto-N-fucopentaose I (LNFP-I), difucosyllactose (DFL),
lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and
combinations thereof, the amount effective for increasing a
relative abundance of Bifidobacterium adolescentis in the
gastrointestinal microbiota of the non-infant human; regulating
serotonin synthesis in the non-infant human by increasing the
relative abundance of Bifidobacterium adolescentis in the
gastrointestinal tract non-infant human by administering the
effective amount of the selected HMOs to the non-infant human for
an initial treatment period of at least 14 days, thereby treating
the migraines in the non-infant human.
16. The method of claim 15, wherein during the initial treatment
period the selected total amount of the one or more HMOs that is
effective to increase the relative abundance of Bifidobacterium
adolescentis in the gastrointestinal tract of the non-infant human
is from 3 g to 10 g per day.
17. The method of claim 15, wherein the selected amount of the one
or more HMOs is effective to increase the level of serotonin in the
blood of the non-infant human.
18. The method of claim 15, wherein administering to the non-infant
human the selected amount of the one or more HMOs further
comprises: administering, during a maintenance treatment period of
one or more days following the initial treatment period, a reduced
amount, relative to the selected amount administered during the
initial treatment period, of the one or more HMOs that is effective
for increasing the relative abundance of one or both of
Bifidobacterium longum and Bifidobacterium bifidum in the
gastrointestinal tract of the non-infant human.
19. The method of claim 15, wherein the maintenance treatment
period is at least one month.
20. The method of claim 16, wherein the selected amount of the one
or more HMOs is effective to decrease the concentration of one or
both of IFN-.gamma. and TNF-.alpha. in the blood of the non-infant
human.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This is a continuation of and claims priority to U.S. patent
application Ser. No. 16/303,131 titled "SYNTHETIC COMPOSITION" and
filed on Nov. 19, 2018, which is a National Stage Entry of
PCT/DK2017/050165 filed on May 19, 2017 and claims priority to
Denmark Application No. PA 2014 70333 filed on May 19, 2016, all of
which are incorporated herein by reference to the extent legally
allowable.
FIELD
[0002] This invention relates to a method and composition for
improving gastrointestinal motility, secretion and sensation by
regulating serotonin synthesis in humans.
BACKGROUND
[0003] A regulated communication between the gut and the brain
plays a very important role in the wellbeing of a human, and normal
gastrointestinal (GI) health relies on a properly functioning
brain-gut axis. Serotonin, which functions as a neurotransmitter,
facilitates communication between the enteric nervous system and
the rest of the gut, and plays an important role in regulation of
gut motility and secretion. Serotonin can activate neurons causing
either bowel contraction or relaxation, can initiate secretion, and
contribute to the dilution and neutralization of luminal contents,
causing protective responses to eliminate luminal pathogens.
However, in various studies, the effects of HMOs on serotonin
levels and gut motility in non-infant humans is unknown and for
non-infants, there remains a need for a safe, well tolerated, and
effective way of managing serotonin synthesis and thereby improve
gut motility.
SUMMARY
[0004] One or more methods and compositions for improving
gastrointestinal motility, secretion, and sensation by regulating
serotonin synthesis and/or tryptophan levels in non-infant humans
are disclosed. A method of increasing the relative abundance of
Bifidobacterium adolescentis in the gastrointestinal tract and
regulating levels of one or both of tryptophan and serotonin in a
non-infant human is disclosed where the method includes
administering for an initial treatment period an amount of one or
more human milk oligosaccharides (HMOs) selected from
2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL),
lacto-N-fucopentaose I (LNFP-I), difucosyllactose (DFL),
lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT), where the
amount is effective to increase the relative abundance of
Bifidobacterium adolescentis in the gastrointestinal tract of the
non-infant human.
[0005] In various examples, the non-infant human is an irritable
bowel syndrome (IBS) patient. During the initial treatment period,
in some examples, the selected total amount of the one or more HMOs
that is effective to increase the relative abundance of
Bifidobacterium adolescentis in the gastrointestinal tract of the
non-infant human is from 3 g to 10 g per day. In certain examples,
the selected amount of the one or more HMOs is effective to
increase the level of serotonin in the blood of the non-infant
human.
[0006] In certain examples, administering to the non-infant human
the selected amount of the one or more HMOs further comprises:
administering, during a maintenance treatment period of one or more
days following the initial treatment period, a reduced amount,
relative to the selected amount administered during the initial
treatment period, of the one or more HMOs that is effective for
increasing the relative abundance of one or both of Bifidobacterium
longum and Bifidobacterium bifidum in the microbiota in the
gastrointestinal tract of the non-infant human.
[0007] In some examples, the maintenance treatment period is at
least one month. In various examples, the selected amount of the
one or more HMOs is effective to decrease the concentration of one
or both of IFN-.gamma. and TNF-.alpha. in the blood of the
non-infant human.
[0008] A method is disclosed for increasing the relative abundance
of Bifidobacterium adolescentis and regulating serotonin to improve
gut motility in a non-infant human with irritable bowel syndrome
(IBS). The method includes: selecting an effective amount of one or
more human milk oligosaccharides (HMOs) chosen from the group
consisting of 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL),
difucosyllactose (DFL), lacto-N-fucopentaose I (LNFP-I),
lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and
combinations thereof; and increasing the relative abundance of
Bifidobacterium adolescentis in the gastrointestinal tract of the
non-infant human and regulating serotonin to improve gut motility
by administering to the non-infant human the selected amount of the
one or more HMOs during an initial treatment period.
[0009] In various examples, during the initial treatment period,
the selected total amount of the one or more HMOs that is effective
to increase the relative abundance of Bifidobacterium adolescentis
in the gastrointestinal tract of the non-infant human is from 3 g
to 10 g per day.
[0010] In some examples, the selected amount of the one or more
HMOs is effective to increase the level of serotonin in the blood
of the non-infant human. In various examples, administering to the
non-infant human the selected amount of the one or more HMOs
further comprises: administering, during a maintenance treatment
period of one or more days following the initial treatment period,
a reduced amount, relative to the selected amount administered
during the initial treatment period, of the one or more HMOs that
is effective for increasing the relative abundance of one or both
of Bifidobacterium longum and Bifidobacterium bifidum in the
microbiota in the gastrointestinal tract of the non-infant
human.
[0011] In some examples, the non-infant human is an IBS-D patient
and the method further comprises decreasing stool frequency and
improving stool consistency by administering to the non-infant
human the selected amount of the mixture. In various examples, the
non-infant human is an IBS-C patient and the method further
comprises increasing stool frequency and softening stool
consistency by administering to the non-infant human the selected
amount of the mixture. In certain examples, the non-infant human is
an IBS-A patient and the method further comprises normalizing stool
frequency and stool consistency by administering to the non-infant
human the selected amount of the mixture.
[0012] A method is disclosed for treating migraines associated with
serotonin dysregulation in a non-infant human. The method includes
selecting an effective amount of one or more human milk
oligosaccharides (HMOs) chosen from the group consisting of
2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL),
difucosyllactose (DFL), lacto-N-fucopentaose I (LNFP-I),
lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), and
combinations thereof, the amount effective for increasing a
relative abundance of Bifidobacterium adolescentis in the
gastrointestinal microbiota of the non-infant human. The method
also includes regulating serotonin synthesis in the non-infant
human by increasing the relative abundance of Bifidobacterium
adolescentis in the gastrointestinal microbiota of non-infant human
by administering the effective amount of the selected HMOs to the
non-infant human for an initial treatment period of at least 14
days, thereby treating the migraines in the non-infant human.
[0013] In some examples, during the initial treatment period, the
selected total amount of the one or more HMOs that is effective to
increase the relative abundance of Bifidobacterium adolescentis in
the gastrointestinal tract of the non-infant human is from 3 g to
10 g per day.
[0014] In certain examples, the selected amount of the one or more
HMOs is effective to increase the level of serotonin in the blood
of the non-infant human. In some examples, administering to the
non-infant human the selected amount of the one or more HMOs
further includes administering, during a maintenance treatment
period of one or more days following the initial treatment period,
a reduced amount, relative to the selected amount administered
during the initial treatment period, of the one or more HMOs that
is effective for increasing the relative abundance of one or both
of Bifidobacterium longum and Bifidobacterium bifidum in the
microbiota in the gastrointestinal tract of the non-infant
human.
[0015] In some examples, the maintenance treatment period is at
least one month. In various examples, the selected amount of the
one or more HMOs is effective to decrease the concentration of one
or both of IFN-.gamma. and TNF-.alpha. in the blood of the
non-infant human.
DETAILED DESCRIPTION OF THE PRESENT DISCLOSURE
[0016] The GI tract is the largest producer of serotonin in the
human body. Since 95 percent of the serotonin is found in the gut,
an imbalance in its signaling can cause alterations in motility and
secretion. This has been shown in a study, where disruption of
serotonin signaling resulted in loss of peristaltic motility
(McGill et al., FASEB J. 30, Supplement 1254.6 (2016)). Tryptophan
is the precursor of a wide array of metabolites, which are involved
in a variety of aspects of human nutrition and metabolism.
Tryptophan can be metabolized to serotonin; however, it can also
enter the kynurenine pathway.
[0017] The kynurenine pathway is regulated by pro-inflammatory
cytokines such as IFN-.gamma. and TNF-.alpha., and metabolites from
this pathway have been shown to be able to negatively modulate
immune responses. Hence, accumulating evidence indicates that
shunting tryptophan down the kynurenine pathway at the expenses of
serotonin biosynthesis may cause abnormalities in serotonin
signaling and could lead to functional GI disorders such as
irritable bowel syndrome (IBS), celiac disease and inflammatory
bowel disease (IBD).
[0018] Studies have shown that IBS patients have impaired levels of
serotonin compared to controls, with higher levels in IBS-D and
lower levels in IBS-C(Yu et al., World J. Gastroenterol. 22, 3451
(2016); Wang et al., ibid. 13, 6041 (2007)). Additionally, abnormal
tryptophan catabolism has been found to correlate with severe IBS
symptoms (Faure et al., Gastroenterology 139, 249 (2010);
Fitzgerald et al., Neurogastroenterol. Motil. 20, 1291 (2008)).
[0019] Since tryptophan is an essential amino acid with an
estimated dietary requirement of 5 mg/kg/day and it is the limiting
amino acid in nearly all protein sources, dietary sources of
tryptophan may not be enough to obtain a regulated serotonin
synthesis, and maintain normal GI function. Therefore, there is a
need for a safe, effective intervention for obtaining sufficient
tryptophan for regulating serotonin synthesis.
[0020] In addition to tryptophan and serotonin metabolism, an
optimal functioning GI tract requires a healthy bacterial
population of beneficial bacteria. The human intestinal microbiota
is a complex and very dynamic microbial ecosystem that consists of
various populations, which are important to preserve human health.
Selective stimulation of specific beneficial intestinal bacteria to
promote their growth and metabolic activity could be a helpful
approach in regulating the tryptophan and serotonin metabolic
pathways, resulting in improvement of gut motility and
secretion.
[0021] Human milk oligosaccharides (HMOs) are a heterogeneous
mixture of soluble glycans found in human milk. They are the third
most abundant solid component after lactose and lipids in human
milk and are present in concentrations of 5-25 g/l (Gabrielli et
al., Pediatrics 128, e1520 (2011)). HMOs are resistant to enzymatic
hydrolysis in the small intestine and are thus largely undigested
and unabsorbed (ten Bruggencate et al., Nutrition Reviews 72, 377
(2014); Rudloff et al., Glycobiology 16, 477 (2006)).
[0022] The majority of HMOs that reach the colon serve as a
substrate to shape the gut ecosystem by selectively stimulating the
growth of specific beneficial bacteria. HMOs are believed to
substantially modulate the infant gut microbiota and play a
decisive role in the differences in the microbiota of formula-fed
and breast-fed infants. These differences include the predominance
of Bifidobacterium in the gut of breast-fed infants compared to a
more diverse gut microbiota in formula-fed infants (Bezirtzoglou et
al., Anaerobe 17, 478 (2011)).
[0023] This is viewed as beneficial for the infant because strains
of Bifidobacterium species and their metabolites are believed to
have a positive effect on human health (Rudloff et al.,
Glycobiology 16, 477 (2006); Bezirtzoglou et al., Anaerobe 17, 478
(2011)). Recently, it has also been demonstrated that some
sialylated and fucosylated HMOs has a positive effect on the growth
of certain strains of Bifidobacterium species that are typically
found in both infant and adult microbiota (WO 2013/154725). WO
2013/054001 described a positive correlation between the level of
serotonin in the blood and proportion of bifidobacteria and/or
clostridia species in the intestine flora of tested
individuals.
[0024] The effect of HMOs on serotonin level and gut motility has
never been studies and is therefore unknown. Therefore, there
remains a need for a safe, well tolerated and effective way of
managing serotonin synthesis and thereby improve gut motility.
[0025] This disclosure provides synthetic compositions comprising
one or more human milk oligosaccharides (HMOs) that can be
advantageously used for regulating serotonin synthesis, and
improving gut motility and secretion, in a human individual.
[0026] Accordingly:
[0027] a first aspect of this disclosure relates to one or more
human milk oligosaccharides for use in the prophylaxis or treatment
of a symptom or disease associated with serotonin and/or tryptophan
dysregulation in a human;
[0028] a second aspect of this disclosure relates to one or more
human milk oligosaccharides for use in the prophylaxis or treatment
of serotonin and/or tryptophan dysregulation in a human, in
particular in an irritable bowel symptom (IBS) patient;
[0029] a third aspect of this disclosure relates to one or more
human milk oligosaccharides for use in improving physiological
and/or psychological conditions associated with serotonin and/or
tryptophan dysregulation in a human;
[0030] a fourth aspect of this disclosure relates to a synthetic
composition for use in the prophylaxis or treatment of a symptom or
disease associated with serotonin and/or tryptophan dysregulation
in a human, the composition comprising an effective amount of one
or more human milk oligosaccharides;
[0031] a fifth aspect of this disclosure relates to a synthetic
composition for use in the prophylaxis or treatment of serotonin
and/or tryptophan dysregulation in a human, in particular in an
irritable bowel symptom (IBS) patient, the composition comprising
an effective amount of one or more human milk oligosaccharides;
[0032] a sixth aspect of this disclosure relates to a synthetic
composition for use in improving physiological and/or psychological
conditions associated with serotonin and/or tryptophan
dysregulation in a human, the composition comprising an effective
amount of one or more human milk oligosaccharides;
[0033] a seventh aspect of this disclosure provides a method for
the prophylaxis or treatment of a symptom or disease associated
with serotonin and/or tryptophan dysregulation in a human, the
method comprising administering to the human an effective amount of
one or more human milk oligosaccharides;
[0034] an eighth aspect of this disclosure provides a method for
the prophylaxis or treatment of serotonin and/or tryptophan
dysregulation in a human, the method comprising administering to
the human an efficient amount of one or more human milk
oligosaccharides;
[0035] a ninth aspect of this disclosure provides a method for
improving physiological and/or psychological conditions associated
with serotonin and/or tryptophan dysregulation in a human, the
composition comprising an effective amount of one or more human
milk oligosaccharides.
[0036] Preferably, the human milk oligosaccharide is 2'-FL, 3-FL,
DFL, 3'-SL, 6'-SL, LNnT, LNT or LNFP-I or a mixture thereof. For
example, the composition can comprise a mixture of a fucosylated
HMO such as 2'-FL and/or DFL and a non-fucosylated neutral HMO such
as LNnT or LNT, or both. In one preferred example, the human milk
oligosaccharide is a mixture of 2'-FL and LNnT and/or LNT. In this
example, the 2'-FL and LNnT/LNT may be present in a mass ratio of
about 5:1 to 1:1; more preferably about 4:1 to 2:1. In another
preferred example, the human milk oligosaccharide is a mix of 2'-FL
and/or DFL and LNnT and/or LNT. In this example, the 2'-FL/DFL and
LNnT/LNT may be present in a mass ratio of about 5:1 to 1:1; more
preferably about 4:1 to 2:1.
[0037] Preferably, the amount of HMOs is effective to increase the
abundance of bifidobacteria in the gastrointestinal tract and
improve gut motility of the concerned human in need. For example,
the amount may be about 1 g to about 15 g per day; for example,
about 3 g to about 10 g per day. More preferably an amount of about
3 g to about 7 g is administered per day.
[0038] In an example, the synthetic composition can be a
nutritional or pharmaceutical composition. Preferably, synthetic
composition of the present disclosure is administered daily.
Furthermore, the synthetic composition of the present disclosure is
preferably administered for a period of at least one month, such as
at least 2 months or for a longer period of time, for example
chronically on an ongoing basis.
[0039] Synthetic composition of the present disclosure may be
administered to the human or patient as a daily dose of about 1 g
to about 15 g, such as from about 3 g to about 10 g, more
preferably about 3 g to about 7 g of HMOs.
[0040] Preferably, the one or more HMOs are administered to a
human, preferably a non-infant human, in need in two steps: (a) in
a first step, during an initial treatment period of about 14 days,
to increase the relative abundance of bifidobacteria of the
phylogenetic Bifidobacterium adolescentis group; and (b) in a
second step, during an additional period of treatment of 1 or more
days following the initial treatment period, to increase the
relative abundance of Bifidobacterium longum and/or Bifidobacterium
bifidum, in the microbiota in the gastro-intestinal tract of said
human.
[0041] It has now been surprisingly found that administration of
human milk oligosaccharides (HMOs) to humans, in particular
non-infant humans, preferentially increases the abundance of
bifidobacteria in the gastrointestinal tract and improves gut
motility. The increase of bifidobacteria could lead to regulation
of serotonin synthesis, and hence affect gut motility and
secretion. Since species of bifidobacteria are able to synthesize
tryptophan, and in addition, can inhibit production of
pro-inflammatory cytokines such as TNF-.alpha. and IFN-.gamma.,
selective stimulation of these bacteria can affect tryptophan
metabolism shunting it down the serotonin pathway instead of the
kynurenine pathway causing improvement of gut motility and
secretion. Furthermore, fucosylated HMOs can directly regulate gut
motor contraction.
[0042] Thus, it has been discovered that HMOs can, by oral or
enteral ingestion, modulate the intestinal microbiota in humans, in
particular non-infant humans, by preferentially promoting the
growth of bifidobacteria. As an outcome, the synthesis of serotonin
is up-regulated and improvement of gut motility and secretion is
obtained.
Terms and Definitions
[0043] The term "human milk oligosaccharide" or "HMO" preferably
means a complex carbohydrate found in human breast milk 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 backbone, fucosylated and sialylated oligosaccharides.
Backbone HMOs consists of Glu, Gal and GlcNAc and are devoid of Fuc
and sialic acid. Examples of backbone HMOs include lacto-N-tetraose
(LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexaose (LNnH) and
lacto-N-hexaose (LNH). Fucosyl HMOs are fucosylated lactoses or
fucosylated backbone 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 1 (FLNH-I),
fucosyl-lacto-N-hexaose III (FLNH-III) and
fucosyl-para-lacto-N-neohexaose (F-pLNnH). Sialyl HMOs are
sialylated lactoses or sialylated backbone 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). HMOs containing both sialyl and fucosyl groups may be
considered to belong to either of the latter two groups. Examples
for sialyl and fucosyl HMOs include di
sialyl-fucosyl-lacto-N-hexaose II (DSFLNH-II),
fucosyl-sialyl-lacto-N-neohexaose 1 (FSLNnH-1),
fucosyl-sialyl-lacto-N-hexaose 1 (FSLNH-I) and
3-fucosyl-3'-sialyllactose (FSL). In context of the present
disclosure, lactose is not included in the group of HMOs.
[0044] The terms "microbiota", "microflora" and "microbiome"
preferably mean a community of living microorganisms that typically
inhabits a bodily organ or part. 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 the microorganisms of Bacteroides, Faecalibacterium,
Bifidobacterium, Roseburia, Alistipes, Collinsella, Blautia,
Coprococcus, Ruminococcus, Eubacterium, and Dorea; and at species
level microorganisms of Bacteroides uniform is, 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.
In some instances, 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.
[0045] The term "bifidobacteria" means a member of the
Bifidobacterium genus commonly found in the human gastro-intestinal
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 Bifidobacterium
longum, Bifidobacterium bifidum, and the phylogenetic
Bifidobacterium adolescentis group, for example Bifidobacterium
pseudocatenulatum and/or Bifidobacterium adolescentis.
[0046] The term "serotonin" refers to the compound having the IUPAC
name 3-(2-aminoethyl)-5-hydroxyindole, and the following structural
formula:
##STR00001##
[0047] Serotonin is also known as 5-hydroxytryptamine, enteramine,
thrombocytin, 3-(3-aminoethyl)-5-hydroxyindole, thrombotonin and
5-HT.
[0048] The term "serotonin dysregulation" means that the level of
serotonin in blood of a human subject deviates from the level which
is typically regarded to be normal, i.e. it is lower or higher than
101-283 ng/ml.
[0049] The term "tryptophan" refers to the amino acid having the
IUPAC name (2S)-2-amino-3-(1H-indol-3-yl) propanoic acid, and the
following structural formula:
##STR00002##
[0050] The term "tryptophan dysregulation" means that the level of
tryptophan in blood of a human subject deviates from the level
which is typically regarded to be normal, i.e. it is lower or
higher than 25-73 .mu.mol/l.
[0051] The term "symptom or disease associated with serotonin
and/or tryptophan dysregulation" refers to a symptom or disease
preferably selected from the following group: depression, anxiety,
anger, being unusually sensitive to pain, carbohydrate cravings and
binge eating, constipation, digestive disorders, feeling glum from
lack of sunlight, feeling overly dependent on others, feeling
overwhelmed, hypervigilance, insomnia, joylessness, low
self-esteem, migraines, poor cognitive function and tinnitus.
[0052] The term "synthetic composition" designates 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 examples a
synthetic composition of the present disclosure may be, but
preferably is not, identical with a naturally occurring
composition. The synthetic composition of the present 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 examples, 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
examples, 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 examples of a synthetic composition of the
present disclosure are also described below.
[0053] The term "relative abundance of bifidobacteria", preferably,
means the abundance of bifidobacteria relative to other genus in
the microbiota of the gastro-intestinal tract.
[0054] The term "relative growth of bifidobacteria", preferably,
means the growth of bifidobacteria relative to other genus in the
microbiota in the gastro-intestinal tract.
[0055] The term "human" preferably means a non-infant human
individual. The term "non-infant human" or "non-infant" means in
the present context a human of 3 years of age and older. A
non-infant human can be a child, a teenager, an adult or an
elderly.
[0056] The term "enteral administration" preferably 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.
[0057] The term "oral administration" preferably means any
conventional form for the delivery of a composition to a human
through the mouth thereby depositing the composition in the
gastrointestinal tract (including the stomach) of the patient.
Accordingly, oral administration includes swallowing of composition
by the patient, enteral feeding through a nasogastric tube, and the
like. Oral administration is a form of enteral administration.
[0058] The term "effective amount" preferably means an amount of a
composition that provides an HMO in a sufficient amount to render a
desired treatment outcome in a human. An effective amount can be
administered in one or more doses to achieve the desired treatment
outcome.
[0059] The term "irritable bowel syndrome" (also abbreviated herein
as IBS) means a clinically heterogeneous disorder of human,
particularly adult human individuals, wherein the patients have
chronic symptoms such as abdominal pain, abdominal discomfort,
abdominal bloating, fatigue, and changes in bowel movement
patterns, such as patterns of loose or more frequent bowel
movements, diarrhoea and constipation. Routine clinical tests on
IBS patients typically show no abnormalities, although their bowels
may be more sensitive to certain stimuli, such as balloon
insufflation testing. Individuals having the later and other
symptoms (than listed above) are also contemplated as subjects of
the present disclosure. There are at least three forms of IBS,
depending on which symptom predominates: (1) diarrhoea-predominant
(IBS-D); (2) constipation-predominant (IBS-C); and (3) IBS with
alternating stool pattern (IBS-A or IBS-M). There are also various
clinical subtypes of IBS, such as post-infectious IBS (IBS-PI) or
unsubtyped IBS (IBS-U).
[0060] The term "prophylaxis" refers to a measure taken to maintain
health and prevent occurrence of undesirable physiological or
psychological conditions or spread of symptoms of a disease.
[0061] The term "treatment" refers to an attempt to remediate a
health problem, e.g., a physiological or psychological condition or
symptom of a disease, usually following a diagnosis.
[0062] The term "initial period" of treatment with an HMO or an HMO
mixture is about 14 days from the start of the treatment;
"additional period" of treatment means 1 or more days following the
initial period of treatment.
[0063] The term "about" in the present context means up to 2.5%
deviation from the corresponded value.
[0064] The term "preferably" is used herein to indicate a best mode
that does not limit the scope of invention.
[0065] Implementations include:
[0066] HMOs for use in
[0067] the prophylaxis or treatment of a symptom or disease
associated with serotonin and/or tryptophan dysregulation in a
human,
[0068] the prophylaxis or treatment of serotonin and/or tryptophan
dysregulation in a human, in particular in an irritable bowel
symptom (IBS) patient, or
[0069] improving physiological and/or psychological conditions
associated with serotonin and/or tryptophan dysregulation in a
human,
[0070] the administered compositions may be a single HMO, or a
mixture of any HMOs suitable for the purposes of the disclosed
methods.
[0071] Preferably, the HMO is a fucosylated or a non-fucosylated
neutral HMO. More preferably, the HMOs for prophylaxis or treatment
of non-infectious diarrhoea in a human is a mixture of at least a
first HMO and at least a second HMO, where the first HMO is a
fucosylated neutral HMO and the second HMO is a non-fucosylated
neutral HMO. Particularly, the mixture contains 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 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/or DFL and
LNnT and/or LNT. In some implementation, the mixture 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.
[0072] 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 example the mixture
essentially consists of 2'-FL and LNnT, in another preferred
example the mixture essentially consists of 2'-FL and LNT. The
mixture may also contain sialylated HMOs such as 3'-SL and
6'-SL.
[0073] 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.
[0074] 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 2010/100979, 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.
[0075] 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 01/04341
and WO 2007/101862 describe how to make core human milk
oligosaccharides optionally substituted by fucose or sialic acid
using genetically modified E. coli.
[0076] The synthetic composition for use in
[0077] the prophylaxis or treatment of a symptom or disease
associated with serotonin and/or tryptophan dysregulation in a
human,
[0078] the prophylaxis or treatment of serotonin and/or tryptophan
dysregulation in a human, in particular in an irritable bowel
symptom (IBS) patient, or
[0079] improving physiological and/or psychological conditions
associated with serotonin and/or tryptophan dysregulation in a
human,
[0080] may comprise a single HMO or a mixture of any HMOs suitable
for the purpose of the present disclosure. The HMOs may be selected
as described above.
[0081] A synthetic composition of this disclosure comprising one or
more human milk oligosaccharides, 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, preferably where the other
carbohydrate is lactose and/or a carbohydrate different than an
HMO. The synthetic composition can also be a pharmaceutical
composition.
[0082] In one example, the synthetic composition may be a
pharmaceutical composition. The pharmaceutical composition can
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.
[0083] 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, flavoring 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.
[0084] The pharmaceutical compositions can also be administered by
rectal suppository, aerosol tube, nasogastric tube or direct
infusion into the GI tract or stomach.
[0085] The pharmaceutical compositions can also include therapeutic
agents such as antiviral agents, antibiotics, probiotics,
analgesics, and anti-inflammatory agents. The proper dosage of
these compositions for a human can be determined in a conventional
manner, based upon factors such as severity of the lactose
intolerance, immune status, body weight and age. The required
amount would generally be in the range from about 200 mg to about
20 g per day, in certain examples from about 1 g to about 15 g per
day, from about 3 g to about 10 g per day, in certain examples from
about 3 g to about 7 g per day. Appropriate dose regimes can be
determined by conventional methods.
[0086] In another example, the synthetic composition may be a
nutritional composition. The nutritional composition can contain
sources of protein, lipids and/or digestible carbohydrates and can
be in powdered or liquid forms. The composition can be designed to
be the sole source of nutrition or a nutritional supplement.
[0087] Suitable protein sources include milk proteins, soy protein,
rice protein, pea protein and oat protein, or mixtures thereof.
Milk proteins can be in the form of milk protein concentrates, milk
protein isolates, whey protein or casein, or mixtures of both. The
protein can be whole protein or hydrolyzed protein, either
partially hydrolyzed or extensively hydrolyzed. Preferably the
protein source contains no or low concentrations of lactose. The
protein can also be provided in the form of free amino acids. The
protein can comprise about 5% to about 30% of the energy of the
nutritional composition, normally about 10% to 20%.
[0088] The protein source can be a source of glutamine, threonine,
cysteine, serine, proline, tryptophan or a combination of these
amino acids. The glutamine source can be a glutamine dipeptide
and/or a glutamine enriched protein. Glutamine can be included due
to the use of glutamine by enterocytes as an energy source.
Threonine, serine and proline are important amino acids for the
production of mucin. Mucin coats the GI tract and can reduce
permeability. Cysteine is a major precursor of glutathione, which
is key for the antioxidant defenses of the body.
[0089] Suitable digestible carbohydrates include maltodextrin,
hydrolyzed or modified starch or corn starch, glucose polymers,
corn syrup, corn syrup solids, high fructose corn syrup,
rice-derived carbohydrates, pea-derived carbohydrates,
potato-derived carbohydrates, tapioca, glucose, fructose, maltose,
trehalose, sucrose, honey, sugar alcohols (e.g., maltitol,
erythritol, sorbitol), or mixtures thereof. Preferably the
carbohydrate source is free from lactose. Generally, digestible
carbohydrates may provide about 35% to about 55% of the energy of
the nutritional composition. A particularly suitable digestible
carbohydrate is a low dextrose equivalent (DE) maltodextrin. In
some examples, the composition may also comprise indigestible
saccharides, such as e.g., lactulose.
[0090] Suitable lipids may include medium chain triglycerides (MCT)
and long chain triglycerides (LCT). Preferably the lipid is a
mixture of MCTs and LCTs. For example, MCTs can comprise about 30%
to about 70% by weight of the lipids, more specifically about 50%
to about 60% by weight. MCTs offer the advantage of easier
digestion which can be important for non-infants with inflamed GI
tracts. Generally, the lipids may provide about 35% to about 50% of
the energy of the nutritional composition. The lipids can contain
essential fatty acids (omega-3 and omega-6 fatty acids).
Preferably, these polyunsaturated fatty acids provide less than
about 30% of total energy of the lipid source. Decreasing the
levels of these polyunsaturated fatty acids is believed to decrease
sensitivity to peroxidation; which can be beneficial for
non-infants having inflammatory conditions.
[0091] Suitable sources of long chain triglycerides may be selected
from rapeseed oil, sunflower seed oil, palm oil, soy oil, milk fat,
corn oil, high oleic oils, and soy lecithin. Fractionated coconut
oils can be a suitable source of medium chain triglycerides. The
lipid profile of the nutritional composition is preferably designed
to have a polyunsaturated fatty acid omega-6 (n-6) to omega-3 (n-3)
ratio of about 4:1 to about 10:1. For example, the n-6 to n-3 fatty
acid ratio can be about 6:1 to about 9:1.
[0092] The nutritional composition can also include 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, but not limited to,
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, but not limited to, calcium,
iron, zinc, magnesium, iodine, copper, phosphorus, manganese,
potassium, chromium, molybdenum, selenium, nickel, tin, silicon,
vanadium and boron.
[0093] 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 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 mg/ml to about 5 g/ml of lycopene.
Beta-carotene can comprise from about 0.001 .mu.g/ml to about 10
mg/ml, for example about 0.034 .mu.g/ml to about 5 .mu.g/ml of
beta-carotene.
[0094] The nutritional composition can also contain various other
conventional ingredients such as preservatives, emulsifying agents,
thickening agents, buffers, fibres and probiotics, especially
probiotics which can help to reduce symptoms in IBS patients (e.g.,
VSL #3, B. infantis 35624, B. animalis subsp. lactis BB-12, B.
lactis Bi-07, L. rhamnosus GG, L. rhamnosus Lc705, L. plantarum DSM
9843, L. plantarum CECT7484, L. plantarum CECT7485, L. acidophilus
NCFM, L. fermentum CECT5716, B. breve Bb99, Propionibacterium
freundenreichii ssp. Shermanii JS, P. acidilactici CECET7483,
Streptococcus faecium), 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, flavors, and stabilizers, lubricants, and
so forth.
[0095] The nutritional composition can be in the form of a soluble
powder, a liquid concentrate, or a ready-to-use formulation. The
composition can be fed to a human via a nasogastric tube or orally.
Various flavors, fibers and other additives can also be
present.
[0096] 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,
flavors are added, and water is added to achieve the desired total
solid level.
[0097] 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.
[0098] 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 0.02% to about 2.0%, including from about
0.1% to about 1.5%, including from about 0.3% 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 0.04% to about 4.0%, including from about 0.2% to
about 3.0%, including from about 0.6% to about 2.0%.
[0099] The synthetic composition can also be in a unit dosage form
such as a capsule, tablet or sachet. For example, the synthetic
composition can be in a tablet form comprising the HMOs, and one or
more additional components to aid formulation and administration,
such as diluents, excipients, antioxidants, lubricants, colorants,
binders, disintegrants, and the like.
[0100] Suitable diluents, excipients, lubricants, colorants,
binders, and disintegrants include, but not limited to,
polyethylene, polyvinyl chloride, ethyl cellulose, acrylate
polymers and their copolymers, hydroxyethyl-cellulose,
hydroxypropylmethyl-cellulose (HPMC), sodium
carboxymethylcellulose, polyhydroxyethyl methylacrylate (PHEMA),
polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyethylene
oxide (PEO), or polyacrylamide (PA), carrageenan, sodium alginate,
polycarbophil, polyacrylic acid, tragacanth, methyl cellulose,
pectin, natural gums, xanthan gum, guar gum, karaya gum,
hypromellose, magnesium stearate, microcrystalline cellulose, and
colloidal silicon dioxide. Suitable antioxidants are vitamin A,
carotenoids, vitamin C, vitamin E, selenium, flavonoids,
polyphenols, lycopene, lutein, lignan, coenzyme Q10 ("CoQ10") and
glutathione.
[0101] The unit dosage forms, especially those in sachet form, can
also include various nutrients including macronutrients.
[0102] The unit dosage forms 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,
flavoring 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.
[0103] The unit dosage forms can also be administered by rectal
suppository, aerosol tube, nasogastric tube or direct infusion into
the GI tract or stomach.
[0104] The unit dosage forms can also include therapeutic agents
such as antiviral agents, antibiotics, probiotics, analgesics, and
anti-inflammatory agents. The synthetic composition in unit dosage
form may be a pharmaceutical composition or a nutritional
supplement.
[0105] For reducing or preventing serotonin and/or tryptophan
dysregulation, as well as a disease or symptoms associated with
serotonin and/or tryptophan dysregulation in a patient, the amount
of HMO(s) required to be administered to the patient will vary
depending upon factors such as the risk and severity of the
disease, the age of the patient, the form of the composition, and
other medications being administered to the patient. However, the
required amount can be readily set by a medical practitioner and
would generally be in the range from about 200 mg to about 20 g per
day, in certain examples from about 1 g to about 15 g per day, from
about 3 g to about 10 g per day, in certain examples from about 3 g
to about 7.5 g per day. An appropriate dose can be determined based
on several factors, including, for example, body weight and/or
condition, the severity of the condition, being treated or
prevented, other ailments and/or diseases, the incidence and/or
severity of side effects and the manner of administration.
Appropriate dose ranges may be determined by methods known to those
skilled in the art. During an initial treatment phase, the dosing
can be higher or lower depending upon the need to boost
bifidobacteria abundance or initial tolerance to HMOs. During a
maintenance phase, the dosing can be set for chronic long term
use.
[0106] The duration of the HMO administration will vary depending
upon factors such as the risk and severity of the medical
condition, age, the form of the composition, the dose and other
medications being administered. However, the duration can be
readily set by a medical practitioner. For example, the duration
may be for 1 to 3 months. The administration can continue
chronically for an indefinite period.
[0107] Although examples may be described with reference to a
preferred implementation, it will be appreciated that various
modifications are possible within the scope of the present
disclosure.
EXAMPLE
[0108] The following example is described to illustrate an
implementation and should not be considered as limiting the scope
of the claims.
Example 1--Human Trial
[0109] A total of 60 male and female IBS patients are recruited to
participate in the study. After a screening visit and run-in period
of 1-2 weeks, the patients are selected. The patients are
randomized into three groups, each of 20 patients, with two groups
consuming the treatment product and one group the placebo product
for 4 weeks. The treatment product contains either 5 or 10 grams of
a combination of 2'-FL and LNnT in a 4:1 ratio, while the placebo
product contains 5 grams glucose. Both products are in powder form
in a unit dosage container.
[0110] The patients are eligible to participate if they are at an
age between 18-60 years, fulfil definition of IBS-D, IBS-C or
IBS-A/M according to the Rome IV criteria for IBS and have a global
IBS-SSS score of >174 during the 2 weeks run-in period. All
recruited patients are able and willing to understand and comply
with the study procedures.
[0111] Patients are excluded if: they have any known
gastrointestinal disease(s) that may cause symptoms or interfere
with the trial outcome, in particular lactose intolerance and
coeliac disease; they have participated in a clinical study one
month prior to screening visit; they have abnormal results in the
screening tests which are clinically relevant for study
participation; they are suffering for a severe disease such as
malignancy, diabetes, severe coronary disease, kidney disease,
neurological disease, or severe psychiatric disease or any
condition which can confound the results of the study; used highly
dosed probiotic supplements (yoghurt allowed) for 1 months prior to
the study; consumed antibiotic drugs 1 months prior to the study;
consumed on a regular basis any medication that might interfere
with symptom evaluation 2 weeks prior to the study; diagnosed with
and treated for IBS for more than 10 years; and pregnant or
lactating.
[0112] At the screening visit, clinical and medical history and
concomitant medication is registered. IBS diagnostic criteria will
be assessed and part 2 of the IBS-SSS questionnaire will be
completed.
[0113] A fecal sample kit is distributed together with the Bristol
Stool Form Scale (BSFS) and Bowel Movement Diary (BMD) to be filled
in during the 7 days just prior to visit 2. Patients will be asked
to register their diet 3 days just prior to visit 2, and will be
reminded not to change their usual diet during the study.
[0114] At the second visit, eligibility criteria are checked and
eligible subjects are randomized to the three arms in the trial. A
physical examination is done and a number of questionnaires
(GSRS-IBS, IBS-SSS, HADS, NRS-11, VSI, IBS-QOL and PHQ-15 scales)
are answered. Questionnaires are filled in electronically. Those
who are unable or unwilling to use the electronic system fill out
the questionnaires on paper. Based on clinical symptoms and data
from questionnaires, patients are characterized into one of the
three following groups; diarrhoea predominant (IBS-D), constipation
predominant (IBS-C) or alternating/mixed (IBS-A/M). This enables
allocation of patients from each subgroup into the intervention
groups. When allocated to the groups patients are provided with
either treatment or placebo products. Sigmoidoscopy is performed
and mucosal biopsies and fecal aspirates taken. Patients are asked
about any adverse events and any changes in their usual medication.
The BSFS and BMD is collected and new forms, to be filled in daily
during the intervention period, are distributed. Fecal samples are
collected and equipment for new samples are distributed. Blood
samples are collected for routine clinical chemistry and hematology
and biomarker analysis and a saliva sample is collected to analyze
FUT2 secretor status. Diet records are collected, and patients are
asked to register their diet for 3 days just prior to visit 3.
Patients are reminded not to change their usual diet during the
study.
[0115] At the third visit, a physical examination is performed and
a number of questionnaires (GSRS-IBS, IBS-SSS, HADS, NRS-11, VSI,
IBS-QOL and PHQ-15 scales) are answered. Questionnaires are filled
in electronically. Those who are unable or unwilling to use the
electronic system fill out the questionnaires on paper. Remaining
study products and compliance diaries are collected to check
compliance. Blood samples are collected for routine clinical
chemistry and hematology and biomarker analysis, and sigmoidoscopy
is performed and mucosal biopsies and fecal aspirates taken.
Patients are asked about any adverse events and any changes in
their usual medication. Fecal samples are collected and equipment
for collecting new samples distributed. The BSFS and BMD is
collected and new forms, to be filled in during the 7 days just
prior to visit 4, are distributed. Diet records are collected, and
patients are reminded not to change their usual diet during the
study.
[0116] The treatment period lasts 4 weeks, the patients are
administered 5 or 10 g of mix of 2'-FL+LNnT or 5 g of glucose
daily. Patients are instructed to consume the products in the
morning with breakfast. Compliance is monitored through the
interactive internet enabled system.
[0117] At the end of the study, each patient has an exit visit with
the medical team. Fecal samples and blood samples are collected and
analyzed as before. A number of questionnaires (GSRS-IBS, IBS-SSS,
HADS, NRS-11, VSI, IBS-QOL and PHQ-15 scales) are answered.
Questionnaires are filled in electronically. Those who are unable
or unwilling to use the electronic system fill out the
questionnaires on paper. Patients are asked about any adverse
events and any changes in their usual medication or diet, and the
BSFS and BMD is collected.
[0118] To assess the microbiota profile, DNA is extracted from
fecal samples using a 96-well PowerSoil DNA Isolation Kit (MO-BIO).
A minimum of one sample-well per plate is kept empty to serve as a
negative control during PCR. PCR is done with the forward primer
S-D-Bact-0341-b-S-17 and reverse primer S-D-Bact-0785-a-A-21 with
Illumina adapters attached (Klindworth et al. Nucleic Acids Res.
41, el (2013)). These are universal bacterial 16S rDNA primers,
which target the V3-V4 region. Following PCR program is used:
98.degree. C. for 30 sec, 25.times. (98.degree. C. for 10 s,
55.degree. C. for 20 s, 72.degree. C. for 20 s), 72.degree. C. for
5 min. Amplification is verified by running the products on a 1%
agarose gel. Barcodes are added in a nested PCR using the Nextera
Index Kit V2 (Illumina) with the following PCR program: 98.degree.
C. for 30 sec, 8.times. (98.degree. C. for 10 s, 55.degree. C. for
20 s, 72.degree. C. for 20 s), 72.degree. C. for 5 min. Attachment
of primers is verified by running the products on a 1% agarose gel.
Products from the nested PCR are normalized using the SequalPrep
Normalization Plate Kit and pooled. Pooled libraries are
concentrated by evaporation and the DNA concentration of pooled
libraries is measured on a Qubit fluorometer using the Qubit High
Sensitivity Assay Kit (Thermo Fisher Scientific). Sequencing is
done on a MiSeq desktop sequencer using the MiSeq Reagent Kit V3
(IIlumina) for 2.times.300 bp paired-end sequencing. The 64-bit
version of USEARCH is used for bioinformatical analysis of the
sequence data.
[0119] Plasma tryptophan and serotonin are determined using High
Performance Liquid Chromatography (HPLC). The mobile phase
consisted of 50 mmol/1 acetic acid, 100 mmol/1 Zinc Acetate with 3%
(v/v) acetonitrile is filtered through a 0.45 .mu.m Millipore
filter (AGB, Dublin, Ireland) and vacuum degassed prior to use.
Separations are achieved by isocratic elution at 0.3 mL/min. The
fluorescent detector is set to an excitation wavelength of 254 nm
and an emission wavelength of 404 nm. The PDA detector start
wavelength is 210 nm and the end wavelength is 400 nm with
chromatogram extraction at 330 nm. Working standard dilutions are
prepared from millimolar stock solutions of each standard and
stored at -80.degree. C. until required for analysis. Plasma
samples are deproteinized by the addition of 20 .mu.l of 4 mol/1
perchloric acid to 200 .mu.l of plasma spiked with
3-nitro-L-tyrosine as internal standard. Twenty microliters of
either sample or standard is injected onto the HPLC system and
chromatograms generated are processed using Waters Empower
software.
[0120] Biopsies are fixed and stained with hematoxylin eosin and
immunostained by use of the avidin biotin complex (ABC) method
using the Vectastain ABC kit (Vector Laboratories). The antibody
used for immunostaining for serotonin is monoclonal mouse
anti-serotonin (DakoCytomation, code no. 5HT-209).
[0121] Measurement of plasma IFN-.gamma. and TNF-.alpha. was
performed using an electro-chemiluminescence multiplex system
Sector 2400 imager from Meso Scale Discovery (Gaithersburg, Md.,
USA) where antibodies labelled with Sulfo-tag reagents emitted
light upon electrochemical stimulation.
[0122] The results show that oral ingestion of HMOs modulate the
intestinal microbiota, and specifically stimulate the abundance of
bifidobacteria. The abundance of bifidobacteria resulted in an
increase in plasma tryptophan. Additionally, serotonin level both
in plasma and the biopsies correlated positively with the
tryptophan level, and negatively with the IFN-.gamma. and
TNF-.alpha. concentration in the IBS patients given HMOs.
Interestingly, the results show that supplementing with HMOs
affected gut motility shown by decrease in stool frequency and
improvement of consistency in the IBS-D patients; and increase in
stool frequency and softening stool consistency in IBS-C patients;
and normalization of frequency and consistency in IBS-A patients.
Collectively, HMOs are able to increase bifidobacteria and
tryptophan levels, and hereby affect the regulation of the
serotonin pathway which contribute to improvement in gut motility
in IBS patients.
* * * * *