NZ613432B2 - Disialyllacto-n-tetraose (dslnt) or variants, isomers, analogs and derivatives thereof to prevent or inhibit bowel disease - Google Patents
Disialyllacto-n-tetraose (dslnt) or variants, isomers, analogs and derivatives thereof to prevent or inhibit bowel disease Download PDFInfo
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- NZ613432B2 NZ613432B2 NZ613432A NZ61343212A NZ613432B2 NZ 613432 B2 NZ613432 B2 NZ 613432B2 NZ 613432 A NZ613432 A NZ 613432A NZ 61343212 A NZ61343212 A NZ 61343212A NZ 613432 B2 NZ613432 B2 NZ 613432B2
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- gal
- neu5ac
- dslnt
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- glcnac
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P10/00—Shaping or working of foodstuffs characterised by the products
- A23P10/20—Agglomerating; Granulating; Tabletting
- A23P10/28—Tabletting; Making food bars by compression of a dry powdered mixture
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/06—Gastro-intestinal diseases
- G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/06—Gastro-intestinal diseases
- G01N2800/067—Pancreatitis or colitis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/38—Pediatrics
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7095—Inflammation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
Abstract
Disclosed is an infant formula, baby food, or fortified breast milk, comprising a formulation comprising an isolated disialyllacto-N-tetraose (DSLNT) or isomer thereof and a probiotic, wherein the isolated DSLNT or isomer is free of non-DSLNT oligosaccharides and is present in the formulation at 300 ?M or greater, wherein DSLNT is biologically active ?-Neu5Ac-(2?3)-?-Gal-(1?3)-[?-Neu5Ac-(2?6)]-?-GlcNAc-(1?3)-?-Gal-(1?4)-Glc; and wherein the isomer is any of ?-Neu5Ac-(2?3)- ?-Gal-(1?3)-[?-Neu5Ac-(2?6)]-?-GlcNAc-(1?3)-?-Gal-(1?4)-Glc, ?-Neu5Ac-(2?3)-?-Gal-(1?3)-[?-Neu5Ac-(2?6)]- ? -GlcNAc-(1?3)-?-Gal-(1?4)-Glc, ?-Neu5Ac-(2?3)-?-Gal-(1?3)-[?-Neu5Ac-(2?6)]-?-GlcNAc-(1?3)- ?-Gal-(1?4)-Glc, ?-Neu5Ac-(2?3)-?-Gal-(1?3)-[?-Neu5Ac-(2?6)]-?-GlcNAc-(1?3)-?-Gal-(1?4)-Glc, or ?-Neu5Ac-(2?3)-?-Gal-(1?3)-[ ?-Neu5Ac-(2?6)]-?-GlcNAc-(1?3)-?-Gal-(1?4)-Glc. Also disclosed is a formulation in the form of a tablet, caplet or powder comprising ?-Neu5Ac-(2?3)- ?-Gal-(1?3)-[?-Neu5Ac-(2?6)]-?-GlcNAc-(1?3)-?-Gal-(1?4)-Glc, ?-Neu5Ac-(2?3)-?-Gal-(1?3)-[?-Neu5Ac-(2?6)]- ? -GlcNAc-(1?3)-?-Gal-(1?4)-Glc, ?-Neu5Ac-(2?3)-?-Gal-(1?3)-[?-Neu5Ac-(2?6)]-?-GlcNAc-(1?3)- ?-Gal-(1?4)-Glc, ?-Neu5Ac-(2?3)-?-Gal-(1?3)-[?-Neu5Ac-(2?6)]-?-GlcNAc-(1?3)-?-Gal-(1?4)-Glc, or ?-Neu5Ac-(2?3)-?-Gal-(1?3)-[ ?-Neu5Ac-(2?6)]-?-GlcNAc-(1?3)-?-Gal-(1?4)-Glc. ?M or greater, wherein DSLNT is biologically active ?-Neu5Ac-(2?3)-?-Gal-(1?3)-[?-Neu5Ac-(2?6)]-?-GlcNAc-(1?3)-?-Gal-(1?4)-Glc; and wherein the isomer is any of ?-Neu5Ac-(2?3)- ?-Gal-(1?3)-[?-Neu5Ac-(2?6)]-?-GlcNAc-(1?3)-?-Gal-(1?4)-Glc, ?-Neu5Ac-(2?3)-?-Gal-(1?3)-[?-Neu5Ac-(2?6)]- ? -GlcNAc-(1?3)-?-Gal-(1?4)-Glc, ?-Neu5Ac-(2?3)-?-Gal-(1?3)-[?-Neu5Ac-(2?6)]-?-GlcNAc-(1?3)- ?-Gal-(1?4)-Glc, ?-Neu5Ac-(2?3)-?-Gal-(1?3)-[?-Neu5Ac-(2?6)]-?-GlcNAc-(1?3)-?-Gal-(1?4)-Glc, or ?-Neu5Ac-(2?3)-?-Gal-(1?3)-[ ?-Neu5Ac-(2?6)]-?-GlcNAc-(1?3)-?-Gal-(1?4)-Glc. Also disclosed is a formulation in the form of a tablet, caplet or powder comprising ?-Neu5Ac-(2?3)- ?-Gal-(1?3)-[?-Neu5Ac-(2?6)]-?-GlcNAc-(1?3)-?-Gal-(1?4)-Glc, ?-Neu5Ac-(2?3)-?-Gal-(1?3)-[?-Neu5Ac-(2?6)]- ? -GlcNAc-(1?3)-?-Gal-(1?4)-Glc, ?-Neu5Ac-(2?3)-?-Gal-(1?3)-[?-Neu5Ac-(2?6)]-?-GlcNAc-(1?3)- ?-Gal-(1?4)-Glc, ?-Neu5Ac-(2?3)-?-Gal-(1?3)-[?-Neu5Ac-(2?6)]-?-GlcNAc-(1?3)-?-Gal-(1?4)-Glc, or ?-Neu5Ac-(2?3)-?-Gal-(1?3)-[ ?-Neu5Ac-(2?6)]-?-GlcNAc-(1?3)-?-Gal-(1?4)-Glc.
Description
DISIALYLLACTO-N-TETRAOSE (DSLNT) OR VARIANTS, ISOMERS,
S AND TIVES THEREOF TO PREVENT OR INHIBIT
BOWEL DISEASE
Throughout this application various publications are referenced. The disclosures of these,
publications in their entirety are hereby incorporated by reference into this application in
order to more fully describe the state of the art to which this invention pertains.
This invention was made with government support under Grant No. K99/R00 DK078668
awarded by NIHINIDDK. The government has n rights in the invention.
BACKGROUND OF THE INVENTION
Necrotizing Enterocolitis (NBC) is one of the most frequent and fatal intestinal disorders
in preterm infants. Almost 10% of very—low—birth-weight infants (<1,500g birth weight)
develop NBC. More than 25% of them die from the er. The ors are often
faced with long-term neurological impairment. In 1990, Lucas and Cole (Lancet,
336:1519-23) had already reported that formula—fed infants are at a 6— to 10—fold higher
risk to develop NBC when compared to breast-fed infants. Since then several molecules
in human milk (e.g. LC-PUFA, PAF—AH, BGF) have been associated with NBC
tion, mostly based on animal studies. r, despite improvements in formula
composition over the past 10—15 years, formula-fed infants are still at a 6- t0 d
higher risk than -fed infants. The data suggests that human milk ns something
else that is missing in formula and protects breast-fed infants from NBC. Identifying the
protective ent in human milk as well as its mechanisms of action would pave the
way for the development of desperately needed additional options to treat and maybe
even prevent this devastating disorder.
We discovered that certain Human Milk Oligosaccharides (HMO) protect the breast—fed
infant from NBC. Thus, we provide formulations containing such HMOS, methods and
means for inhibiting bowel disease such as NBC.
SUMMARY OF THE INVENTION
In accordance with the present invention, novel formulations have been discovered that are
useful for a variety of therapeutic applications. The invention provides formulations comprising
isolated Disialyllacto-N-tetraose (DSLNT) or variants, isomers, analogs and derivatives thereof
(DSLNT of the invention).
The ion also provides methods for preventing or treating a subject having a bowel disease
and/or inflammation by administering Disialyllacto-N-tetraose (DSLNT) or variants, isomers,
analogs and derivatives thereof in an amount ient to prevent or treat the bowel disease
and/or inflammation in the t.
The invention also provides methods of identifying whether a -fed infant is at risk of
ping Necrotizing Enterocolitis (NEC) comprising measuring the concentration of
Disialyllacto-N-tetraose (DSLNT) in the mother’s milk, a low level of DSLNT being indicative
that the breast-fed infant is at risk of developing NEC.
According to a first aspect of the present invention, there is provided a formulation in the form
of a tablet, caplet or powder comprising
α-Neu5Ac-(2→3)--Gal-(1→3)-[α-Neu5Ac-(2→6)]-β-GlcNAc-(1→3)-β-Gal-(1→4)-Glc, α-
Neu5Ac-(2→3)-β-Gal-(1→3)-[α-Neu5Ac-(2→6)]--GlcNAc-(1→3)-β-Gal-(1→4)-Glc, α-
Neu5Ac-(2→3)-β-Gal-(1→3)-[α-Neu5Ac-(2→6)]-β-GlcNAc-(1→3)--Gal-(1→4)-Glc, -
Neu5Ac-(2→3)-β-Gal-(1→3)-[α-Neu5Ac-(2→6)]-β-GlcNAc-(1→3)-β-Gal-(1→4)-Glc, or α-
Neu5Ac-(2→3)-β-Gal-(1→3)-[-Neu5Ac-(2→6)]-β-GlcNAc-(1→3)-β-Gal-(1→4)-Glc.
According to a second aspect of the present invention, there is provided use of isolated
disialyllacto-N-tetraose (DSLNT) or an isomer thereof in the manufacture of a medicament for
the ent or tion of a bowel disease in a subject,
wherein the isolated DSLNT or isomer is free of non-DSLNT accharides and is
present in the formulation at 300 µM or r,
wherein DSLNT is biologically active α-Neu5Ac-(2→3)-β-Gal-(1→3)-[α-Neu5Ac-
(2→6)]-β-GlcNAc-(1→3)-β-Gal-(1→4)-Glc; and
AH26(11361247_1):JIN
wherein the isomer is any of α-Neu5Ac-(2→3)--Gal-(1→3)-[α-Neu5Ac-(2→6)]-β-
GlcNAc-(1→3)-β-Gal-(1→4)-Glc, α-Neu5Ac-(2→3)-β-Gal-(1→3)-[α-Neu5Ac-(2→6)]--
GlcNAc-(1→3)-β-Gal-(1→4)-Glc, α-Neu5Ac-(2→3)-β-Gal-(1→3)-[α-Neu5Ac-(2→6)]-β-
GlcNAc-(1→3)--Gal-(1→4)-Glc, -Neu5Ac-(2→3)-β-Gal-(1→3)-[α-Neu5Ac-(2→6)]-β-
GlcNAc-(1→3)-β-Gal-(1→4)-Glc, or α-Neu5Ac-(2→3)-β-Gal-(1→3)-[-Neu5Ac-(2→6)]-β-
GlcNAc-(1→3)-β-Gal-(1→4)-Glc.
According to a third aspect of the present invention, there is provided an infant formula, baby
food, or fortified breast milk, comprising a formulation comprising an isolated disialyllacto-N-
tetraose (DSLNT) or isomer thereof and a tic,
wherein the isolated DSLNT or isomer is free of non-DSLNT oligosaccharides and is
present in the formulation at 300 µM or greater,
wherein DSLNT is biologically active Ac-(2→3)-β-Gal-(1→3)-[α-Neu5Ac-
(2→6)]-β-GlcNAc-(1→3)-β-Gal-(1→4)-Glc; and
wherein the isomer is any of Ac-(2→3)- α-Gal-(1→3)-[α-Neu5Ac-(2→6)]-β-
GlcNAc-(1→3)-β-Gal-(1→4)-Glc, Ac-(2→3)-β-Gal-(1→3)-[α-Neu5Ac-(2→6)]- α -
GlcNAc-(1→3)-β-Gal-(1→4)-Glc, α-Neu5Ac-(2→3)-β-Gal-(1→3)-[α-Neu5Ac-(2→6)]-β-
GlcNAc-(1→3)- α-Gal-(1→4)-Glc, β-Neu5Ac-(2→3)-β-Gal-(1→3)-[α-Neu5Ac-(2→6)]-β-
GlcNAc-(1→3)-β-Gal-(1→4)-Glc, or α-Neu5Ac-(2→3)-β-Gal-(1→3)-[ β-Neu5Ac-(2→6)]-β-
GlcNAc-(1→3)-β-Gal-(1→4)-Glc.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. HMO and GOS. Lactose (Gal1-4Glc) forms the reducing end of Human Milk
Oligosaccharides (HMO, left) and can be elongated at the ducing end by one or more
lactosamine disaccharides (Gal1-3/4GlcNAc), generating HMO of varying sizes. Lactose or
the polylactosamine backbone can be ed by addition of fucose and/or by the addition of
sialic acid (N-acetyl-neuraminic acid in humans) in various linkages. Each sialic acid
contributes one negative charge to the HMO. Galactooligosaccharides (GOS, right) which are
urally very different from HMO, are elongated by galactose and lack fucose and sialic
acid.
Figure 2. HMO prevent NEC in neonatal rats A: Four-day survival. HMO (10 mg/mL)
restored survival rates while GOS (8 mg/mL) had no effect. Note that x-axis
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intersects at 50% survival. B: Pathology scores of H&E—stained ileum sections (0:
healthy; 4: complete ction). Addition of HMO at low (1 mg/mL) and high
concentrations (10 mg/mL) significantly reduced . G08 (8 mg/mL) had no effect.
Each point represents one animal. Lines represent mean and standard ion. [BF:
breast-fed; FF: formula-fed].
Figure 3. TWO-dimensional chromatography identifies most effective HMO. A: Rat
NEC after HMO fractionation by charge. Pooled HMOs were fractionated by QAE
based on charge. HMOS that contain no sialic acid carry no charge (0). I-IMOs with one
or more sialic acids carry one or more negative charges (-1, -2,.. .). The QAE—2 fraction
was most effective in preventing NEC. B: HPLC—FL showed QAE—2 contained only 4
major oligosaccharides. #1 is a monosialylated HMO, which was ered an irrelevant
spill-over from the QAE-l fraction, which was ineffective. C: FPLC P2
subfractionation by size. Fractions containing only #2 (31—40) were pooled and
separated from fiactions containing only #3 and #4. #3 and #4 could not be ted
from each other. Loss in the excluded ons was considered accordingly. D: Rat
NEC after FPLC subfractionation. While #3 and #4 had no effect, #2 significantly
reduced NEC. [BF: breast-fed; FF: formula—fed].
Figure 4. Protective HMO #2 identified as Disialyllacto—N—tetraose. Glycan structure
ation identified HMO #2 as a ic isomer of disialyllacto-N—tetraose (DSLNT).
Figure 5. Human milk oligosaccharides (HMO) and galacto-oligosaccharides (GOS) are
structurally different. (A) Fluorescence high-performance liquid chromatography (HPLC-
FL) chromatogram of ZAB-labelled HMO isolated from pooled human milk. Most
cormnon HMO are annotated and listed in panel B. *Disialyllacto-N—tetraose (DSLNT),
which was later identified as the NEC-protective HMO. (B) Schematic representation of
the most common oligosaccharides found in the isolated pooled HMO. s in
brackets correspond to the annotated peaks in panel A. 2’FL, 2’-fucosyllactose; 3FL, 3-
llactose; 3’SL, 3’-sialyllactose; LNT, lacto—N—tetraose; LNnT, lacto—N—neotetraose;
LNFPl, lacto-N—fucopentaose 1; LNFPZ, lacto—N—fucopentaose 2; LSTb, sialyllacto—N—
tetraose b; LSTc, sialyllacto-N—tetraose c. Monosaccharide key: dark circle, glucose
(Glc); light circle, galactose (Gal); square, N—acetyl~glucosamine (GlcNAc); triangle,
fucose (Fuc); diamond, N—acetyl-neuraminic acid (NeuAc). (C) L chromatogram
of Vivinal GOS. Peak clusters represent structural isomers of oligosaccharides with the
same degree of polymerization and depend on the number of galactose residues per GOS
molecule. Comparison of the HMO and GOS chromatograms confirmed a clear
difference in the structural composition.
Figure 6. Pooled human milk oligosaccharides (HMO), but not galacto-oligosaccharides
(GOS) improve survival and reduce necrotizing enterocolitis (NBC) in neonatal rats. (A)
Survival of neonatal rats within the first 96 h post-partum. DF, dam—fed; FF, a-fed;
FF+HMO, fed formula with HMO (10 ; FF+GOS, fed formula with G03 (8
mg/ml). (B) Macroscopic evaluation of rat intestines at 96 h post-partum. Compared to
DF (left) and FF+HMO (right) animals, the intestines of FF animals (center) were darker
with patchy necrosis and evidence of hemorrhagic intestine as well as ural gas
cysts (Pneumatosis intestinalis). (C) Microscopic evaluation of H&E-stained rat ileum
sections. Based on the presence or absence of histological ies (three examples are
shown in the bottom panel), ileum sections were graded from 0 (normal) to 4 (complete
destruction). (D) Ileum pathology scores at 96 h post-partum. Each intervention was
tested in a total of 10-20 animals in three independent experiments. Each symbol
represents the pathology score for an individual . Horizontal lines represent mean
pathology scores. ***p<0.001.
Figure 7. EXposure to human milk oligosaccharides (HMO) in the first 24 h artum
is ed, but not ent to reduce necrotizing enterocolitis. Neonatal rats were dam-
fed (DF), fed HMO-free formula for the entire first 96 h post—partum (FF), fed HMO-free
formula for the first 24 h and then switched to HMO-containing formula (10 mg/ml) for
the remaining 72 h O End), 01' fed HMO-containing formula for the first 24 h
and then switched to HMO—free formula (FF+HMO . Each intervention was tested
in a total of 9—12 s in two independent ments. ***p<0.001.
2012/023866
Figure 8. A single, disialylated human milk oligosaccharide (HMO) reduces necrotizing
enterocolitis. (A) Ileum pathology scores in response to adding charge—fractionated HMO
to formula. Anion exchange chromatography was used to fi'actionate pooled HMO by
charge based on whether HMO contained no (0), one (—1), two (-2), three (~3) or four (-4)
sialic acid residues. The —2 charged HMO on, containing oligosaccharides with two
sialic acids (two negative charges) had the most pronounced effect. (B) HPLC-FL
chromatogram of -2 charged HMO fraction. (C) MALDI-TOF mass spectra and ial
composition of the four major HMO peaks in the -2 charged HMO fraction. The
predicted number of hexoses (circles), hexosamines e), N—acetylneuramic acid
(NeuAc, diamond) and fucose (triangle) per le are listed above each mass
spectrum. Loss of NeuAc during analysis reduces the mass by 291 Da. (D) Fast protein
liquid chromatography (FPLC) with a gel exclusion column was used to separate the four
major HMO peaks in the -2 charged HMO fraction by size. FPLC fractions ning
mostly HMO peak 2 were pooled together (HMO 2). HMO peaks 3 and 4 could not be
separated by gel ion and were pooled in one fraction (HMO 3+4). (E) Ileurn
pathology scores in response to adding size-fractionated HMO to formula. Each
intervention was tested in a total of 11-14 animals in two independent experiments.
***p<0.001.
Figure 9. The necrotizing enterocolitis-protective hmnan milk oligosaccharide (HMO) is
disialyllacto-N—tetraose (DSLNT). (A) Linkage c neuraminidase treatment shows
the presence of one a2 and one linked N-acetyl—neuraminic acid (NeuAc).
Fluorescence high—performance liquid chromatography (HPLC—FL) chromatogram a:
protective HMO 2; b: HMO 2 after treatment with d2-3 fic neuraminidase; c: HMO
2 after treatment with e promiscuous neuramidase. (B) The underlying HMO
backbone has a type I structure —3GlcNAc). HPLC-FL chromatogram d: asialo-
HMO 2 (after treatment with 0L2-3/6 neuraminidase, product 0); e: asialo-HMO 2 after
treatment with |31-3—specific galactosidase; f: asialo-HMO 2 after treatment with [51—4-
specific galactosidase. (C) The subterminal sugar in the HMO backbone is N—acetyl—
glucosamine (GlcNAe). HPLC—FL chromatogram g: asialo—agalacto-HMO 2 (after
WO 06665
ent with d2-3/6 neuraminidase and 131-3 galactosidase, product e); h: asialo-
agalacto-HMO 2 after treatment with GlcNAcase. (D) Gas chromatography mass
spectrum (GC-MS) of lly methylated alditol acetate (PMAA) derivatives of HlVIO
2. (E) Schematic representation of DSLNT based on the results from sequential
exoglycosidase digestion and GC—MS PMAA linkage analysis.
Figure 10. cially available DSLNT shows necrotizing colitis (NBC)
protective effects. Ileum pathology scores in response to adding commercially available
DSLNT to a. Commercially available DSLNT (300 uM) significantly reduced
NBC pathology scores. Each intervention was tested in a total of 11~26 animals in three
independent experiments. * * *p<0.001.
DETAILED DESCRIPTION OF THE ION
Unless defined otherwise, all technical and scientific terms used herein have the same
meaning as is commonly understood by one of ordinary skill in the art to which this
invention belongs, All patents, applications, published applications and other
publications referred to herein are orated by reference in their entirety.
As used herein, the term "cemprising" when placed before the recitation of steps in a
method means that the method encompasses one or more steps that are additional to those
expressly recited, and that the additional one or more steps may be performed before,
between, and/or after the recited steps. For example, a method comprising steps a, b, and
c encompasses a method of steps a, b, x, and c, a method of steps a, b, c, and x, as well as
a method of steps x, a, b, and c. rmore, the term ising” when placed before
the recitation of steps in a method does not (although it may) require sequential
performance of the listed steps, unless the content clearly dictates otherwise. For
example, a method comprising steps a, b, and c encompasses, for example, a method of
performing steps in the order of steps a, c, and b, the order of Steps 0, b, and a, and the
order of steps 0, a, and b. Unless ise indicated, all numbers expressing quantities
of ingredients, properties such as molecular weight, reaction conditions, and so forth as
used herein, are to be understood as being modified in all instances by the term ”abou '."
Accordingly, unless indicated to the contrary, the numerical parameters herein are
approximations that may vary depending upon the desired properties sought to be
obtained by the present invention. At the very least, and without limiting the application
of the doctrine of lents to the scope of the claims, each numerical parameter should
at least be construed in light of the number of reported significant digits and by applying
ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters
describing the broad scope of the invention are approximations, the numerical values in
the specific examples are reported as ely as possible. Any numerical value,
however, inherently contains standard deviations that necessarily result from the errors
found in the numerical value's testing measurements.
As used herein, the terms "subject" and ”patient” refer to any animal, such as a mammal.
s include but are not limited to, humans, murines, s, felines, canines,
equines, bovines, pcrcines, ovines, es, rabbits, marmnalian farm animals,
ian sport animals, and mammalian pets. In many ments, the hosts will be
humans.
Isolated DSLNT and/or its variant, isomer, analog and/0r derivative may be obtained by
ing DSLNT from nature or synthesized using known chemical or mical
principles and methods. As used herein, the term “isolated” in reference to DSLNT of
the invention does not require absolute purity.
I. FORMULATIONS OF THE INVENTION
The invention provides formulations comprising isolated yllacto-N-tetraose
(DSLNT) or variants, isomers, analogs and derivatives thereof (also referred to herein as
DSLNT or DSLNT of the invention).
As used herein, Disialyllacto-N-tetraose (DSLNT) is also known as (28,43,5R,6R)-5—
acetamido-Z-[(2R,3R,4S,5S,6R)—2-[(2R,3S,4R,5R,6S)—5-acetamido—2—[[(2R,4S,5R,6R)~5—
2012/023866
acetamidocarboxylatohydroxy—6-[(1 R,2R)—1 ,2,3-trihydroxypropy1]exam-2—
yl]oxymethyl]—6-[(2R,3R,4S,5R,6S)—3,5-dihydroxy(liydroxymethyl)
[(2R,3S,4R,5R)—4,5,6-trihydroxy—2-(hydroxymethyl)oxan~3—y1]oxy~oxan—4-yl]oxy—3~
hydroxy-oxany1]oxy—3,5~dihydroxy(hydroxymethyl)oxanyl]oxy—4—hydroxy
[(1R,2R)—1,2,3-trihydroxypropyl]oxane—2—carboxylate, 01' its synonyms, O-(N-acetyl-
alpha-neuraminosy1)—(2-6)—O—(0—(N-acetyl-alpha—neuraminosyl)—(2-3)-beta—D-
galactopyranosyl-(l ~3))—O—2-(acety1amino)deoxy—beta-D~glucopyranosyl—(1 —3)-D-
Glucose, d—NeuSAc~(2——>3)-B-Gal—(1 ~—>3)—[d—NeuSAc—(2—>6)]«B—GlcNAc-(l —>3)-B-Gal-
(1—>4)—Glc, or Di-N-Acetylneuraminosyllacto-N—tetraose, with CAS registry number
612784.
An embodiment ofDSLNT is shown in Figures 1, 4, 9E, and 10.
Derivatives, isomers, analogs and variants ofDSLNT include accharides having (1)
at least four sugar residues as in the lacto-N-tetraose (GalB1—3GlcNAcI31-3Gall31-4Glc)
ne (e.g., as shown in Figure 4), wherein the sugar can be any of D—glueose (Glc),
D—galactose (Gal), L-fucose (Fuc), D-fructose (Fru), marmose (Man), N-
acetylgalactosamine (GalNAc) or N—acetyl-glucosamine (GleNAc) and (2) a sialic acid
(N—aoetylneuraminic acid or NeuSAc) residue at any two or more of the sugar residues.
For example, a sialic acid e may be attached at each of the first and second sugar
es with the al non-reducing sugar residue designated as the first sugar residue
ofthe oligosaccharide. In another example, a sialic residue may be attached at each of the
first and third sugar residues with the terminal non-reducing sugar e designated as
the first sugar residue of the oligosaccharide. However, the invention also plates
that a sialic acid residue may be attached at any position including at the ng end,
e.g., Glc of DSLNT.
Merely by way of example, the DSLNT derivative may have four sugar residues such as
a combination of glucose, galactose and N—acetyl-glucosamine and be modified with at
least two sialic acid residues at any of the first three sugar subtmits within the
oligosaccharide chain in which the last final sugar would be glucose in the case of
WO 06665 2012/023866
DSLNT or the corresponding sugar at this position in DSLNT variants, isomers, s
and derivatives.
In another example, the DSLNT tive may have four sugar residues, including a
combination of glucose, galactose and N—acetyl-glucosamine (e.g., Figure 4) and be
modified with at least two sialic acid residues at any of the first three sugar subunits
within the accharide chain.
In another example, the DSLNT tive may have four sugar residues including a
combination of glucose, fructose, galactose and N-acetyl—glucosamine and be modified
with at least two sialic acid residues at any of the first three sugar subunits within the
oligosaccharide chain.
In another example, the DSLNT variant, isomer, analog and derivative may have four
glucose residues and be modified with at least two sialic acid residues at any of the first
three sugar subunits within the oligosaccharide chain. In another e, the DSLNT
derivative may have four fi-uctose es and be modified with at least two sialic acid
residues at any of the first three sugar subunits within the oligosaccharide chain.
In another example, the DSLNT derivative may have 'four galactose residues and be
modified with at least two sialic acid residues at any of the first three sugar subunits
within the oligosaccharide chain.
In another example, the DSLNT derivative may have four N—acetyl-glucosarnine residues
and be modified with at least two sialic acid residues at any of the first three sugar
subunits within the oligosaccharide chain.
In another example, the DSLNT derivative may have three galactose residues followed
by a glucose residue, e.g., Gal-Gal—Gal-Glc, and be modified with at least two sialic acid
residues at any of the first three sugar subunits within the oligosaccharide chain.
2012/023866
In another example, the DSLNT derivative may have three se residues followed by
a glucose residue, e.g., Fru—Fru-Fru-Glc, and be modified with at least two sialic acid
residues at any of the first three sugar subunits within the oligosaccharide chain.
While the derivatives may have any chemically permitted linkages for forming a covalent
chemical bond or bonds between any two sugar molecules or monosaccharides, the
preferred chemical linkages are: NeuSAc residue linked in 0t2-3 or 0L2—6 e to Gal or
GlcNAc residue; Fuc residue linked in Oil-2, 0t1-3, or Oil—4 linkage to Gal, GlcNAc, or
Glc residue; Fru residue linked in [31—2, Oil-2, to Fru, Gal, GlcNAc or Glc; GlcNAc
residue linked in [31—3, [31—4, or 131—6 linkage to a Gal residue; and Gal residue linked in
[31-3 or [51—4 linkage to a GlcNAc, Gal, or Glc residue. It is also preferred that the
terminal sugar is a e residue, preferably linked to a galactose as in the disaccharide
lactose.
Further, derivatives of DSLNT may be made by covalent linking of DSLNT to any other
chemical nd or polymer, using methods known in the art of c and synthetic
chemistry or through enzymatic methods. These derivatives include but are not limited to
attaching or nt linking DSLNT to other oligosaccharides, amino acids,
polypeptides, and nucleic acids.
Further, DSLNT variant, isomer, analog and derivatives may be made by substituting a
sugar e within DSLNT with a sugar analog. For example, galactose may be
substituted with its analogs, including but not limited to 2—desoxy—D-galactose, 2—desoxy—
2—fluoro-D—galactose and 2-desoxy-2—amino—D—galactose. For example, glucose may be
substituted with its analogs, including but not limited to y-D—glucose, 2,2-
o-deoxy—D—glucose, 2-deoxyfluoroiodo—D—glucose, l-O-methyl-D—glucose,
2-O-methyl—D-glucose, 2-deoxy—2—chloro—D-glucose, 2-deoxybromo-D-glucose, 3-O-
11C—methyl-D-glucose, 6-deoxy-D—glucose, 6-deoxy—6-fluoro—D—glucose, and 6-deoxy-6—
iodo-D-glucose, and 2-deoxy18Fnfluoro—D—glucose. For example, N-
acetylglucosamine may be substituted with its analogs, N—acetylglucosaminylasparagine,
N—acetylglucosamine 6-sulfate, N—acetylglucosamine—l—phosphate, N~acetylglucosamine
2012/023866
6-phosphate, methyl-2~acetamido-2—deoxy-D—glucopyranoside, N—acetylglucosaminitol,
N—bromoacetylglucosamine, 2-acetamido-1,3,6-tri—O-acetyl~4—deoxy
fluoroglucopyranose, N—acetylglucosamine thiazoline, N—fluoroacetyl—D-glucosamine, 2-
acetamido—2-deoxy~D-glucono-(1,5)—lactone, and amido-3,6—dideoxyglucose.
Furthermore isomers of DSLNT may be obtained based on a chiral center, such that D-
glucose as a six member ring can exist either as lucopyranose or [3—D—
glucopyranose, depending on the orientation of the hydroxyl group at the C-1 position
with respect to the rest of the ring. Similarly, D—galactose, N—acetylglucosamine and
sialic acid rings may exist in either or- or [By-conformation based on the yl group at
the C-1 (for D—galactose and N—acetylglucosamine) and 02 (for sialic acid) position with
respect to the rest of the ring. The isomers of DSLNT may differ based on oc— or [3-
position of the acetal functional groups. For example, the glycosidic linkage between
galactose and glucose may be oc-acetal functional group instead of B—acetal functional
group as is normally found in lactose moiety of DSLNT. Thus, a number of isomers of
DSLNT may exist based on the orientation of the hydroxyl- group at the 0—1 or C-2
on of the six member rings. Some DSLNT isomers may include but are not limited
to: u—NeuSAc-(2—>3)-oc-Gal—(ln+3)—[a—NeuSAc-(ZA6)]-B-GlcNAc-(1——+3)-[3-Gal-(l——>4)-
Glc, u-NeuSAc-(2—>3)-|3~Gal-(l—>3)—[u—NeuSAc~(2——>6)]-or-GlcNAc-(l —>3)-[3—GalGlc, a-NeuSAc-(2—->3)—[3—Gal—(l—>3)-[u—NeuSAc-(Za6)]-|3-GlcNAc-(l——>3)—or-
Gal-(144)~Glc, B-NeuSAc-(2—>3)u|3-Gal-(1—>3)—[ct—Neu5Ac—(2—>6)]—B—GlcNAc-(1-—>3)—
B-Gal—(l—>4)—Glc, or u~Neu5Ac—(2—)3)-B-Gal-(l—>3)—[[3-Neu5Ac—(2—>6)]-B-GlcNAc-
(163)—B-Gal-(1—94)-Glc.
Since modification by sialic acid uces a negative charge in form of a carboxyl-
group (COO-), other ccharides also contain carboxyl-groups and may substitute
for sialic acid in DSLNT variant, isomer, analog and derivatives. These sugars could be
glucoronic acid, galacturonic acid, iduronic acids, y—D—manno-oct—2—ulosonic
acid, neuraminic acid, or any other carboxyl-group containing monosaccharides or
derivatives thereof.
WO 06665
Variants, analogs and derivatives including its isomers and lites can be ed
by modifying DSLNT through tutions, ations, and conjugations that
such as, for
preserve the biological activity of preventing or inhibiting a bowel disease,
example, necrotizing enterocolitis, in a t, e.g., a pediatric subject including infants,
children, or adolescents. The subject may be a human or animal subject including a
monkey, rat, mouse, dog, cat, pig, goat, sheep, horse or cow.
As used herein, suitable amounts of DSLNT (or variants, isomers, analogs and
derivatives thereof) means an amount sufficient to inhibit a bowel disease. Examples of
suitable amounts include, but are not limited to, an amount of about at least 30 uM, at
least 300 M, at least 600 uM, at least 800 uM, greater than 800 uM, in the range of
approximately 10 uM-10,000 uM, approximately 600-1500 uM or approximately 500—
800 MM dosage forms or compositions containing active ingredient (DSLNT of the
invention) of about at least 38.7 mg/L, at least 387 mg/L, in the range of approximately
12.9 mg to 12.9 g per liter, approximately 774 mg/L to 1,935 mg/L, approximately 645
mg/L to 1,032 mg/L, or approximately 200-500 mg/L of DSLNT or derivative thereof
with the balance made up from non~toxic carrier may be prepared. In some
embodiments, these amounts or ranges may vary by about 10%. In other embodiments,
the amounts or ranges may vary by about 20%. In still other embodiments, these
amounts or ranges may vary by about 25%. Methods for ation of these
compositions are known to those skilled in the art.
In one embodiment, the present formulation ses about 387 mg/L (~300 uM)
DSLNT ofthe invention.
The formulation of the invention preferably comprises other ents, such as
vitamins and/or ls, preferably according to intemational directives for infant
formulae.
The concentration of DSLNT of the invention in the formulation will depend on
absorption, inactivation and excretion rates of the DSLNT of the invention, the dosage
le, and amount administered as well as other factors known to those of skill in the
art. The concentrations of the DSLNT of the invention are effective for delivery of an
amount, upon administration, that treats, prevents, or ameliorates one or more of the
symptoms of diseases or disorders to be d.
In the formulations, DSLNT of the invention can also be mixed with other mammalian or
plant proteins. For example, mammalian proteins include proteins from mammalian milk
(e.g., either intact or l protein hydrolysates of Whole or fractionated mammalian
milk). Plant proteins include intact protein or protein hydrolysate from pea, soy, almond,
and/or rice proteins. In the formulation, the weight fraction of the DSLNT of the
invention may be dissolved, suspended, dispersed or otherwise mixed in a selected r
at an effective concentration such that the d condition is relieved, prevented, or one
or more symptoms are ameliorated.
In one embodiment, the ation of the invention is an enteral ation. l
formulations of the invention may be embodied in an infant formula, breast milk, water,
juices, or baby food. Additionally, enteral formulations of the invention may be
embodied in a nutritional supplement.
In the formulations of the invention, DSLNT can also be mixed with other mammalian or
plant proteins. For example, mammalian proteins include proteins from mammalian milk
e.g., either intact or partial protein hydrolysates of whole or fractiouated mammalian
milk. Plant proteins include intact protein or protein hydrolysate from pea, soy, almond,
and/or rice proteins.
In a further embodiment, the formulation of the ion may be added to any liquid for
consumption. Liquids include, but are not limited to, water or juices.
In another embodiment, the formulation of the invention is used to supplement or fortify
the ’s own milk or human donor milk (human milk f01tif1er) with DSLNT and/or
its derivatives, isomers, analogs. Commercial pasteurized human donor milk may be
obtained from Prolacta Bioscience (Monrovia, CA) under the name Prolact+ HEMF such
as Prolact+4®, Prolact+6®, Prolact+8®, and Prolact+10®. Fortification of mother’s milk
may be performed with 01' without prior knowledge of the DSLNT content of the
mother’s milk and would be warranted in the case of 10W DSLNT levels in either
mother’s own milk or donor milk.
The present invention additionally provides pharmaceutical formulations (also known as
ceutical compositions or dosage forms) comprising isolated DSLNT and/or its
derivatives, isomers, analogs and/or variants, and a pharmaceutically acceptable excipient
or e.
In one embodiment, the formulation of the ion ses isolated DSLNT or
variants, s, analogs and derivatives thereof and a pharmaceutical acceptable
excipient.
In another embodiment, the formulation of the invention ts of isolated DSLNT or
variants, isomers, analogs and derivatives thereof and a pharmaceutical acceptable
excipient;
In yet another embodiment, the formulation of the invention ses isolated DSLNT
or variants, isomers, analogs and derivatives thereof and a pharmaceutical acceptable
excipient but is ntially free of other oligosaccharides (e.g. non-DSLNT
oligosaccharides).
Pharmaceutically acceptable ent or vehicle refers to a non-toxic solid, semisolid
(also referred to herein as softgel) or liquid filler, diluent, encapsulating material or
formulation auxiliary of any type.
Further, isolated DSLNT and/or its derivatives, isomers, analogs and/or variants of the
invention can be pegylated, phosphorylated, esterified, tized with amino acids
and/or peptides, to improve solubility for both formulation and bioavailability.
The isolated DSLNT and/or its derivatives, isomers, analogs and/or variants of the
present invention may be mixed with pharmaceutically acceptable excipients. Examples
of excipients include but are not limited to binders, diluents, adjuvants, or vehicles, such
as preserving agents, fillers, polymers, disintegrating agents, glidants, wetting agents,
emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming
agents, ating , acidifying agents, coloring agent, dyes, preservatives and
dispensing agents, or compounds of a similar nature depending on the nature of the mode
of administration and dosage forms. Such ingredients, including pharmaceutically
acceptable carriers and excipients that may be used to formulate oral dosage forms, are
described in the Handbook of Pharmaceutical ents, American ceutical
Association (1986), incorporated herein by reference in its entirety.
Pharmaceutically acceptable excipients are lly non-toxic to recipients at the
dosages and trations ed and are compatible with other ients of the
formulation. Examples of pharmaceutically acceptable excipients include water, saline,
Ringer’s solution, dextrose on, l, s, vegetable oils, fats, ethyl oleate,
lipOSOmes, waxes polymers, including gel forming and non-gel forming polymers, and
suitable mixtures thereof. The carrier may contain minor amounts of additives such as
substances that enhance isotonicity and chemical stability. Such als are non-toxic
to recipients at the dosages and trations employed, and include buffers such as
phosphate, citrate, succinate, acetic acid, and other organic acids or their salts;
antioxidants such as ascorbic acid; low molecular weight (less than about ten residues)
polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, n,
or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids,
such as glycine, glutamic acid, aspartic acid, or argim'ne; monosaccharides, disaccharides,
and other carbohydrates including cellulose or its derivatives, glucose, mannose, or
dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol;
counterions such as sodium; and/or nonionic surfactants such as polysorbates,
poloxamers, or PEG. ably the r is a parenteral carrier, more preferably a
solution that is ic with the blood of the recipient.
WO 06665
Examples of binders include, but are not limited to, microcrystalline cellulose and
cellulose derivatives, gum tragacanth, glucose on, acacia mucilage, gelatin solution,
molasses, polvinylpyrrolidine, ne, crospovidones, sucrose and starch paste.
Examples of diluents e, but are not limited to, lactose, sucrose, starch, kaolin, salt,
mannitol and dicalcium phosphate.
Examples of excipients include, but are not limited to, starch, surfactants, lipophilic
vehicles, hydrophobic vehicles, pregelatinized starch, Avicel, lactose, milk sugar, sodium
citrate, calcium carbonate, dicalcium phosphate, and lake blend . Typical
excipients for dosage forms such as a softgel include gelatin for the capsule and oils such
as soy oil, rice bran oil, canola oil, olive oil, corn oil, and other similar oils; glycerol,
polyethylene glycol liquids, Vitamin E TPGS as a surfactant and absorption enhancer
els: Manufacturing Considerations; Wilkinson P, Foo Sog Horn, Special Drug
Delivery Systems; Drugs and the Pharmaceutical Sciences Vol 41 Praveen Tyle Editor,
Marcel Dekker 1990, 409-449; Pharmaceutical Dosage Forms and Drug ry by
Ansel, ch and Allen 1995, Williams and Wilkins, Chapter 5 pp 155-225).
Examples of disintegrating agents include, but are not limited to, complex silicates,
croscarmellose , sodium starch glycclate, alginic acid, corn starch, potato starch,
bentonite, methylcellulose, agar and carboxymethylcellulose.
Examples of glidants include, but are not limited to, colloidal silicon dioxide, talc, corn
.
Examples of wetting agents include, but are not limited to, propylene glycol
earate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene
laural ether.
Examples of sweetening agents include, but are not limited to, sucrose, lactose, mannitol
and artificial sweetening agents such as saccharin, and any number of spray dried flavors.
Examples of flavoring agents include, but are not limited to, natural flavors extracted
from plants such as fruits and synthetic blends of compounds which produce a pleasant
sensation, such as, but not limited to peppermint and methyl salicylate.
Examples of lubricants include ium or calcium stearate, sodium lauryl sulphate,
talc, starch, diurn and c acid as well as high molecular weight polyethylene
glycols.
Examples of coloring agents include, but are not limited to, any of the approved certified
water soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes
ded on alumina hydrate.
The artisan of ordinary skill in the art Will recognize that many ent ingredients can
be used in formulations ing to the t invention, in addition to the active
agents (DSLNT of the invention), while maintaining effectiveness of the formulations in
ng the bowel disease e.g., NBC. The list provided herein is not exhaustive.
The formulation of the invention may be administered orally (e.g., in liquid form within a
solvent such as an aqueous or non-aqueous liquid, or within a solid r), rectally,
parenterally, intracisternally, intravaginally, intraperitoneally, lly (as by powders,
ointments, lotion, gels, drops, transdermal patch or transcutaneous , bucally, in
bronchial form or as an oral or nasal spray. The term "parenteral" as used herein refers to
modes of administration which include intravenous (cg, within a dextrose or saline
solution), intramuscular, intrasternal, subcutaneous, intracutaneous, intrasynovial,
intrathecal, periostal, intracerebroventricularly, intra—articular injection and/or infusion.
Administration can be performed daily, weekly, monthly, every other month, quarterly or
any other schedule of stration as a single dose injection or infusion, multiple
doses, or in continuous dose form. The administration of the formulation of the present
2012/023866
invention can be intermittent or at a gradual, continuous, constant or controlled rate to a
subject. In addition, the time of day and the number of times per day that dosage form(s)
is administered can vary.
The appropriate dose of the compound will be that amount ive to prevent
occurrence of a bowel disease. By “effective amount", "therapeutic " or "effective
dose" is meant that amount sufficient to elicit the desired pharmacological or therapeutic
effects, thus resulting in ive prevention or treatment of the disorder or condition.
The ed DSLNT or variants, isomers, analogs and derivatives thereof of the
invention may be also formulated into suitable pharmaceutical preparations such as
solutions, suspensions, tablets, dispersible tablets, pills, capsules, s, sustained
release formulations or elixirs, for oral administration.
Oral formulations may be solid, gel or liquid. The solid dosage forms include tablets,
capsules, granules, and bulk powders. Liquid formulations can, for example, be prepared
by dissolving, dispersing, or otherwise mixing isolated DSLNT of the invention as
defined above and pharmaceutical adjuvants in a carrier, such as, for example, water,
saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to thereby form a
solution or suspension. If desired, the ation of the invention to be administered
may also contain minor amounts of nontoxic auxiliary substances such as wetting ,
emulsifying agents, solubilizing agents, pH buffering agents and the like, for example,
acetate, sodium citrate, cyclodextrine derivatives, an monolaurate, triethanolamine
sodium acetate, triethanolamine oleate, and other such agents.
In certain embodiments, the formulations are solid dosage forms, in one embodiment,
capsules or tablets. The tablets, pills, capsules, troches and the like can contain one or
more of the following ingredients, or compounds of a similar : a ; a
lubricant; a diluent; a glidant; a disintegrating agent; a coloring agent; a sweetening
agent; a flavoring agent; a wetting agent; an emetic coating; and a film coating. es
of s include microcrystalline cellulose, gum tragacanth, glucose solution, acacia
mucilage, gelatin solution, molasses, polvinylpyrrolidine, povidone, crospovidones,
sucrose and starch paste. Lubricants include talc, starch, magnesium or calcium stearate,
lycopcdium and stearic acid. Diluents include, for example, lactose, sucrose, ,
, salt, and marmitol and dicalcium phosphate.
Glidants include, but are not limited to, colloidal silicon dioxide. Disintegrating agents
include crosscarmellose sodium, sodium starch glycolate, alginic acid, corn , potato
starch, ite, methylcellulose, agar and carboxymethylcellulose. Coloring agents
include, for example, any of the ed certified water soluble FD and C dyes,
mixtures thereof; and water insoluble FD and C dyes suspended on alumina hydrate.
ning agents include sucrose, lactose, mannitol and artificial sweetening agents
such as saccharin, and any number of spray dried flavors.
Flavoring agents include natural flavors extracted from plants such as fruits and synthetic
blends of compounds which produce a pleasant sensation, such as, but not limited to
peppermint and methyl salicylate. Wetting agents include propylene glycol earate,
sorbitan monooleate, lene glycol monolaurate and polyoxyethylene laural ether.
Emetic—coatings include fatty acids, fats, waxes, shellac, ammuoniated shellac and
cellulose acetate phthalates. Film coatings include yethylcellulose, sodium
carboxymethylcellulose, polyethylene glycol 4000 and cellulose acetate phthalate.
When the dosage unit fonn is a capsule, it can contain, in addition to material of the
above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain
various other materials which modify the physical form of the dosage unit, for example,
coatings of sugar and other c agents. The compounds can also be stered as a
component of an elixir, suspension, syrup, wafer, sprinkle, chewing gum or the like. A
syrup may contain, in addition to the active nds, sucrose as a sweetening agent
and certain preservatives, dyes and colorings and flavors.
The active material, isolated DSLNT of the invention, can also be mixed with other
mammalian or plant proteins. For e, mammalian proteins e proteins from
mammalian milk (e.g., either intact or partial protein hydrolysates of Whole or
fractionated ian milk). Plant proteins include intact protein or protein
hydrolysate from pea, soy,_.almond, and/or rice proteins.
Examples of preservatives include glycerin, methyl and propylparaben, benzoic acid,
sodium benzoate and alcohol. es of non-aqueous liquids ed in emulsions
include mineral oil and cottonseed oil. Examples of emulsifying agents include gelatin,
acacia, tragacanth, bentonite, and tants such as polyoxyethylene sorbitan
monooleate, Suspending agents include sodium carboxymethylcellulose, pectin,
tragacanth, Veegum and acacia. Sweetening agents include sucrose, syrups, glycerin and
artificial sweetening agents such as rin. Wetting agents include propylene glycol
monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene
lauryl ether. Organic acids include citric and tartaric acid. s of carbon dioxide
e sodium bicarbonate and sodium carbonate. Coloring agents include any of the
approved certified water soluble FD and C dyes, and mixtures thereof. Flavoring agents
include natural flavors extracted from plants such fruits, and synthetic blends of
compounds which produce a pleasant taste sensation.
For a solid dosage form, the solution or suspension, in for example propylene carbonate,
vegetable oils or cerides, is in one embodiment ulated in a gelatin capsule.
Such solutions, and the preparation and encapsulation thereof, are well known in the art.
For a liquid dosage form, the solution, e.g., for example, in a polyethylene glycol, may be
diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e. g.,
water, to be easily measured for administration.
Alternatively, liquid or semi—solid oral formulations may be prepared by dissolving or
dispersing the active compound or salt in ble oils, s, triglycerides, propylene
glycol esters (e.g., ene carbonate) and other such carriers, and encapsulating these
solutions or suspensions in hard or soft n capsule shells.
Of interest herein are also powders, which can be reconstituted for administration as
solutions, emulsions and other mixtures. They may also be reconstituted and formulated
as solids or gels.
Isolated DSLNT may reduce the risk ofNEC through its action as a prebiotic, promoting
the growth of beneficial ia such as Bifidobacreria and Lacrobacz'lli while reducing
the load of pathogenic bacteria that cause bowel diseases. As such, ed DSLNT and
its variants, isomers, analogs and derivatives may serve as a prebiotic, which selectively
ates the growth or zation of one or more bacterial species in the
gastrointestinal tract of a host and presence of these bacteria are beneficial to the health
ofthe host.
As a prebiotic, isolated DSLNT and/or its derivatives, isomers, analogs and/or variants
be administered with a probiotic, which can be live
may or dead microorganisms
conferring a health benefit to the host when stered in sufficient ty.
Microorganisms considered to have health benefit to its host include but are not d to
those belonging to the genera, Bifidobacz‘eria and Lacrobacz'lli. Although the probiotic
can have either live or dead microorganism, it is generally preferably to live-
rganism ingested by the subject as a probiotic. The probiotic may take on a
number of different forms, such as powder, freeze-dried cells, bar, liquid culture,
concentrated liquid e, paste, yogurts, or combinations f. Enteral
administration or liquid feeding are preferred routes for introducing prebiotic along with
DSLNT and its derivatives, isomers, analogs and variants to newborns.
The probiotic in , liquid or bar fonn may be included into a nutritional formula, as
described hereinafter. The compositions may comprise any amount of Bifidobacreria
and/or Lactobacilli probiotic effective for treating and/01' preventing NEC or bowel
disease when enterally administered to an individual in combination with the prebiotic of
the present disclosure. Typically, the compositions will comprise probiotic in sufficient
amounts to provide a daily dose of e.g. from about 10‘5 colony forming units (cfii) to about
1012 colony forming unit (cfu), or from about 106 cfu-Ito about 1010 cfu, or from about 108
cfu to about 1012 cfu, or from about 108 cfu to about 1010 Cfil of probiotic to an individual
the In some embodiments, the
upon ingestion of composition. itions will
comprise tic sufficient to provide a daily dose of about 106 cfu, or about 107 cfu, or
abbut 108 Cfil, or about 109 cfu, or about 1010 cfu, or about 1011 cfu or about 1012 cfu to an
individual upon ingestion ofthe composition.
In a nutritional formula, the nutritional formula may comprise: 1) DSLNT and/or its
tives, isomers, analogs and/or variants, and 2) tic microorganism from about
104 cfu to about 1010 Ch; of probiotic per gram dry weight of the nutritional formula, or
from about 10"l cfu to about 109 cfu per gram dry weight of the nutritional formula, or
from about 104 cfu to about 108 cfu of probiotic per gram dry weight of the nutritional
formula, or from about 104 cfu to about 107 cfu of probiotic per gram dry weight of the
nutritional formula, or from about 104 cfu to about 106 cfu of tic per gram dry
weight of the nutritional formula, or from about 104 cfu to about 105 cfu of probiotic per
gram dry weight of the ional formula. In another embodiment, the nutritional
formula may comprise from about 10‘5 cfu to about 103 cfu of probiotic per gram dry
weight of the nutritional formula, or from about 106 cfu to about 107 cfu of probiotic per
gram dry weight of the nutritional formula. In another embodiment, the nutritional
formula may comprise about 104 cfu of probiotic per gram dry weight of the nutritional
formula, or about 105 cfu of probiotic per gram dry weight of the nutritional formula, or
about 106 cfu of probiotic per gram dry weight of the nutritional formula, or about 107 cfu
of probiotic per gram dry weight of the nutritional formula, or about 108 cfu of probiotic
per gram dry weight of the nutritional formula, or about 109 cfu of probiotic per gram dry
weight of the nutritional formula, or about 1010 cfu of probiotic per gram dry weight of
the nutritional formula.
DSLNT of the invention can be derived using any number of sources and methods known
to those of skill in the art. Alternatively, DSLNT of the invention can be synthesized by
enzymatic methods, using isolated oligosaccharide thetic enzyme or lic
enzyme that participate in the thesis or catabolism of DSLNT of the invention in
either forward or reverse reaction, tively; or altematively, DSLNT derivatives,
analogs, and variants can be derived by replacing key enzymatic steps with a different
biosynthetic or catabolic enzyme and desired sugar analog to obtain the desired
oligosaccharide. DSLNT of the invention can also be synthesized by chemical methods
and purified to obtain the desired compounds.
The prebiotic and prebiotic combination composition may be in powder or liquid form
and/or may be included into a nutritional or infant formula. The compositions may
comprise any amount of prebiotic DSLNT and/or its derivatives, isomers, s and/or
variants effective for treating and/or preventing NBC or bowel disease when enterally
administered to an dual in combination with a tic, such as bacterial species
from the genera, Bifidobacteria and/or Lacrobaciili.
When the combination of prebiotic DSLNT and/or its derivatives, isomers, analogs
and/or variants and probiotic, such as ial species from the genera, Bzfidobacreria
and/or Lactobacillz', is ated as a nutritional or infant formula, the nutritional or
infant formula may comprise from about 0.35 grams tic per 100 grams nutritional
to about 9.2 grams prebiotic per 100 grams nutritional, or from about 1.5 grams prebiotic
100 grams nutritional, or from
per 100 grams nutritional to about 7.0 grams prebiotic per
about 1.5 grams prebiotic per 100 grams nutritional to about 6.0 grams prebiotic per 100
grams nutritional. And preferably from about 3.0 grams prebiotic per 100 grams
nutritional to about 6.0 grams prebiotic to about 100 grams nutritional.
In one embodiment, DSLNT and/or its derivatives, isomers, analogs and/or variants may
be administered to a subject or patient as a prebiotic to stimulate colonization or growth
of Brfzdobacteria and Lacrobacilli in the gastrointestinal tract and ng the presence
ofpafliogens.
In another embodiment, DSLNT and/or its derivatives, isomers, analogs and/or variants
may be administered to a t or t along with a tic, such as Bifidobacz‘eria
and Lacrobacilli, to help ish a healthy gastrointestinal tract microbial flora.
II. METHODS OF THE INVENTION
The invention also es method of preventing or treating a subject having a bowel
disease and/or inflarmnation by administering isolated Disialyllacto-N—tetraose (DSLNT)
or variants, isomers, analogs and derivatives thereof in an amount ient to prevent or
treat the bowel disease and/or inflammation in the subject.
Examples of bowel diseases and atory diseases include but are not limited to
Necrotizing Enterocolitis (NBC), ulcerative colitis, Crohn‘s disease, collagenous colitis,
lymphocytio colitis, ischaemie colitis, diversion colitis, 's disease, indeterminate
colitis, microscopic colitis, pouchitis, pseudomembranous colitis, ischemic s,
diverticulitis, inflammatory bowel e, appendicitis, and irritable bowel syndrome.
The phrase "treating" or ”treatment" refers to any manner in which one or more of the
ms of a e or disorder are ameliorated or otherwise beneficially altered,
whether in a permanent or temporary manner, which can be attributed to or associated
with administration ofthe formulation herein. The term encompasses any pharmaCeutical
use, including prophylactic uses in which the development of one or more of the
symptoms of a disease or disorder is prevented, delayed or reduced, whether in a
permanent or temporary manner, which can be attributed to or associated with
administration of the formulation of the invention.
The subject may be a human or animal subject including a monkey, rat, mouse, dog, cat,
pig, goat, sheep, horse or cow. Preferably, the subject is a pediatric t including
infants, en, and adolescents. The subject may be suffering from diarrhea, enteritis,
colitis, cramping, abdominal pain, edema, ulcer, gastritis, intestinal disease, digestive
e or inflammatory bowel disease. In one embodiment, the bowel disease is an
infectious disease. In another embodiment, the bowel e is Necrotizing Enterocolitis
(NBC).
In accordance with the practice of the invention, the formula of the invention may be a
liquid a (e.g., an infant formula), or a solid or olid formula (e.g., baby food
means
or nutritional supplement) that is given by mouth. However, other administrations
are possible and encompassed by the invention.
Additionally, DSLNT of the invention may also be administered by means of animal
feed, animal feed supplement, or animal nutritional supplement.
The invention further es methods of identifying Whether a breast-fed infant is at
risk of developing Necrotizing Enterccolitis (NBC). In one embodiment, the method
comprises measuring the concentration of DSLNT in the mother’s milk, a low level of
DSLNT being indicative that the breast-fed infant is at risk of developing NBC. Merely
as an example, low levels of DSLNT may be established by ing the concentration
of DSLNT in mother’s milk and total amount of daily DSLNT , and comparing
these values with the incidence ofNEC in newborns.
III. KITS
According to another aspect of the invention, kits are provided. Kits according to the
invention include e(s) comprising formulations of the invention.
The phrase "package" means any vessel containing compounds or compositions
presented herein. In red embodiments, the package can be a box or wrapping.
Packaging als for use in ing pharmaceutical products are well known to
those of skill in the art. Examples of pharmaceutical packaging materials include, but are
not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers,
syringes, bottles, and any ing material suitable for a selected formulation and
intended mode of administration and treatment.
The kit can also contain items that are not contained within the package but are attached
to the outside of the package, for example, pipettes.
Kits may optionally contain instructions for administering formulations of the present
invention to a subject having a ion in need of treatment. Kits may also se
ctions for approved uses of compounds herein by regulatory agencies, such as the
United States Food and Drug Administration. Kits may optionally contain labeling or
t inserts for the present compounds. The package(s) and/or any product insert(s)
include compounds in
may themselves be approved by regulatory es. The kits can“
the solid phase or in a liquid phase (such as buffers provided) in a package. The kits also
can include buffers for preparing solutions for ting the methods, and pipettes for
transferring liquids from one ner to another.
In a further embodiment, the present invention es kits (i.e., a packaged
combination of reagents with ctions) containing the DSLNT and/or its tives,
isomers, analogs and/or variants of the invention useful for treating a boweldisease (e.g.,
NBC).
The kit can contain a formulation of the invention that includes one or more agents of the
invention effective for treating a bowel disease and an acceptable carrier or adjuvant, e.g.,
pharmaceutically acceptable buffer, such as phosphate—buffered saline, Ringer's solution
or dextrose solution.
It may further include other materials desirable from a corrnnercial and user standpoint,
including other buffers, diluents, filters, needles, syringes, and package inserts with
instructions for use.
The agents may be provided as dry powders, usually lyophilized, including excipients
that upon dissolving will provide a reagent solution having the appropriate concentration.
The kit comprises one or more containers with a label and/or instructions. The label can
provide directions for carrying out the preparation of the DSLNT and/or its derivatives,
isomers, analogs and/or variants for e, dissolving of the dry powders, and/or
treatment for e.g. a bowel disease (such as NBC).
The label and/or the instructions can indicate directions for in viva use of the formulation
of the invention. The label and/or the instructions can te that the formulation of the
invention is used alone, or in combination with another agent to treat e.g., a bowel
disease (such as NBC).
The label can indicate appropriate dosages for the DSLNT and/or its derivatives, isomers,
analogs and/0r variants of the invention as described supra.
Suitable containers include, for example, bottles, vials, and test tubes. The containers can
be formed from a y of materials such as glass or plastic. The container can have a
sterile access port (for e the container can be an intravenous solution bag or a vial
having a stopper pierceable by a needle such as a hypodermic injection needle).
The following examples are provided to further illustrate s of the invention. These
examples are miting and should not be construed as limiting any aspect of the
invention.
EXAMPLES
EXAMPLE 1
Human Milk Oligosaccharides (HMO) Prevent NEC in al Rats
In addition to lactose, one liter of mature human milk ns 5-15 g of unbound
oligosaccharides, which is similar to the total amormt of milk proteins and exceeds the
amount of milk lipids. shows that HMO are a heterogeneous group of
oligosaccharides that vary in charge depending on the number of sialic acids per HMO
molecule as well as in size depending on the length of the polylactosamine ne.
More than 150 different HMO have been identified. In contrast, infant formula contains
much lower amounts of oligosaccharides, which are also structurally less complex. To
compensate for the lack of HMO, formula is now often supplemented with
Galactooligosaccharides (GOS), which partially mimic the prebiotic effect of HMO.
However, as shown in (308 are structurally very ent from HMO and likely
unable to also mimic the more structure-specific effects of HMO.
Although a beneficial effect of HMO on NBC has been hypothesized, the limited
availability of HMO make controlled and statistically powered intervention studies on
human preterm infants unfeasible. Instead, we tested HMO in a well—established NEC
model with neonatal Sprague—Dawley rats. We induced time—pregnant rats by in
and randomized their pups into the different intervention groups. Some pups were left
with the dam to serve as breast-fed controls; others were a-fed by oral gavage
twice daily. All pups were exposed to hypoxia thrice daily. On day—of~life 4, we
sacrificed the pups and analyzed their intestines for macroscopic and microscopic signs
of NBC. The ileum was prepared for H&E staining and evaluated blindly by three
ndent igators to determine NEC pathology scores. While all breast-fed pups
survived until day 4, the survival rate dropped to 72% in formula-fed pups (). In
parallel, NEC pathology scores increased significantly (). We then isolated HMO
from pooled human milk and added them to the formula. Survival rates and pathology
scores significantly improved and were similar to -fed controls. These results show
for the first time that HMO indeed prevent NBC in an animal model. Adding GOS had no
effect on survival and pathology , suggesting that the beneficial s of HMO
might not simply be prebiotic in nature and more structure-specific.
in Neonatal Rats
. One specific HMO Prevents NEC
HMO are a structtn‘ally heterogeneous group of oligosaccharides, triggering the question
whether all HMO have similar effects in ting NEC or whether the beneficial
effects are based on distinct structural features. We separated the pooled HMO by two-
dimensional glycan chromatography, and tested whether the fractions and subfractions
reduce NEC in rats.
In the first dimension, we used anion exchange tography (QAE) to separate the
HMO by charge based on the number of sialic acids per HMO molecule. The neutral
fraction with no sialic acid slightly reduced NEC pathology scores ().
Monosialylated HMO (-1), which comprise about 90% of all sialylated HMO, had no
effect, but the disialylated HMO (-2) significantly reduced scores comparable to breast-
fed controls. Tri~ (-3) and tetrasialylated HMO (-4) were ineffective, probably due to
their extremely low abundance. We used HPLC-FL of fluorescently tagged HMO and
showed that the disialylated HMO contained only four major oligosaccharides ().
One of them (#1) was identified as a minor alylated HMO spill-over, which we
disregarded.
In the second dimension, we used FPLC size ion chromatography to further
separate the disialylated HMO fraction by size (). We were able to separate
HMO #2 from HMO #3 and 4. HMO #3 and 4 did not prevent NBC (). However,
HMO #2 significantly reduced NEC pathology scores.
Each HMO fraction and subtraction was tested at concentrations that were based on their
relative abundance in pooled HMO at 10 mg/mL, the average concentration in mature
human milk. Assuming an average lar weight of 1,000 g/mol for pooled HMO, 10
mg/mL is equivalent to 10 mM. The relative abundance of the protective HMO #2 was
«3%, which ponds to 300 MM and is well within the range of other previously
reported bioactive glycans. In conclusion, we identified one specific HMO that prevents
NEC in a neonatal rat model at biologically relevant concentrations. Next, we elucidated
the structural composition of this ular HMO.
Glycan Structure Elucidation identifies DSLNT as tive HMO
We collaborated with the UC San Diego Glycotechnology Core to elucidate the
monosaccharide ition, sequence and glycosidic linkages of the protective HMO
#2. MALDl-TOF~MS analysis suggested the presence of three s, one mine
and, as expected, two sialic acids. Sequential digestion with linkage-specific
exoglycosidases as well as GC-MS analysis of permethylated derivatives revealed lacto—
aose (GalB1-3G1cNAcBl—3 GalBl-4Glc) as the backbone ( with one sialic
acid d2—3~1inked to the terminal Gal and the other sialic acid 1inked to the
subterminal . Our approach unambiguously identified HMO #2 as a specific
isomer of disialyllacto-N—tetraose (DSLNT).
EXAMPLE 2
MATERIALS AND METHODS
Isolation of pooled HMO
Human milk was obtained from 12 healthy volunteers of preterm infants recruited at the
University of California — San Diego Medical Center, San Diego, California, USA, after
approval by the university’s institutional review board. After centrifugation, the lipid
layer was removed and proteins were precipitated from the aqueous phase by on of
ice-cold l and subsequent centrifilgation. l was removed from the HMO-
containing supernatant by roto—evaporation. e and salts were removed by gel
filtration chromatography over a BioRad P2 column (100 cm X 16 min, Bio-Rad,
Hercules, California, USA) using a semi-automated fast protein liquid chromatography
(FPLC) system. GOS syrup (Vivinal, dry matter 75%) was provided by Friesland
Campina Domo (Amersfoort, The Netherlands). Disialyllacto-N—tetraose (DSLNT) was
purchased from Dextra (Reading, UK).
HMO fractionation by two—dimensional chromatography
Pooled HMO were separated by charge using anion exchange chromatography over QAE
y columns (Sigma Aldrich, St. Louis, Missouri, USA). Lyophilized pooled HMO
were dissolved in 2 111M Tris and d to equilibrated columns. Neutral, —1, —2, —3 and -
4 charged HMO were eluted with 2 mM Tris containing 0, 20, 70, 100 and 400 mM
NaCl, respectively. Tris and NaCl were removed by gel filtration chromatography over a
P2 column. Separation was monitored by cence high-performance liquid
chromatography (HPLC-FL) as described below. Differently charged HMO fractions
were further separated by size using P2 gel filtration chromatography (100 cm x 16 nun)
and red by HPLC-FL. Fractions that contained the same, but no other HMO were
pooled and lyophilized.
Oligosaccharide profiling by HPLC
HMO and GOS were fluorescently labelled with 2—aminobenzarnide (ZAB) and separated
by HPLC on an 80 column (4.6 mm ID x 25 cm, 5 mm Tosoh Bioscience, Tokyo,
Japan) with a 50 mM ammonium formate/acetonitiile buffer system. Separation was
monitored by a fluorescence detector at 360 nm tion and 425 nm emission. Peak
tion was based on standard retention times and mass spectrometric (MS) analysis
on a Thermo LCQ Duo Ion trap mass spectrometer equipped with a Nano—ESI—source.
HMO analysis by MALDI-TOF mass spectromefl
ZAB-labelled HMO peaks were collected, dried and mixed with super-DHB matrix in a
1:1 ratio and spotted on MALDI plates for analysis. Spectra were acquired in positive ion
mode.
HMO analysis by tial exoglycosidase digest
Linkage promiscuous neuraminidase (0(2-3>6,8,9; bacter ureafaciens) was
purchased from Sigma Aldrich (St. Louis, Missouri, USA); specif1c neuraminidase
(Salmonella Diphimurium), [31—3 galactosidase (Xanthomonas mam‘hotis), [31—4
galactosidase (Bacteroides fi‘agilis) and B—N—acetyl-glucosaminidase (GlcNAcase, X
manihotis) were obtained from New England s (Ipswich, Massachusetts, USA).
All enzymes were used at concentrations and incubation times according to the
manufacturers’ protocols.
WO 06665 2012/023866
HMO linka e anal sis b as to a h mass 3 ectrometr GC-MS
The unknown HMO 2 was dissolved in dimethylsulphoxide and par—O-rnethylated by
sequential addition of sodium hydroxide and methyl iodine. Chloroform was added and
the reaction stopped by the addition of water. The ated glycan was extracted in the
chloroform layer, dried and hydrolyzed with 4N trifluoroacetic acid at 100°C for 6 h.
Acids were d with 50% isopropanolzwater under dry nitrogen flush. I-Iydrolyzed
samples were reduced overnight by sodium dride in 1M ammonium hydroxide.
Excess borohydride was neutralized by 30% acetic acid and boric acid was removed as
methyl borate. Samples were treated with 1:1 acetic anhydridezpyridine at 100°C for 1 h.
Pyridine and acetic anhydride were removed by nitrogen flush. Partially methylated
alditol acetates were extracted with dichloromethane, analyzed by GC~MS with a DB-S
capillary column, and identified by a combination of ished retention times and mass
fragmentation patterns.
Induction and evaluation ofNEC in neonatal rats
The NEC model in neonatal rats was originally described by Barlow et a1. (Surgery
1975;77:687—90) and later d (Nadler EP, son E, Knisely A, et al, J Surg
Res 2000;92:71—7). Briefly, pregnant time-dated Sprague-Dawley rats were induced at
term using Pitocin (1-2 U per animal). Immediately after birth, neonates were randomized
into one of the different study groups. Animals in the dam—fed (DF) group remained with
the dam. All other animals were separated from the dam, housed in a temperature— and
humidity-controlled incubator and orally gavaged with a special rodent formula (0.2 ml)
twice daily. The formula approximates the n and caloric content of rat breast milk
and consists of 15 g Similac 60/40 (Ross Pediatrics, Columbus, Ohio, USA) in 75 m1 of
Esbilac canine milk replacer (Pet-Ag, Hampshire, Illinois, USA). All animals, dam-fed
and gavaged, were exposed to 10 min of hypoxia (5% 02, 95% N2) thrice daily in a
modular chamber. All animals were sacrificed 96 h postpartum; their intestines were
collected and inspected for the presence of gross necrotic s or Pneumat‘osz‘s
intestinalis. A 0.5 cm section of the terminal ileum was ed for H&E staining per
standard protocols and scored blindly by three investigators based on morphological
changes that included epithelial sloughing, villus oedema, infiltration of neutrophils,
apoptosis of villus enterocytes, crypt hyperplasia and misaligned nuclei in the epithelium.
If at least one pathology sign was observed, a score of 0.5—1.5 was assigned depending on
severity. Two or three signs together resulted in a score of 2-3. The maximum score of 4
was given in case of complete obliteration of the epithelium with or without inal
perforation. Pathology scores were plotted for each animal and the mean calculated per
of ments with a
group. Each intervention was tested in at least two independent sets
total of 8—26 animals per intervention group. ences n the groups were
calculated by one—way ANOVA with the Kruskal-Wallis test and Dunn’s le
ison test. Significance was defined as p<0.05.
Pooled HMO, but not GOS improve survival and reduce NEC in neonatal rats
The primary objective of this study was to assess whether HMO affect NBC in neonatal
rats. Therefore, we randomized rat pups at birth into different study groups. The first
but was
group stayed with the dam for the entire duration of the study (dam-fed, DF),
exposed to hypoxia thrice a day together with all the other groups. The second group was
fed formula that did not contain HMO (forrnula~fed, FF). The third group was fed
formula mented with HMO at 10 mg/ml (FF+I-IMO), the average HMO
concentration in mature human milk. The fourth group was fed formula supplemented
with GOS at 8 mg/ml S), comparable to the GOS tration in infant formula
with prebiotics. HPLC—FL analysis of the isolated, pooled HMO showed that 2’-
fucosyllactose (2’FL), lacto-N—fiicopentacse 1 (LNFP 1) and lacto—N—tetraose (LNT)
were the major oligosaccharides (figure 5A,5B). In addition, the pooled HMO contained
several complex, fucosylated and/or sialylated oligosaccharides. In comparison and as
expected, the HPLC—FL profile of GOS looked strikingly different (figure 5C) and
contained mostly tri- and tetra-saccharides and hardly any complex glycans.
2012/023866
Comparable to published data derived from the same neonatal rat model,?‘7'2'9 all DF pups,
but only 19 of 26 FF pups (73.1%) survived the first 96 h post—partum (figure 6A)
(Nadler EP, Dickinson E, Knisely A, et al. J Surg Res 2000;92:71—7; Upperman JS,
Potoka D, Grishin A, et a1. Semin r Surg 2005;14:159-66; Guner YS, Franklin AL,
Chokshi NK, et 31. Lab Invest 1:1666—79). Most intriguingly, the addition of HMO
greatly improved survival (19 of 20 pups, . GOS, however, had no effect (13 of 17
pups, 76.5%).
DF pups gained weight faster than FF pups, but the addition of HMO or GOS did not
e weight gain, suggesting that improved survival was independent of weight gain.
Macroscopic evaluation 96 h artum showed that the intestines of most FF and
FF+GOS pups were darker, with patchy necrosis and evidence of hemorrhagic intestine
as well as intramural gas cysts (Pneumarosis intestinalis), which are teristic signs
of NEC (figure 6B) and were absent from the intestines of all DF and most FF+HMO
confirmed the macroscopic
pups. Microscopic evaluation of H&E-stained ileum sections
observations (figure 6C). While the ileum of most DF and FF+HMO pups showed a
normal, healthy microscopic architecture, some of the sections from FF and FF+GOS
pups showed complete destruction. While the mean pathology score (iSD) was
0.15:0.34 in tlie DF group, it increased significantly to 1.98i1.11 in the FF group
(p<0.001) (figure 6D). Pups that received HMO with their formula (10 mg/ml) had a
mean pathology score of 0.44:0.30, which was significantly lower than in the FF group
(p<0.001), but statistically not ent from that of BF pups. Pups that received HMO at
a 10—fold lower concentration (1 mg/ml) had a mean pathology score of 0.64i0.54, which
than
was still cantly lower than that in the FF group (p<0.001), but slightly higher
in the DF controls (p<0.05). GOS had no effect on pathology scores (1.69:0.90). These
results demonstrate for the first time that oligosaccharides isolated from human milk
improve survival and reduce NEC in a neonatal rat model of the disease.
Exposure to HMO in the first 24 h post—partum is reguired, but not sufficient to reduce
To assess whether or not HMO have to be present in all feedings to be protective, we fed
a group of pups with formula that did not contain HMO for the first 24 h and then
switched to formula that was supplemented with HMO for the remaining 72 h (figure 7).
To our surprise, pathology scores 1.06) were not different from pups that received
unsupplelnented formula for the entire duration of the study (1.97i1.15). Another group
of pups received formula with HMO for the first 24 h and formula t HMO for the
remaining 72 11. Again, ogy scores (2.04i0.80) were not different from the group
that received unsupplemented formula for the entire time. Together, these s indicate
that exposure to HMO in the first 24 h post—partum is required, but not sufficient to
protect from NBC.
A single, disialylated HMO reduces NEC
Since more than 150 structurally different HMO have been identified so far, we
wondered whether all HMO are protective or whether the effect depends on a specific
structural epitope. First, we used anion exchange chromatography to separate the pooled
HMO by charge based on the number of sialic acid moieties on the individual HMO. As
confirmed by HPLC-FL, we generated five ct HMO fractions with oligosaccharides
that contained either zero, one, two, three or four sialic acids and had a net charge of 0, ~
1, —2, -3 or -4, respectively. We then tested these fractions in the rat model at their
respective concentrations in pooled HMO at 10 mg/ml (figure 8A). Adding the neutral (0)
HMO fraction to the formula d pathology scores to 1.18:0.50 (p<0.05). While the
-1, ~3 and —4 charged HMO fractions had no effect, the -2 charged on lowered
pathology scores to 0.44i0.42, which was significantly different from the FF group
(p<0.001), but not ent from DF controls. These results showed that not all HMO are
protective and that the effects depend on the presence of two sialic acids.
We analyzed the —2 charged HMO fraction by HPLC-FL and detected four distinct peaks
(figure 8B) which we collected and analyzed by MALDI-TOF—MS (figure 8C). The m/z
value of peak 1 corresponded to the 2AB-labelled sodium adduct of an accharide
containing three hexoses, one N—acetyl-hexosarnine and one N—acetyl—neuraminic acid,
likely a monosialylated lacto-N-tetraose (Gall31-3GlcNAcl31-3Gall31-4Glc) or lacto-N-
neotetraose (GalB1-4GlcNAcB1—3GalB1-4Glc). Since peak 1 contained only one sialic
acid and we had shown that the monosialylated (-1) HMO fraction had no significant
effect on ng NBC pathology scores, we assumed that peak 1 was a spillover from
the -1 charged HMO fraction and disregarded this oligosaccharide in future analyses.
Peak 2 was different from peak 1 only by the addition of one N-acetyl—neuraminic acid
and was likely disialylated N-tetraose or lacto—N—neotetraose. Peaks 3 and 4
contained one additional hexose and one additional N—acctyl-hexosamine, which likely
represent an extension of the HMO backbone by the disaccharides N—acetyl—lactosamine
(GalBl-4GlcNAc) or lacto-N-biose (GalBl—3GlcNAc). Peak 3 was ent from peak 4
only by the addition of a fucose moiety. In the following, the oligosaccharides
ented by peaks 2, 3 and 4 are called HMO 2, HMO 3 and HMO 4, respectively.
Next, we used gel exclusion chromatography to further separate the oligosaccharides in
the -2 charged HMO fraction by size. While we were unable to separate HMO 3 and 4
from each other, we separated HMO 3+4 from HMO 2 (figure 8D). We then pooled the
ctions containing either HMO 2 or HMO 3+4 and tested them in the rat model at
their original concentrations in pooled HMO at 10 mg/ml (figure 8B). While HMO 3+4
had no effect, HMO 2 reduced ogy scores to 0.64:0.41, which was significantly
lower than that of the FF group (p<0.001), but not different from DF controls.
The NBC—protective HMO is DSLNT
The results of our two—dimensional chromatography approach showed that a distinct
disialylated HMO ts neonatal rats from NBC. While TOF—MS provided
the first insights into the overall composition of the protective HMO, we used HPLC-FL
after sequential exoglycosidase digestion to ine the exact positions and linkages of
the different monosaccharide e. First, we determined Whether the two sialic acids
are bound in an 0t2-3 or d2-6 linkage. Ineubating HMO 2 with an d2specific
neuraminidase caused a complete shift of the HMO 2 peak in the HPLC-FL
chromatogram (figure 9A), indicating that at least one sialic acid is bound in an d2—3
position. ting HMO 2 with a linkage promiscuous neuraminidase that cleaves both
d2—3~ and OL2b0111’1d sialic acid resulted in an even bigger shift of the HMO 2 peak
(figure 9A). Together, these results indicate that one sialic acid is bound in an d2—3
e and one in an 0L2-6 linkage. After removal of both sialic acids, we used linkage
specific galactosidases to determine whether the terminal monosaccharide is indeed
galactose and Whether the underlying HMO backbone is a type I (GalBl-B GlcNAc-R) or
type II chain (GalBl-4GlcNAc-R). [31specific galactosidase digestion resulted in a
complete peak shift; [31specific galactosidase digestion had no effect, confirming the
used a B—N—acetyl-
presence of terminal galactose in a type I chain (figure 9B). Next, we
glucosaminidase and confirmed that the subterminal monosaccharide is indeed GlcNAc
(figure 9C). The remaining disaccharide was cleaved by a specific galactosidase,
verifying that lactose forms the reducing end of HMO 2.
After ating the position and some of the linkages in the HMO 2 backbone, we
determined the positions of the two sialic acids. The [31specific galactosidase d
the terminal galactose only after pretreatment with the linkage promiscuous
neuraminidase or the d2—3—specific neuraminidase, suggesting that the terminal galactose
is capped by 0L2linked sialic acid. Removal of the subterminal GlcNAc was only
possible after pretreatment with the linkage promiscuous but not the d2speeific
neuraininidase, suggesting that the second sialic acid is bound to the subterminal GlcNAc
in 0L2-6 linkage.
In on, we used GC—MS analysis of partially methylated alditol acetate (PMAA)
derivatives and confimied the presence of 3-linked galactose, 4—linked glucose and 3,6—
linked GlcNAc (figure 9D). The combined data of tial exoglycosidase digestions
and PMAA linkage is unambiguously identified HMO 2 as DSLNT with the
2012/023866
isomeric configuration NeuAcoc2-3Gall31—3 (NeuAcoc2-6) GlcNAcfi1—3Gall31-4Glc
(figure 9E).
DSLNT has NBC-protective effects
Based on HPLC-FL analysis of the pooled I-IMO (figure 5A), the DSLNT concentration
was about 300 uM in formula that we had mented with pooled HMO at 10 mg/ml.
We purchased commercially available DSLNT, added it to formula at 300 uM, and
confirmed that it significantly reduced NEC pathology scores to 0.60:0.52 compared to
1.90j;1.13 in the FF group (p<0.001) (figure 10).
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I
Claims (27)
1. A formulation in the form of a tablet, caplet or powder sing α-Neu5Ac- (2→3)- α-Gal-(1→3)-[α-Neu5Ac-(2→6)]-β-GlcNAc-(1→3)-β-Gal-(1→4)-Glc, α-Neu5Ac- (2→3)-β-Gal-(1→3)-[α-Neu5Ac-(2→6)]- α -GlcNAc-(1→3)-β-Gal-(1→4)-Glc, α-Neu5Ac- β-Gal-(1→3)-[α-Neu5Ac-(2→6)]-β-GlcNAc-(1→3)- α-Gal-(1→4)-Glc, β-Neu5Ac- (2→3)-β-Gal-(1→3)-[α-Neu5Ac-(2→6)]-β-GlcNAc-(1→3)-β-Gal-(1→4)-Glc, or α-Neu5Ac- β-Gal-(1→3)-[ β-Neu5Ac-(2→6)]-β-GlcNAc-(1→3)-β-Gal-(1→4)-Glc.
2. The ation of claim 1, n the formulation is formulated for addition to an infant formula, baby food, or fortified breast milk.
3. Use of isolated disialyllacto-N-tetraose (DSLNT) or an isomer thereof in the manufacture of a medicament for the treatment or prevention of a bowel disease in a subject, wherein the isolated DSLNT or isomer is free of non-DSLNT accharides and is present in the formulation at 300 µM or greater, wherein DSLNT is biologically active α-Neu5Ac-(2→3)-β-Gal-(1→3)-[α-Neu5Ac- (2→6)]-β-GlcNAc-(1→3)-β-Gal-(1→4)-Glc; and wherein the isomer is any of α-Neu5Ac-(2→3)- α -Gal-(1→3)-[α-Neu5Ac-(2→6)]-β- GlcNAc-(1→3)-β-Gal-(1→4)-Glc, α-Neu5Ac-(2→3)-β-Gal-(1→3)-[α-Neu5Ac-(2→6)]- α- GlcNAc-(1→3)-β-Gal-(1→4)-Glc, α-Neu5Ac-(2→3)-β-Gal-(1→3)-[α-Neu5Ac-(2→6)]-β- GlcNAc-(1→3)- (1→4)-Glc, β-Neu5Ac-(2→3)-β-Gal-(1→3)-[α-Neu5Ac-(2→6)]-β- GlcNAc-(1→3)-β-Gal-(1→4)-Glc, or α-Neu5Ac-(2→3)-β-Gal-(1→3)-[ β-Neu5Ac-(2→6)]-β- GlcNAc-(1→3)-β-Gal-(1→4)-Glc.
4. The use according to claim 3, wherein the subject is a mammal.
5. The use according to claim 4, wherein the mammal is an infant.
6. The use according to any one of claims 3 to 5, wherein DSLNT is administered by means of an infant formula, fortified breast milk, baby food or nutritional supplement.
7. The use according to claim 4, wherein the mammal is a human, monkey, rat, mouse, dog, cat, pig, goat, sheep, horse or cow. AH26(11361247_1):JIN
8. The use according to any one of claims 3, 4 or 7, wherein isolated DSLNT is stered by means of animal feed, animal feed supplement, or animal nutritional ment.
9. The use according to any one of claims 3 to 8, wherein the t has a bowel disease selected from diarrhea, enteritis, colitis, cramping, abdominal pain, edema, ulcer, gastritis, intestinal disease, digestive disease, and inflammatory bowel e.
10. The use according to any one of claims 3 to 8, wherein the bowel disease is necrotizing enterocolitis (NEC).
11. An infant formula, baby food, or fortified breast milk, comprising a formulation comprising an isolated disialyllacto-N-tetraose (DSLNT) or isomer thereof and a probiotic, wherein the isolated DSLNT or isomer is free of LNT oligosaccharides and is present in the ation at 300 µM or greater, wherein DSLNT is biologically active α-Neu5Ac-(2→3)-β-Gal-(1→3)-[α-Neu5Ac- (2→6)]-β-GlcNAc-(1→3)-β-Gal-(1→4)-Glc; and wherein the isomer is any of α-Neu5Ac-(2→3)- α-Gal-(1→3)-[α-Neu5Ac-(2→6)]-β- GlcNAc-(1→3)-β-Gal-(1→4)-Glc, α-Neu5Ac-(2→3)-β-Gal-(1→3)-[α-Neu5Ac-(2→6)]- α - GlcNAc-(1→3)-β-Gal-(1→4)-Glc, α-Neu5Ac-(2→3)-β-Gal-(1→3)-[α-Neu5Ac-(2→6)]-β- GlcNAc-(1→3)- α-Gal-(1→4)-Glc, β-Neu5Ac-(2→3)-β-Gal-(1→3)-[α-Neu5Ac-(2→6)]-β- GlcNAc-(1→3)-β-Gal-(1→4)-Glc, or α-Neu5Ac-(2→3)-β-Gal-(1→3)-[ β-Neu5Ac-(2→6)]-β- GlcNAc-(1→3)-β-Gal-(1→4)-Glc.
12. The infant formula, baby food, fortified breast milk, or nutritional ment of claim 11, wherein the probiotic comprises a bacterial species from the genera Bifidobacteria and/or Lactobacilli.
13. The use of any one of claims 3 to 10, wherein the ed DSLNT is modified with a substitution that preserves the biological activity of preventing or inhibiting a bowel e in a subject.
14. The use of claim 13, wherein the isolated DSLNT is modified by substituting a sugar residue of DSLNT with a sugar analog. AH26(11361247_1):JIN
15. The use of claim 14, wherein the sugar analog is ed from 2-desoxy-D-galactose, 2- desoxyfluoro-D-galactose and 2-desoxyamino-D-galactose.
16. The use of claim 13, wherein the isolated DSLNT is modified by substituting the Nacetylglucosamine of DSLNT with any of N-acetylglucosaminylasparagine, Nacetylglucosamine 6-sulfate, N-acetylglucosaminephosphate, N-acetylglucosamine 6- ate, methylacetamidodeoxy-D-glucopyranoside, N-acetylglucosaminitol, Nbromoacetylglucosamine, 2-acetamido-1,3,6-tri-O-acetyldeoxyfluoroglucopyranose, lglucosamine thiazoline, N-fluoroacetyl-D-glucosamine, 2-acetamidodeoxy-D- o-(1,5)-lactone, or amido-3,6-dideoxyglucose.
17. The use of claim 13, wherein the isolated DSLNT is modified by substituting oligosaccharides of isolated DSLNT to afford an accharide chain with four glucose residues (Glc-Glc-Glc-Glc), four se residues (Fru-Fru-Fru-Fru), four ose residues (Gal-Gal-Gal-Gal), four N-acetyl-glucosamine residues (GlcNAc-GlcNAc-GlcNAc-GlcNAc), three galactose es followed by a glucose residue (Gal-Gal-Gal-Glc), or three fructose residues followed by a glucose residue (Fru-Fru-Fru-Glc) modified with at least two sialic acid residues at any of the first three sugar subunits within the oligosaccharide chain.
18. The use of any one of claims 3 to 10, wherein isolated DSLNT is modified by a conjugation that preserves the biological activity of preventing or inhibiting a bowel disease in a subject.
19. The use of claim 18, wherein isolated DSLNT or an isomer of DSLNT is conjugated to a al compound or a polymer by a covalent bond.
20. The use of claim 19, wherein the chemical compound or the polymer is selected from oligosaccharide, amino acid, polypeptide and nucleic acid.
21. The use of any one of claims 3 to 10, wherein isolated DSLNT and/or its isomer is pegylated, phosphorylated, esterified, derivatized with amino acids, and/or derivatized with peptides.
22. The infant formula, baby food, or fortified breast milk, of any one of claims 11 and 12, wherein the formulation further comprises a vitamin and/or mineral. AH26(11361247_1):JIN
23. The use of any one of claims 3 to 10, wherein the medicament further comprises a vitamin and/or mineral.
24. The use of any one of claims 3 to 10, wherein the medicament is formulated for administration in combination with a vitamin and/or mineral.
25. The use of any one of claim 3 to 10, wherein the isolated DSLNT exerts a protective activity for necrotizing enterocolitis (NEC) at a p-value of about p<0.001.
26. The use of any one of claims 3 to 10 and 13 to 21, wherein the sialic acid of isolated DSLNT is substituted with glucoronic acid, galacturonic acid, ic acids, 3-deoxy-D- mannooctulosonic acid, neuraminic acid, or any other carboxyl-group ning monosaccharide or tive thereof.
27. The use of any one of claims 3 to 10, wherein the amount of the DSLNT in the medicament is at least 800 µM.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201161439705P | 2011-02-04 | 2011-02-04 | |
| US61/439,705 | 2011-02-04 | ||
| PCT/US2012/023866 WO2012106665A2 (en) | 2011-02-04 | 2012-02-03 | Disialyllacto-n-tetraose (dslnt) or variants, isomers, analogs and derivatives thereof to prevent or inhibit bowel disease |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| NZ613432A NZ613432A (en) | 2016-06-24 |
| NZ613432B2 true NZ613432B2 (en) | 2016-09-27 |
Family
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