Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR PROVIDING GLUTAMINE
FIELD OF THE INVENTION
The invention relates to methods for providing glutamine supplementation via
the oral
administration of an effective amount of N-acetyl-L-glutamine, or a
nutritionally acceptable
salt thereof.
BACKGROUND
Glutamine is the most abundant amino acid in the human body. It comprises more
than
60% of the free amino acids in skeletal muscle and more than 20% of the total
circulating
amino acids. Glutamine is involved in many body functions, including
gluconeogenesis,
nucleotide synthesis, acid-base balance and other critical metabolic
processes. Studies have
indicated that glutamine is an important metabolic substrate used by rapidly
replicating cells,
particularly gastrointestinal tract and mucosal cells. Glutamine can be
efficiently absorbed in
the hwnan jejunum (part of the small intestine) i~ vivo.
Glutamine is not considered an essential amino acid because it can be
synthesized by
virtually all tissues of the body. It is believed to be produced in sufficient
quantities to
adequately supply body needs (i.e., glutamine-consuming tissues) when the body
is in a
normal physiologic condition. However, numerous studies have shown that during
abnormal
physiologic conditions i.e., disease and metabolic stress), glutamine
production can become
insufficient to meet the body's needs. Thus, glutamine may be more accurately
considered a
conditionally essential amino acid. For example, several studies have
classified glutamine as
such in cases of gut trauma. Souba, W. W.; Smith, R. J.; and Wilmore, D. J.:
Glutamine
Metabolism by the Intestinal Tract. JPEN 9(5): 608-617 (1985); Furst, P.;
Albers, S and
Stehle, P.: Evidence for a nutritional need for glutamine in catabolic
patients. Kidney Intl. 36
(Suppl. 27): S-287-S-292 (1989); Klimberg, V.S., et al.: Oral glutamine
accelerates healing of
the small intestine and improves outcome after whole abdominal radiation.
Glutamine has
also been suggested as a primary energy source for cultured HeLa cells.
Reitzer, L. J.; Wice,
B. M.; and Kennell, D.: Evidence that glutamine, not sugar, is the major
energy source for
cultured HeLa cells. J. Biol. Chem. 254(8): 2669-2676 (1979). And, it has been
suggested
that glutamine may be preferentially utilized by tumor cells, resulting in
progressive glutamine
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depletion in cancer patients. Souba, W. W.: Glutamine and Cancer. Ann. Surg.
218(6):
715-728 (1993).
Nutritional formulas have previously been supplemented with glutamine. By
supplemented it is meant that additional glutamine (either as the free amino
acid or in another
relatively concentrated form such as hydrolyzed wheat gluten) is added to the
formula. As a
naturally occurring amino acid, glutamine is present in all proteins to a
certain extent, and thus
will be present to some extent in any nutritional formula that contains
protein. However,
glutamine only comprises a certain small amount of most naturally occurring
proteins, and
thus, in order to produce a formula with glutamine over a certain level,
glutamine must be
added in a supplemental form. Some of these glutamine-supplemented formulas
are marketed
towards patients who are metabolically stressed, who have impaired GI function
(such as due
to severe multiple trauma, diarrhea, inflammatory bowel disease, GI surgery,
severe burns or
injury due to chemotherapy or radiation therapy), who have malabsorptive
conditions (such as
Crohn's disease) and/or acute trauma.
1 S The easiest way to produce such glutamine-enriched supplements is via the
hydrolysis
of gluten, as this complex mixture of polypeptides, is characteristically
defined by the large
content of glutamine. These products are operationally considered to be gluten-
free as the
gluten is hydrolyzed in small fragments. There is, however, a potential risk
in using these
glutamine enriched 'gluten-free' compounds, as gluten is the triggering
environmental factor
of Celiac Disease and small fragments of gliadin have been shown to have a
toxic effect for
Celiac patients.
Celiac disease is an autoimmune enteropathy triggered by the ingestion of
gluten-
containing grains in susceptible individuals. It is the gliadin fraction of
wheat gluten and
similar alcohol-soluble proteins in other grains that are the environmental
factors responsible
for the development of the intestinal damage. It is now evident that Celiac
disease is the result
of an inappropriate T cell-mediated immune response against ingested gluten.
The disease is
also associated with human leukocyte antigen (HLA) of the major
histocompatibility complex.
and in the continued presence of gluten the disease is self perpetuating. The
typical intestinal
damage characterized by loss of absorptive villi and hyperplasia of the crypts
completely
resolves upon elimination of gluten-containing grains from the patients diet.
Recent estimates
indicate that up to 1 out of every 100 to 300 individuals in the general
worldwide population
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might be affected by this condition. Celiac disease is further characterized
by a series of
complications that significantly hamper the quality of life and in many cases
are life
threatening such as mucosal lymphomas.
Due to the medical benefits described above, attempts have been made to
incorporate
glutamine into nutritional products. One problem complicating these efforts is
the limited
stability of glutamine in aqueous solutions. Free glutamine is known to
degrade in aqueous
media, forming pyroglutamic acid and glutamic acid. Some studies have shown
that
pyroglutamic acid is a neurotoxin in rodents. C. F. deMello, et al.:
Neurochemical effects of
L-pyroglutamic acid. Neurochem. Res. 20(12): 1437-1441 (1995); McGreer, E. G.
and
Singh, E.: Neurotoxic effects of endogenous materials: quinolinic acid, L-
pyroglutamic acid,
and thyroid releasing hormone (TRH). Exp. Neurol. 16(3-4): 410-413 (1984);
Rieke, G. K.,
et al.: L-Pyroglutamate: an alternative neurotoxin for a rodent model of
Huntington's disease.
Exp. Neurol. 104(2): 147-154 (1989). As well as creating pyroglutamic acid,
such
degradation also decreases the amount of glutamine available for the body when
the nutritional
formula is fed. Thus, the use of free glutamine as a supplemental glutamine
source in
nutritional sources has been mostly restricted to powder formulas, which are
reconstituted with
water immediately or almost immediately (24-48 hours) prior to feeding, and
optimally stored
under refrigeration after reconstitution. Such powder formulas include
AlitraQ~ (Ross
Products Division of Abbott Laboratories), Nu-Immu~ (Enjoy Foods), and Vivonex
Plus~
(Sandoz). These formulas provide approximately 25.4, 20.1 and 14.5 g of
glutamine per 1500
kcal (as analyzed), respectively. Additionally, European Patent Application
No. EP 1097646
to Mawatari et al. discloses the use of modified milk powder composition,
which contains
glutamine and/or a peptide containing glutamine. While such products have made
a significant
contribution to patient care, powdered products are considered less than
optimal by most
health care facilities in the United States. Due to the shortage of trained
medical personnel in
many US communities, health care facilities vastly prefer ready-to-feed
nutritionals (RTF).
Further, these nutritionals must have a shelf life of at least 12 months to be
acceptable in the
market place. Thus free glutamine, due to its limited stability, is
unacceptable in these RTF
products.
Researchers have continued to look for glutamine sources that possess long-
term
stability in solution. For example, U.S. Patent No. 5,561,111 to Guerrant et
al., entitled
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"Stable Glutamine Derivatives for Oral and Intravenous Rehydration and
Nutrition
Therapy" discloses the use of alanine-glutamine for this role. Guerrant et al.
generically states
that acyl protecting groups may be placed on the glutamine, but provides no
biological data to
substantiate this assertion. Further, this reference fails to provide any
guidance on the specific
S formulation of any oral or intravenous. compounds containing such
derivatives in such
amounts.
This failure is particularly important in light of formulating problems with
such
solutions as pointed out by Gandini et al., "HPLC Determination of
Pyroglutamic Acid as a
Degradation Product in Parenteral Amino Acid Formulations"
Chf°omatographia, vol. 36, pp.
75-78 (1993). There, the authors note that in order to overcome the problem of
degradation of
glutamine into pyroglutamic acid, the use of dipeptides had been proposed but
such had the
drawback of making the resulting solution qualitatively unbalanced in amino
acid content.
The authors also note the low bioavailability of the glutamine derivative
acetyl-glutamine.
Gurrant et al's lack of biological data is extremely relevant in light of the
work of other
researchers in this area. Palmerini et al. orally administered radiolabeled N-
acetyl-L-
glutamine to rats. "Uptake of Doubly-Labeled N-Acetyl-L-Glutamine in Rat Brain
and
Intestinal Mucosaln Vivo, Farmaco, vol. 36(7), pp. 347-355 (July 1981).
Palmerini et al.
demonstrated that N-acetyl L-glutamine (NAQ) was absorbed intact across the
intestinal
mucosa. The lack of intestinal hydrolysis of the acetyl function would lead
one skilled in the
art to discount NAQ as a potential source of glutamine in nutritional
products, since one of
glutamine's primary activities is to nourish gut epithelium. This function
occurs
predominantly during the intestinal absorption of the amino acid.
Disadvantages of using N-acetyl-L-glutamine in nutritional formulas were
discussed by
Magnusson et al., "Utilization of Intravenously Administered N-Acetyl-L-
Glutamine in
Humans" Metabolism, vol. 38(8), suppl. 1 (August), pp. 82-88 (1989), who found
that 20-40%
of the dose of N-acetyl-L-glutamine administered intravenously was excreted in
the urine.
Other potential problems, especially in rats, were noted by Wallace et al. who
concluded that
there might be problems with inappetance and inefficient utilization of
acetylated peptides,
such as N-acetyl-(alanine)2. "Uptake of acetylated peptides from the small
intestine in sheep
and their nutritive value in rats" British Jouf°rcal of Nutrition, v.
80, pp. 101-108 (1998).
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SUMMARY
In accordance with the present invention, it has been discovered that N-acetyl
L-
glutamine has utility as an oral glutamine supplement in humans. The inventors
have
discovered that human intestinal tissue can utilize N-acetyl L-glutamine as a
source of
glutamine. Therefore, N-acetyl-L-glutamine can be incorporated into liquid
nutritionals
designed for human consumption. These compositions possess long-term stability
and
provide the N-acetyl-L-glutamine in a form that is bioavailable for humans.
The N-acetyl L-
glutamine may be administered as the acid or as a nutritionally acceptable
salt thereof. This
finding was unexpected in light of the earlier work done in other mammals
besides humans.
The N-acetyl L-glutamine or a nutritionally acceptable salt thereof can be
incorporated
into any liquid composition that is suitable for human consumption. Examples
of suitable
compositions include aqueous solutions such as oral rehydration solutions,
liquid nutritional
formulas (including enteral formulas, oral formulas, formulas for adults,
formulas for pediatric
patients and formulas for infants), etc. The quantity of N-acetyl L-glutamine
or a nutritionally
acceptable salt thereof can vary widely but typically, these compositions will
contain sufficient
N-acetyl-L-glutamine or a nutritionally acceptable salt thereof to provide at
least about 10 mg
of total glutamine per kg of body weight per day for any human.
DESCRIPTION OF THE FIGURES
FIGURE 1 illustrates in graphic form the aqueous stability of N-acetyl-L-
glutamine at
various pH values and ambient temperature. All values for pH 5.0 to pH 8.0
samples were the
same.
FIGURE 2 illustrates in graphic form the degradation products formed in
aqueous N-
acetyl-L-glutamine solutions over a pH range from 2.0 to 8.0 when the
solutions were held at
room temperature for 180 days.
FIGURE 3 illustrates in graphic form the amount of added glutamine or N-acetyl-
L-
glutamine remaining in the intestinal lumen as a function of time after
introduction of the
material to an isolated pig intestinal loop during an Intra-Surgery experiment
as described
herein. The analyte remaining is expressed as a percentage of the analyte
present at time zero.
FIGURE 4 illustrates in graphic form the amount of added glucose remaining in
the
intestinal lumen as a function of time after introduction of the material to
an isolated pig
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intestinal loop during an Intra-Surgery experiment as described herein.
Glucose
remaining is expressed as a percentage of the amount present at time zero.
FIGURE 5 illustrates in graphic form the amount of glutamine in the portal
blood (in
mcg/mL) in pigs where different materials (glucosaline control, glutamine
in.glucosaline or N-
acetyl-L-glutamine in glucosaline) were introduced to an isolated intestinal
loop versus time
after administration.
FIGURE 6 illustrates in graphic form the amount of glutamine and glutamate in
the
jejunum mucosa (expressed in mcg/gram wet mucosa) of pig intestine measured
after an Intra-
Surgery Experiment as described herein
FIGURE 7 shows electron transmission micrographs of jejunum enterocytes from
healthy pigs (A, B panels) fed with ENSURE PLUS formula and protein-energy
malnourished
pigs fed with the same formula supplemented with caseinate (C, ~D panels),
glutamine (E, F
panels) or NAQ (G, H panels) for 30 days were analyzed for signs of
inflammation, such as
clear cytoplasmic spaces and lymphocyte infiltration.
FIGURE 8 shows electron transmission micrographs of ileum enterocytes from
healthy
pigs (A, B panels) fed with ENSURE PLUS formula and protein-energy
malnourished pigs fed
with the same formula supplemented with caseinate (C, D panels), glutamine (E,
F panels) or
NAQ (G, H panels) for 30 days were analyzed for signs of inflammation, such as
clear
cytoplasmic spaces and lymphocyte infiltration.
FIGURE 9 shows the effect of different products/compounds on the occurrence of
apoptosis and inflammation on the mucosal of untreated celiac disease
patients.
FIGURE 10 shows the pattern of epithelial TLTNEL expression in mucosal samples
treated with N-acetyl glutamine (A) and PS (B).
FIGURE 11 shows the pattern of CD25 expression in the sub-epithelial
compartment
in mucosal samples treated with N-acetyl glutamine (A) and PS (B).
DETAILED DESCRIPTION
As used in this application the following terms have the meanings described
below:
a) "total glutamine" refers to the total amount of biologically available or
potentially
available glutamine from any source expressed as glutamine. This can include
glutamine supplied as free glutamine, glutamine found as part of a peptide or
intact
protein, and other biologically available glutamine sources, such as N-acetyl-
L-
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glutamine. Byproducts of glutamine degradation (e.g., pyroglutamic acid and
the like) are not included. As an example of this calculation, a hypothetical
product
is described below.
A nutritional product contains 60 grams/liter of protein system containing
intact
and lightly hydrolyzed proteins, including the following:
i. Free glutamine at 1.1 grams/liter, as determined by methods well known
to one skilled in the art.
ii. A blend of intact and lightly hydrolyzed proteins containing 50.0 grams
/liter protein, which has been analyzed by published methodology (e.g.,
by methods such as Fouques, et al., "Study of the Conversion of
Asparagine and Glutamine of proteins into Diaminopropionic and
Diaminobutyric Acids Using [Bis(trifluoroacetoxy)iodo] benzene Prior
to Amino Acid Determination." Analyst, Volume 116, (May), pp 529 -
531 (1991)) to contain 3.4 grams glutamine / 100 grams protein.
iii. N-Acetyl-L-glutamine at 11.6 grams/liter, which contains 9.0 grams of
glutamine as calculated below:
11.6 g NAQ x 1 mole NAQ x 1 mole Gln x 146.1 ~ Gln = 9.0 g Gln
188.2 g NAQ 1 mole NAQ 1 mole Gln
"Total Glutamine" is therefore the sum of these three sources, as: l .l
grams/L
(free) + (3.4 g/100 g protein x 50 g protein/L) + 9.0 grams/L (NAQ) = 11.8
grams.
b) "mmoles" refers to millimoles (i.e. 1/1000 of a mole)
c) The term "nutritionally acceptable salt," means those salts of N-acetyl-L-
glutamine
which are acceptable for use in a liquid composition that is suitable for
administration to humans. Nutritionally acceptable salts of N-acetyl-L-
glutamine
are salts where the hydrogen of the carboxyl group has been replaced with
another
positive cation. Such salts can be prepared during the final isolation and
purification of the N-acetyl-L-glutamine or separately by reacting the
carboxylic
group with a suitable base such as the hydroxide, carbonate, or bicarbonate of
a
metal canon or with ammonia or an organic primary, secondary or tertiary
amine.
Nutritionally acceptable salt cations may be based on alkali metals or
alkaline earth
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metals such as lithium, sodium, potassium, calcium, magnesium, and
aluminum and nontoxic quaternary ammonia and amine cations such as
ammonium, tetramethylammonium, tetraethylammonium, methylamine,
dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine,
tributylamirie, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-
methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-
dibenzylphenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine. Other
representative organic amines useful for the formation of base addition salts
include
ethylenediamine, ethanolamine, diethanolamine, piperidine, and piperazine.
d) Any reference in the specification or claims to a quantity of an
electrolyte should be
construed as referring to the final concentration of the electrolyte in the
oral
rehydration solution. Tap water often contains residual sodium, chlorine, etc.
A
value of 40 mEq of sodium, in this application, means that the total sodium
present
in the oral rehydration solution equals 40 mEq, taking into account both added
sodium as well as the sodium present in the water used to manufacture the oral
rehydration solution.
e) Any reference to a numerical range in this application should be considered
as
being modified by the adjective "about". Further, any numerical range should
be
considered to provide support for a claim directed to a subset of that range.
For
example, a disclosure of a range of from 1 to 10 should be considered to
provide
support in the specification and claims to any subset in that range (i.e.
ranges of
2-9, 3-6, 4-5, 2.2-3.6, 2.1-9.9, etc.).
The present invention provides methods and compositions for providing
glutamine
supplementation to a human by the oral administration of an effective amount
of N-acetyl-L-
glutamine or a nutritionally acceptable salt thereof. A suitable N-acetyl-L-
glutamine for use in
the nutritional formulas can be produced using well established, standard
chemical synthesis
techniques, such as incubating free L-glutarnine with acetic anhydride in the
presence of a
suitable base catalyst (e.g., pyridine), following synthesis, suitable
purification by
recrystallization would produce a suitably pure compound for food - grade
status. Indeed,
several chemical companies well versed in amino acid chemistries provide a
food - grade N-
acetyl-L-glutamine (e.g., Kyowa Hakko Kogyo Co, Ltd., Tokyo, Japan or Flamma,
s.p.a.,
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Italy). Alternatively, other methods (e.g., microbial fermentation, c.f., JP
51038796, JP
57001994, JP 57016796) could be utilized to produce a suitable food - grade N-
acetyl-L-
glutamine. Nutritionally acceptable salts of N-acetyl-L-glutamine are salts
where the hydrogen
of the carboxyl group has been replaced with another positive ration. Such
salts can be
prepared during the final isolation and purification of the N-acetyl-L-
glutamine or separately
by reacting the carboxylic group with a suitable base such as the hydroxide,
carbonate, or
bicarbonate of a metal ration or with ammonia or an organic primary, secondary
or tertiary
amine. Nutritionally acceptable salt rations may be based on alkali metals or
alkaline earth
metals such as lithium, sodium, potassium, calcium, magnesium, and aluminum
and nontoxic
quaternary ammonia and amine rations such as ammonium, tetramethylammonium,
tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine,
diethylamine, ethylamine; tributylamine, pyridine, N,N-dimethylaniline, N-
methylpiperidine,
N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-
dibenzylphenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine. Other
representative organic amines useful for the formation of base addition salts
include
ethylenediamine, ethanolamine, diethanolamine, piperidine, and piperazine. If
desired
pharmaceutical grade N-acetyl-glutamine from Sigma may be used.
Methods of providing glutamine supplementation to a human comprises orally
administering an effective amount of N-acetyl-glutamine or a nutritionally
acceptable salt
thereof. Typically, the N-acetyl-L-glutamine will be administered via liquid
such as an oral
rehydration solution, a sports drink, or a part of an enteral formula.
An effective amount of N-acetyl-glutamine or a nutritionally acceptable salt
thereof is
preferably an amount sufficient to provide approximately 10-50 g of total
glutamine per day or
alternatively at least about 140 mg total glutamine per kg of body weight per
day, more
preferably at least 250 mg total glutamine per kg of body weight per day
(mg/kg/day). The N-
acetyl-L-glutamine will provide from about 1-100% of the total glutamine that
the patient
consumes on a daily basis, preferably from about 10-95%, and more preferably
from about 75-
90% of the total glutamine that the patient consumes on a daily basis.
When N-acetyl-L-glutamine or a nutritionally acceptable salt thereof provides
the sole
source of glutamine that the patient consumes, an effective amount of N-acetyl-
L-glutamine or
a nutritionally acceptable salt thereof is preferably at least about 0.7
mmoles/kg/day. More
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preferably, an effective amount of N-acetyl-L-glutamine or a nutritionally
acceptable salt
thereof may be at least about 1.0 mmoleslleg/day. Even more preferably, an
effective amount
of N-acetyl-L-glutamine may be at least about 1.5 mmoles/kg/day.
As noted above, the amount of N-acetyl-L-glutamine or a nutritionally
acceptable salt
thereof needed to provide total glutamine of 250 mg/kglday will vary depending
upon the
amount of glutamine present in any other protein components the patient is
consuming. As a
general guideline, the patient should consume at least about .7 to about 4.0
mmoles of N-
acetyl-L-glutamine or a nutritionally acceptable salt thereof per kg per day
to obtain the full
benefits of this invention. Lesser amounts may be beneficial, depending on the
total glutamine
content of the other components of the protein system. In general, sufficient
N-acetyl-L-
glutamine should be provided to the patient deliver at least about 140 mg of
total glutamine
per kg of body weight per day, more preferably at least about 250 mg total
glutamine per kg of
body weight per day.
The method may be utilized to provide glutamine supplementation to adults,
children
and infants. The term child refers to a human aged one year up to about 16
years (i.e.
adulthood). The term infant is meant to include all humans less than one year
in age, and
includes premature infants and micro-preemie infants. The term premature
infants is meant to
describe infants born before 37 weeks of gestation and/or less than 2500 grams
at birth, and
the term micro-preemie is meant to describe infants born between 23 and 28
weeks of
gestation. As used herein, the term non-adult includes children and infants.
The concentration of glutamine equivalents that is fed to adults, children and
infants
may vary. One reason for this is the wide variation of caloric density
requirements in various
stressed situations. One example of this situation arises when only a very
small volume of
enteral nutrition can be tolerated, such as in severe trauma or in the
premature infant. In such
cases, the majority of nutrition may initially be provided via parenteral
feeding. In these cases,
very small amounts of enteral nutrition might be acceptable, and it would be
of benefit to
supply as much glutamine equivalents as possible. Therefore, a very high
concentration of N-
acetyl-glutamine or a nutritionally acceptable salt thereof might be used. In
another
application, a standard infant formulation might be supplemented with N-acetyl-
glutamine or a
nutritionally acceptable salt thereof to support gut function, in which case a
substantially lower
concentration would be used
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The N-acetyl-L-glutamine may be utilized for any condition in which glutamine
may be beneficial. Such conditions include at least: enhanced recovery from
gastrointestinal
surgery, gastrointestinal resection, small bowel transplant, and other post
surgical traumas
starvation, critical illnesses and injuries such as multiple trauma, short
bowel syndrome, burns,
bone marrow transplant, AIDS, oral mucositis, Celiac disease, Crohn's disease,
necrotizing
enterocolitis, prematurity of the gut, and prevention or reduction of severity
of infections of
opportunity such as sepsis. Glutamine supplementation may also be helpful in
preventing gut
deterioration associated with particular treatments (such as chemotherapy or
radiation therapy)
or in situations where oral feeding is severely restricted (such as extreme
prematurity). Also
included axe combinations of any of the above.
The N-acetyl-L-glutamine of this invention can be administered using any
liquid
solution that is suitable for human consumption. For example, the N-acetyl-L-
glutamine may
simply be dissolved in water. If desired, it can be incorporated into flavored
drinks to enhance
its palatability. For example, it can be incorporated into Kool-Aid, or sodas
such as Pepsi or
Cola. In a further embodiment, the N-acetyl-L-glutamine can be incorporated
into sports
drinks such as Gator-Aid.
Typically however, the N-acetyl-L-glutamine will be administered via an oral
rehydration solution (ORS) or a liquid nutritional formula. The quantity of N-
acetyl-L-
glutamine that may be incorporated into an aqueous solution, such as ORS, can
vary widely.
Typically, the ORS will contain at least about 5.0 mmoles of N-acetyl-L-
glutamine or a
nutritionally acceptable salt thereof per liter of solution, and further
contain at a minimum,
water, glucose, and sodium. More preferably, the ORS will contain about 20 to
about 300
mmoles per liter of N-acetyl-L-glutamine or a nutritionally acceptable salt
thereof, and more
typically from about 25 to about 200 mmoles. If a liquid such as Kool-Aid or
Gator-Aid is
utilized, then the quantity of N-acetyl-L-glutamine will be comparable to that
described for the
ORS.
Oral rehydration solutions are well known to those skilled in the art. The
ORS's
utilized in this invention will typically contain all the electrolytes and
levels thereof required
by the Food and Drug Administration for oral rehydration formulations sold in
the United
States. In addition to sodium (Nay), potassium (K+), chloride (Cl-) and
citrate ions, the oral
rehydration solutions contain a source of carbohydrate, such as glucose,
fructose, or dextrose.
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Typically, the ORS comprise water, carbohydrate, sodium ions, potassium ions,
chloride
ions, and citrate ions.
The quantity of sodium ions used in the ORS can vary widely, as is known to
those
. skilled in the art. Typically, the ORS will contain from about 30 mEq/L to
about 95 mEq/L of
sodium. In a further embodiment, sodium content can vary from about 30 mEq/L
to about 70
mEq/L, most preferably from about 40 mEq/L to about 60 mEq/L. Suitable sodium
sources
include but are not limited to sodium chloride, sodium citrate, sodium
bicarbonate, sodium
carbonate, sodium hydroxide, and mixtures thereof. As used herein, one
milliequivalent
(mEq) refers to the number of ions in solution as determined by their
concentration in a given
volume. This measure is expressed as the number of milliequivalents per liter
(mEq/L).
Milliequivalents may be converted to milligrams by multiplying mEq by the
atomic weight of
the mineral and then dividing that number by the valence of the mineral.
The ORS will also contain a source of potassium ions. The quantity of
potassium can
vary widely. However, as a general guideline, the ORS will typically contain
from about 10
mEq/L to about 30 mEq/L of potassium. In a further embodiment, they may
contain from
about 15 mEq/L to about 25 mEq/L of potassium. Suitable potassium sources
include, but are
not limited to, potassium citrate, potassium chloride, potassium bicarbonate,
potassium
carbonate, potassium hydroxide, and mixtures thereof.
The ORS will also contain a source of carbohydrate. The quantity of
carbohydrate
utilized is important as described above. The quantity must be maintained at
less than about 3
w/w, and more preferably less than about 2.5 % w/w. Quantities ranging from
about 3%
w/w to about 2.0% w/w are suitable. Excessive carbohydrate will exacerbate the
fluid and
electrolyte losses associated with diarrhea.
Any carbohydrate used in prior art oral rehydration solutions may be used.
Suitable
carbohydrates include, but are not limited to, simple and complex
carbohydrates; glucose,
dextrose, fructooligosacchaxides, fructose and glucose polymers, corn syrup,
high fructose
corn syrup, sucrose, maltodextrin, and mixtures thereof.
The ORS will also typically include a source of base to replace diarrheal
losses.
Typically citrate will be incorporated into the oral rehydration solutions to
accomplish this
result. Citrate is metabolized to an equivalent amount of bicarbonate, the
base in the blood that
12
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helps maintain acid-base balance. While citrate is the preferred source of
base, any base
routinely incorporated into rehydration solutions may be used.
The quantity of citrate can vary as is known in the art. Typically, the
citrate content
ranges from about 10 mEq/L to about 40 mEq/L, more preferably from about 20
mEq/L to
about 40 mEq/L, and most preferably from about 25 mEq/L to about 35 mEq/L.
Suitable
citrate sources include, but are not limited to, potassium citrate, sodium
citrate, citric acid and
mixtures thereof.
The ORS will also typically contain a source of chloride. The quantity of
chloride can
vary as is known in the art. Typically the ORS will contain chloride in the
amount of from
about 30 mEq/L to about 80 mEq/L, more preferably from about 30 mEq/L to about
75
mEq/L, and most preferably from about 30 mEq/L to about 70 mEq/L. Suitable
chloride
sources include but are~not limited to, sodium chloride, potassium chloride
and mixtures
thereof.
Optionally, indigestible oligosaccharides may be incorporated into the ORS.
Indigestible oligosaccharides have a beneficial impact on the microbial flora
of the GI tract.
They help to suppress the growth of pathogenic organisms such as Clostridium
difficile.
These oligosaccharides selectively promote the growth of a nonpathogenic
microbial flora.
Such oral rehydration solutions have been described in United States Patent
5,733,759, filed
April 5, 1995, the contents of which are hereby incorporated by reference.
Typically, the
oligosaccharide will be a fructooligosaccharide, an inulin such as raftilose,
or a
xylooligosaccharide. The quantity can vary widely, but may range from 1 to 100
grams per
liter, and more typically from 3 to 30 grams per liter of aqueous solution.
The ORS will also typically include a flavor to enhance its palatability,
especially in a
pediatric population. The flavor should mask the salty notes of the aqueous
solutions. Useful
flavorings include, but are not limited to, peach, butter pecan, blueberry,
banana, cherry,
orange, grape, fruit punch, bubble gum, apple, raspberry and strawberry.
Artificial sweeteners
may be added to complement the flavor and mask the salty taste. Useful
artificial sweeteners
include saccharin, nutrasweet, sucralose, acesulfane-K (ace-K), etc.
Preservatives may be added to help extend shelf life. Persons knowledgeable in
the art
will be able to select the appropriate preservative, in the proper amount, to
accomplish this
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result. Typical preservatives include, but are not limited to, potassium
sorbate and
sodium benzoate.
In addition to the carbohydrate described above, the ORS may also contain rice
flour,
or any other component of rice that is beneficial in the treatment of
diarrhea. Numerous rice
supplemented oral rehydration solutions have been described in the literature.
Methods for
using such rice supplemented oral rehydration solutions are well known to
those skilled in the
art. Examples of such rice supplemented oral rehydration solutions include
those described in
United States Patent No. 5,489,440 issued February 6, 1996.
The ORS can be manufactured using techniques well known to those skilled in
the art.
As a general guideline, all the ingredients may be dry blended together;
dispersed in water
with agitation; and optionally heated to the appropriate temperature to
dissolve all the
constituents. The ORS is then packaged and sterilized to food grade standards
as is known in
the art.
ORS may be administered in different forms, depending upon patient preference,
as is
known in the art. For example, some children will consume oral rehydration
solutions more
readily if frozen, such as in the form of a Popsicle. Oral rehydration
solution Popsicles are
described in detail in United States Patent No. 5,869,459. Oral rehydration
solutions have
also been formed into gels in order to enhance patient compliance, especially
in a pediatric
population. Gelled rehydration compositions are described in United States
Patent Application
Serial No. 09/368,388 filed August 4, 1999. These gels have also been
described in PCT
Application No. 99/15862. As a general overview, the aqueous solutions may be
formed into
a flowable gel. Alternatively, it may also be formed into a self supporting
gel structure. Such
a result rnay be accomplished by incorporating suitable gelling agents into
the aqueous
solution.
Suitable gelling agents for use in the aqueous solution include but are not
limited to
agar, alginic acid and salts, gum arabic, gum acacia, gum talha, cellulose
derivatives, curdlan,
fermentation gums, furcellaran, gelatin, gellan gum, gum ghatti, guar gum,
iota carrageenan,
Irish moss, kappa carrageenan, konjac flour, gum karaya, lambda carrageenan,
larch
gum/arabinogalactan, locust bean gum, pectin, tamarind seed gum, tara gum, gum
tragacanth,
native and modified starch, xanthan gum and mixtures thereof. Usage rates of
said gelling
agents range from about 0.05 to about 50 wt./wt.%.
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As noted above, the N-acetyl-L-glutamine, or its nutritionally acceptable
salts may
be administered via liquid nutritional products. The quantity of N-acetyl-
glutamine that is
incorporated into the liquid nutritional can vary widely, but will fit into
the dosage guidelines
described above. The amount of N-acetyl-L-glutamine or a nutritionally
acceptable salt
thereof utilized in a liquid nutritional formula will be dependent upon
various factors including
whether the formula provides a majority or sole source of nutrition, whether
the formula
contains other sources of glutamine, the amount of formula consumed on a daily
basis, and the
type of patient for whom the formula is intended (which will also influence
the amount of
formula consumed daily). The formula will preferably contain N-acetyl-L-
glutamine or a
nutritionally acceptable salt thereof in an amount sufficient, when combined
with the
glutamine contained in the other protein components, to provide at least 140
mg of total
glutamine per kg of body weight per day. The amount of N-acetyl-L-glutamine or
a
nutritionally acceptable salt thereof may also be expressed as providing a
percentage of the
protein calories. According to such an expression, nutritional formulas would
contain N-
acetyl-L-glutamine or a nutritionally acceptable salt thereof as about 1 to
about 100% of the
protein calories. The percentages are calculated based on the protein portion
of N-acetyl-L-
glutamine or a nutritionally acceptable salt thereof (i.e., the glutamine
portion), and do not take
into account any caloric contribution from the non-protein portion of N-acetyl-
glutamine or a
nutritionally acceptable salt thereof (i.e., the acetate or salt portion).
Preferably, when a
nutritional formula is for adults, it would contain N-acetyl-L-glutamine or a
nutritionally
acceptable salt thereof sufficient to supply about 10 to about 95% of the
protein calories. If the
nutritional formula is being designed for non-adults, then the N-acetyl-L-
glutamine would be
present in sufficient quantities to supply from about 1 to about 12% of the
protein calories.
Liquid nutritional formulas include enteral formulas, oral formulas, formulas
for adults,
formulas for pediatric patients and formulas for infants. Enteral formulas and
nutritional
formulas, for example, represent an important component of patient care in
both acute care
hospitals and long-term care facilities (i.e., nursing homes). These formulas
can serve as the
sole source of nutrition for a human being over an extended period of time,
though
supplemental use to enhance sub-optimal nutrition status is common.
Accordingly, the
formulas must contain significant amounts of protein, fat, minerals,
electrolytes, etc., if they
are to meet their primary goal of preventing malnutrition. These formulas are
typically
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administered to the patient as a liquid, since a significant proportion of the
patients
targeted are incapable of consuming solid foods. While some patients are
capable of drinking
a formula, there are significant numbers that receive enteral formulas via a
nasogastric tube
(NG tube or tube feeding).
Liquid nutritional formulas contain a protein component, providing from 14 to
35% of
the total caloric content of the formula, a carbohydrate component providing
from 36 to 76%
of the total caloric content, and a lipid component providing from 6 to 51 %
of the total caloric
content. Liquid nutritional formulas may be adult formulas, pediatric formulas
or infant
formulas (just as the aqueous solutions may be administered to either adults,
pediatric patients
or infants). For high glutamine applications, liquid nutritional formulas
preferably provide at
least a majority source of nutrition. The liquid nutritional formulas
described herein, however,
may be used as other than an at least majority source of nutrition,
particularly in the case
where mostly parenteral nutrition is the standard of practice (e.~., in
extremely premature
infants, who are slowly weaned to oral feedings over the first several weeks
ex utero). The
term at least a majority source of nutrition means that the formula is
intended to be fed in an
amount sufficient to provide at least half of the total caloric and
nutritional requirements for a
patient receiving the formula. Encompassed within this definition are formulas
and the
feeding of formulas as a sole source of nutrition, thereby providing all of
the total caloric and
nutritional requirements for a patient receiving the formula. The amount of
calories and
nutrients required will vary from patient to patient, dependent upon such
variables as age,
weight, and physiologic condition. The amount of nutritional formula needed to
supply the
appropriate amount of calories and nutrients may be determined by one of
ordinary skill in the
art, as may the appropriate amount of calorie and nutrients to incorporate
into such formulas.
As examples, when the formula is an adult formula, the protein component may
comprise from
about 14 to about 35 % of the total caloric content of said liquid nutritional
formula; the
carbohydrate component may comprise from about 36 to about 76 % of the total
caloric
content of said liquid nutritional formula; and the lipid component may
comprise from about 6
to about 41 % of the total caloric content of said liquid nutritional formula.
The nutritional
formula may be a formula for oral feeding or a formula for enteral feeding. As
another
example, when the formula is a non-adult formula, the protein component may
comprise from
about 8 to about 25 % of the total caloric content of said liquid nutritional
formula; the
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carbohydrate component may comprise from about 39 to about 44% of the total
caloric
content of said liquid nutritional formula; and the lipid component may
comprise from about
45 to about 51 % of the total caloric content of said liquid nutritional
formula. These ranges
are provided as examples only, and are not intended to be limiting.
As a practical matter, such products would contain an amount of N-acetyl-L-
glutamine or a nutritionally acceptable salt thereof sufficient to provide
about half or more of
the total glutamine content. Alternatively, an effective amount of N-acetyl-L-
glutamine or a
nutritionally acceptable salt thereof may be expressed in mmoles per 1000
kcal. According to
such an expression, if a target amount of glutamine is approximately 300 mg of
glutamine per
day/kg/day, a nutritional formula would preferably contain for an adult, at
least about 35
mmoles of N-acetyl-L-glutamine or a nutritionally acceptable salt thereof per
1000 kcal of
nutritional formula, and for a child, infant or premature infant (a non-adult)
at least about 5.0
mmoles of N-acetyl-L-glutamine or a nutritionally acceptable salt thereof per
1000 kcal of
nutritional formula. More preferably, such nutritional formula for an adult
would contain
about 35 to about 160 mmoles of N-acetyl L-glutamine or a nutritionally
acceptable salt
thereof per 1000 kcal of nutritional formula, for a child about 5.0 to about
32 mmoles of N-
acetyl-L-glutamine or a nutritionally acceptable salt thereof per 1000 kcal of
nutritional
formula, and for an infant or premature infant about 5.0 to about 26 mmoles of
N-acetyl-L-
glutamine or a nutritionally acceptable salt thereof per 1000 kcal of
nutritional formula.
In addition to the N-acetyl-glutamine, the nutritional formulas will contain
suitable
carbohydrates, lipids and proteins as are known to those skilled in the art of
making nutritional
formulas. Suitable carbohydrates include, but are not limited to, hydrolyzed,
intact, naturally
and/or chemically modified starches sourced from corn, tapioca, rice or potato
in waxy or non
waxy forms; and sugars such as glucose, fructose, lactose, sucrose, maltose,
high fructose corn
syrup, corn syrup solids, fructooligosaccharides, and mixtures thereof.
Maltodextrins are
polysaccharides obtained from the acid or enzyme hydrolysis of starches (such
as those from
corn or rice). Their classification is based on the degree of hydrolysis and
is reported as
dextrose equivalent (DE). The DE of any maltodextrins utilized in the
nutritional formulas is
preferably less than about 18-20.
Suitable lipids include, but are not limited to, coconut oil, soy oil, corn
oil, olive oil,
safflower oil, high oleic safflower oil, MCT oil (medium chain triglycerides),
sunflower oil,
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high oleic sunflower oil, palm oil, palm olefin, canola oil, cottonseed oil,
fish oil, palm
kernel oil, menhaden oil, soybean oil, lecithin, lipid sources of arachidonic
acid and
docosahexaneoic acid, and mixtures thereof. Lipid sources of arachidonic acid
and
docosahexaneoic acid include, but are not limited to, marine oil, egg yolk
oil, and fungal or
algal oil. Numerous commercial sources for these fats are readily available
and known to one
practicing the art. For example, soy and canola oils are available from Archer
Daniels
Midland of Decatur, Illinois. Corn, coconut, palm and palm kernel oils are
available from
Premier Edible Oils Corporation of Portland, Organ. Fractionated coconut oil
is available
from Henkel Corporation of LaGrange, Illinois. High oleic safflower and high
oleic sunflower
oils are available from SVO Specialty Products of Eastlake, Ohio. Marine oil
is available from
Mochida International of Tokyo, Japan. Olive oil is available from Anglia Oils
of North
Humberside, United Kingdom. Sunflower and cottonseed oils are available from
Cargil of
Minneapolis, Minnesota. Safflower oil is available from California Oils
Corporation of
Richmond, California.
In addition to these food grade oils, structured lipids may be incorporated
into the
nutritional if desired. Structured lipids are known in the art. A concise
description of
structured lipids can be found in INFORM, Vol.. 8, no. 10, page 1004, entitled
Structured
lipids allow fat tailoring (October 1997). Also see United States Patent No.
4,871,768.
Structured lipids are predominantly triacylglycerols containing mixtures of
medium and long
chain fatty acids on the same glycerol nucleus. Structured lipids and their
use in enteral
formula are also described in United States Patent Nos. 6,194,37 and
6,160,007.
Suitable protein sources include, but not limited to, milk, whey and whey
fractions,
soy, rice, meat (e.g., beef), animal and vegetable (e.g., pea, potato), egg
(egg albumin), gelatin
and fish. Suitable intact protein sources include, but are not limited to, soy
based, milk based,
casein protein, whey protein, rice protein, beef collagen, pea protein, potato
protein, and
mixtures thereof. Suitable protein hydrolysates include, but are not limited
to, soy protein
hydrolysate, casein protein hydrolysate, whey protein hydrolysate, rice
protein hydrolysate,
potato protein hydrolysate, fish protein hydrolysate, egg albumen hydrolysate,
gelatin protein
hydrolysate, a combination of animal and vegetable protein hydrolysates, and
mixtures thereof.
Hydrolyzed proteins (protein hydrolysates) are proteins that have been
hydrolyzed or broken
down into shorter peptide fragments and amino acids. Such hydrolyzed peptide
fragments and
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free amino acids are more easily digested. In the broadest sense, a protein
has been
hydrolyzed when one or more amide bonds have been broken. Breaking of amide
bonds may
occur unintentionally or incidentally during manufacture, for example due to
heating or shear.
For purposes of this disclosure, hydrolyzed protein means a protein that has
been processed or
treated in a manner intended to break amide bonds. Intentional hydrolysis may
be affected, for
example, by treating an intact protein with enzymes or acids. The hydrolyzed
proteins that are
preferably utilized in the liquid nutritional formulas described herein are
hydrolyzed to such an
extent that the ratio of amino nitrogen (AN) to total nitrogen ranges from
about 0.1 AN to
about 1.0 TN to about 0.4 AN to about 1.0 TN, preferably about 0.25 AN to 1.0
TN to about
0.4 AN to about 1.0 TN. (AN:TN ratios are given for the hydrolysate protein
alone and do not
represent the AN:TN ratios in the final nutritional formulas.)
Protein may also be provided in the form of free amino acids. The nutritional
formulas may be supplemented with various amino acids in order to provide a
more
nutritionally complete and balanced formula. Examples of suitable free amino
acids include,
but are-not limited to, all free L-amino acids usually considered a part of
the protein system,
but especially those considered essential or conditionally essential from a
nutritional
standpoint, namely: tryptophan, tyrosine, cyst(e)ine, methionine, arginine,
leucine, valine,
lysine, phenylalanine, isoleucine, threonine, and histidine. Other (non-
protein) amino acids
typically added to nutritional products include carnitine and taurine. In some
cases, the D-
forms of the amino acids are considered as nutritionally equivalent to the L-
forms, and isomer
mixtures are used to lower cost (for example, D,L-methionine).
The nutritional formulas preferably also contain vitamins and minerals in an
amount
designed to supply the daily nutritional requirements of the patient receiving
the formula.
Those skilled in the art recognize that nutritional formulas often need to be
over fortified with
certain vitamins and minerals to ensure that they meet the daily nutritional
requirements over
the shelf life of the product. These same individuals also recognize that
certain
microingredients may have potential benefits for people depending upon any
underlying
illness or disease that the patient is afflicted with. For example, diabetics
benefit from such
nutrients as chromium, carnitine, taurine and vitamin E. Formulas preferably
include, but are
not limited to, the following vitamins and minerals: calcium, phosphorus,
sodium, chloride,
magnesium, manganese, iron, copper, zinc, selenium, iodine, chromium,
molybdenum,
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conditionally essential nutrients m-inositol, carnitine and taurine, and
Vitamins A, C, D,
E, K and the B complex, and mixtures thereof.
If the liquid nutritional is intended for an infant, then specific nutritional
guidelines for
may be found in the Infant Formula Act, 21 U.S.C. section 350(a). The
nutritional guidelines
found in these statutes continue to be refined as further research concerning
nutritional
requirements is completed. The nutritional formulas claimed are intended to
encompass
formulas containing vitamins and minerals that may not currently be listed.
The liquid nutritional formulas also may contain fiber and stabilizers.
Suitable sources
of fiber/and or stabilizers include, but are not limited to, xanthan gum, guar
gum, gum arabic,
gum ghatti, gum karaya, gum tracacanth, agar, furcellaran, gellan gum, locust
bean gum,
pectin, low and high methoxy pectin, oat and barley glucans, carrageenans,
psyllium, gelatin,
microcyrstalline cellulose, CMC (sodium carboxymethylcellulose),
methylcellulose
hydroxypropyl methyl cellulose, hydroxypropyl cellulose, DATEM (diacetyl
tartaric acid
esters of mono- and diglycerides), dextran, carrageenans, FOS
(fructooligosaccharides), and
mixtures thereof. Numerous commercial sources of soluble dietary fibers are
available. For
example, gum arabic, hydrolyzed carboxymethylcellulose, guar gum, pectin and
the low and
high methoxy pectins are available from TIC Gums, Inc. of Belcamp, Maryland.
The oat and
barley glucans are available from Mountain Lake Specialty Ingredients, Inc. of
Omaha,
Nebraska. Psyllium is available from the Meer Corporation of North Bergen, New
Jersey
while the carrageenan is available from FMC Corporation of Philadelphia,
Pennsylvania.
The fiber incorporated may also be an insoluble dietary fiber representative
examples
of which include oat hull fiber, pea hull fiber, soy hull fiber, soy cotyledon
fiber, sugar beet
fiber, cellulose and corn bran. Numerous sources for the insoluble dietary
fibers are also
available. For example, the corn bran is available from Quaker Oats of
Chicago, Illinois; oat
hull fiber from Canadian Harvest of Cambridge, Minnesota; pea hull fiber from
Woodstone
Foods of Winnipeg, Canada; soy hull fiber and oat hull fiber from The Fibrad
Group of
LaVale, Maryland; soy cotyledon fiber from Protein Technologies International
of St. Louis,
Missouri; sugar beet fiber from Delta Fiber Foods of Minneapolis, Minnesota
and cellulose
from the James River Corp. of Saddle Brook, New Jersey.
A more detailed discussion of examples of fibers and their incorporation into
formula
may be found in United States Patent No. 5,085,883 issued to Garleb et al.
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The quantity of fiber utilized in the formulas can vary. The particular type
of fiber
that is utilized is not critical. Any fiber suitable for human consumption and
that is stable in
the matrix of a nutritional formula may be utilized.
In addition to fiber, the nutritionals may also contain oligosaccharides such
as
fructooligosaccharides (FOS) or glucooligosaccharides (GOS). -Oligosaccharides
are rapidly
and extensively fermented to short chain fatty acids by anaerobic
microorganisms that inhabit
the large bowel. These oligosaccharides are preferential energy sources for
most
Bifidobacterium species, but are not utilized by potentially pathogenic
organisms such as
Clostridium perfngens, C. di~cile, or E. coli.
The liquid nutritional formulas may also contain a flavor to enhance its
palatability.
Useful flavorings include, but are not limited to, chocolate, vanilla, coffee,
peach, butter pecan,
blueberry, banana; cherry, orange, grape, fruit punch, bubble gum, apple,
raspberry and
strawberry. Artificial sweeteners may be added to complement the flavor and
mask salty taste.
Useful artificial sweeteners include saccharin, nutrasweet, sucralose,
acesulfane-K (ace-K),
etc..
Liquid nutritional formulas can be manufactured using techniques well known to
those
skilled in the art. Various processing techniques exist. Typically these
techniques include
formation of a slurry from one or more solutions that may contain water and
one or more of
the following: carbohydrates, proteins, lipids, stabilizers, vitamins and
minerals. The slurry is
emulsified, homogenized and cooled. Various other solutions may be added to
the slurry
before processing, after processing or at both times. The processed formula is
then sterilized
and may be diluted to be utilized on a ready-to-feed basis or stored in a
concentrated liquid
form. When the resulting formula is meant to be a ready-to-feed liquid or
concentrated liquid,
an appropriate amount of water would be added before sterilization.
The present invention is also directed to a method of decreasing the
intestinal
mucosal inflammation of patients suffering from Celiac Disease by
administering NAQ
incorporated in the aqueous solutions and liquid nutritionals described above.
In Example 5,
the Inventors utilized the markers TUNEL, to monitor epithelial apoptosis, and
CD25, to
monitor sub-epithelial inflammation. They found compounds/products that
induced both or
one of the markers and considered those compounds/products toxic for the
mucosa of celiac
patients. N-acetyl-glutamine, however, had a clear trophic effect on the
biopsies of untreated
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celiac patients. This trophism was defined by a generalized improvement of the
mucosa in
particular of the epithelia, which were significantly ameliorated compared to
the other
samples. This unexpected aspect of the trophic activity of the N-acetyl-
glutaxnine appeared to
improve the overall condition of the mucosa even compared to the negative
control (medium
alone).
EXAMPLES
Method for Preparing Liquid Nutritional Formulas
Liquid nutritional formulas falling within the scope of the claims can be
prepared by
the following procedures. These examples are being presented as illustrations
and should not
be interpreted as limiting. Other carbohydrates, lipids, proteins,
stabilizers, vitamins and
minerals may be used without departing from the scope of the invention.
EXAMPLE 1
Method for Preparing Liquid Nutritional Formulas containing N-acetyl-L-
glutamine
A ready-to-feed liquid product was made containing N-acetyl-L-glutamine using
the
materials listed in Table 1. The procedure used to produce the product is
outlined below.
30
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TABLE 1: Bill of Materials for Vanilla Flavored Product
Ingredient Name ~ Amount
er 1000 kg)
Water to final mass
Maltodextrin 77.88 kg
Sucrose 52.80 kg
Soy Protein Hydrolysate 30.11 kg
Fish oil / Medium Chain Structured16.14 kg
lipid
sodium caseinate 14.74 kg
Fructooligosaccharide 5.792 kg
Canola oil 4.842 kg
Soybean oil 4.842 kg
45% Potassium Hydroxide 3.653 kg
Tri-calcium Phosphate 2.866 kg
N-Acetyl-L-glutamine 10.03 kg
L-Arginine 2.425 kg
Sodium citrate 2.293 kg
Artificial Carmel 1.500 kg
N&A Vanilla Flavor 1.000 kg
Emulsifier 1.076 kg
Magnesium phosphate 0.948 kg
Magnesium chloride 0.860 kg
Potassium citrate 0.838 kg
Ascorbic acid 0.697 kg
Choline chloride 0.474 kg
Gellan gum 0.250 kg
Vitamin D, E, K Premix 0.203 kg
Taurine w 0.139 kg
Carnitine 0.130 kg
Vitamin E (R, R, R) (81 %) 0.123 kg
Trace Mineral Premix 0.101 kg
Water Soluble Vitamin Premix 0.0882 kg
30% beta Carotene 15.5 grams
Vitamin A (55%) 5.07 grams
Potassium Iodide 0.194 grams
Sodium Selenite 0.132 grams
Vitamin K 0.0617 grams
1. The vitamin D, E, K premix includes vitamin D3 (0.0980 grams), d-alpha-
tocopheryl acetate (55.93
grams), and vitamin I~1 (0.0338 grams) in a coconut oil (146.77 grams)
carrier.
2. The trace mineral premix delivers (per 1000 kg Finished Product) zinc
sulfate (46.3 grams), ferrous sulfate
(39.2 grams), manganese sulfate (11.4 grams), copper sulfate (3.89 grams).
3. The water soluble vitamin premix includes niacinamide (33.07 grams), d-
calcium pantothenate (21.43
grams), folic acid (0.742 grams), thiamine chloride HCL (5.47 grams),
riboflavin (4.27 grams),
pyroxidine HCL (5.26 grams), cyanocobalamin (0.0147 grams) and biotin (0.644
grams) in a dextrose
(17.29 grams) carrier.
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PROCEDURE: The liquid nutritional product described above is manufactured
by preparing three slurries which are blended together, combined with the
marine oil/MCT
structured lipid, heat treated, standardized, packaged and sterilized. A
process for
manufacturing is described in detail below.
A carbohydrate/mineral slurry is prepared by first heating an appropriate
amount of
water to a temperature between about 65° C and about 71° C with
agitation. The required
amount of minerals are then added,in the order listed, under high agitation:
sodium citrate,
trace mineral premix, potassium citrate, magnesium chloride, magnesium
phosphate,
tricalcium phosphate and potassium iodide. Next, the required amount of
maltodextrin
(Maltrin~ M-100 distributed by Grain Processing Corporation of Muscatine,
Iowa) is added to
the slurry under high agitation, and is allowed to dissolve while the
temperature is maintained
at about 71° C. The required amount of sucrose and
Fructooligosaccharide (Nutriflora-P~
Fructo-oligosaccharide Powder distributed by Golden Technologies Company of
Golden,
Colorado) are then added under high agitation. The required amount of gellan
gum
(KelcogelC~ distributed by Kelco, Division of Merck and Company Incorporated
of San Diego,
California) is then dry blended with sucrose in a 1:5 (gellan gumlsucrose
ratio), and added to
the slurry under high agitation. Next, sodium selenite that has been dissolved
in warm water is
added to the slurry under agitation. The completed carbohydrate/mineral slurry
is held with
high agitation at a temperature between about 65 ° C and about 71
° C for not longer than
twelve hours until it is blended with the other slurries.
An oil blend is prepared by combining and heating the required amounts of
soybean oil
and canola oil to a temperature between about 55 ° C and about 65
° C with agitation. The
required amount of emulsifier, diacetyl tartaric acid esters of
monodiglycerides, (Panodan~
distributed by Grindsted Products Incorporated of New Century, Kansas) is then
added under
agitation and allowed to dissolve. The Vitamin D, E, K premix, 55% Vitamin A
Palmitate, D-
alpha-a-tocopherol acetate (R,R,R form), phylloquinone and 30% beta-carotene
are then added
with agitation. The completed oil blend is held under moderate agitation at a
temperature
between about 55 ° C and about 65 ° C for a period of no longer
than twelve hours until it is
blended with the other slurries.
A protein in water slurry is prepared by first heating an appropriate amount
of water to
a temperature between about 60 ° C and about 71 ° C with
agitation. Soy protein hydrolysate
24
CA 02501540 2005-04-07
WO 2004/032653 PCT/US2002/032172
(distributed by MD Foods of Viby J., Denmark ) is added with agitation. The
required
amount of N-acetyl-L-glutamine (obtained from Ajinomoto) is added with
agitation.
Potassium hydroxide solution (45%) is added to raise pH to about 5.6. L-
arginine is slowly
added, with agitation, and the solution stirred until clarified (pH > 6.2).
The required amount
of partially hydrolyzed sodium caseinate (Alanate~ 167 distributed by New
Zealand Milk
Products Incorporated of Santa Rosa, California) is then blended into the
slurry. This
completed protein-in-water slurry is held under moderate agitation at a
temperature between
about 60 ° C and about 71 ° C for a period of no longer than two
hours until it is blended with
the other slurries.
The protein-in-water slurry and oil blend are mixed with agitation and the
resultant
blended slurry is maintained at a temperature between about 55 ° C and
about 65 ° C. After
waiting for at least one minute, the carbohydrate/mineral slurry is added with
agitation and the
resultant blended slurry is maintained at a temperature between about 55
° C and about 65 ° C.
The marine oil/MCT structured lipid is then added to the blended slurry with
agitation.
Desirably, the marine oil/MCT structured lipid is slowly metered into the
product as the blend
passes through a conduit at a constant rate. After waiting for a period of not
less than one
minute nor greater than two hours, the blend slurry is subjected to
deaeration, ultra-high-
temperature treatment, and homogenization, using techniques known to one
skilled in the art.
The blend is then cooled to a temperature between about 1 ° C and about
7 ° C, stored at a
temperature between about 1 ° C and about 7 ° C with agitation.
Preferably, after the above
steps have been completed, appropriate analytical testing for quality control
is conducted.
Based on the analytical results of the quality control tests, an appropriate
amount of water is
added to the batch with agitation for final dilution (standardization).
The vitamin solution is prepared by heating a small amount of water to a
temperature
between about 43 ° C and about 66 ° C with agitation, and
thereafter adding the following
ingredients with agitation: ascorbic acid, 45% potassium hydroxide, taurine,
water soluble
vitamin premix, choline chloride, and L-camitine. The vitamin slurry is then
added to the
blended slurry under agitation.
A flavor solution is prepared by adding the natural and artificial vanilla
flavor and
artificial caramel flavor to an appropriate amount of water with agitation.
The flavor slurry is
then added to the blended slurry under agitation.
CA 02501540 2005-04-07
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The product pH may be adjusted to achieve optimal product stability. The
completed product is then placed in suitable containers (in this case, 8 oz.
metal cans) and
subjected to terminal sterilization (in this case, retort sterilization).
EXAMPLE 2
Aqueous N-acetyl-L-glutamine Stability Studies
Studies were conducted to assess the stability of aqueous N-acetyl-glutamine
upon
heating, at various pH values, and in a matrix similar to that found in a
liquid nutritional type
product.
Aqueous N-acetyl-L-glutamine and Glutamine Heat Stability
In order to test the stability of aqueous N-acetyl-glutamine upon heating, the
following
procedure was followed. Aqueous solutions of N-acetyl-L-glutamine (obtained
from Sigma,
catalog no. A-9125) and glutamine (obtained from Aldrich, catalog no. G-320-2)
at
approximately 1 mg/mL (5.3 mM and 6.8 mM, respectively) were prepared without
pH
adjustment. The pH of the resulting N-acetyl-L-glutamine solution was 2.9 and
the pH of the
glutamine solution was 6Ø The solutions were heated at 100°C using a
Reacti-ThermTM
stirring heat block with sealed 4 mL vessels, one for each time point: 15
minutes, 30 minutes,
1 hour and 2 hours. The samples were removed from the heat block and
immediately placed
into ice until cool. An aliquot of each sample was filtered through 0.45
micrometer filters
(Millipore Millex~-HV, 25 mm) for assessment by HPLC.
HPLC analysis was conducted using an Inertsil~ C8, 5 micrometer, 4.6 x 250 mm
column (obtained from Keystone Scientific, Inc., Bellefonte, PA). The mobile
phase was
water adjusted to pH 2.2 with HCl (isocratic at 1 mL/minute). The injection
volume was 10
microliters. Ultraviolet detection was at 214 nm.
Results are provided in Table 2. Glutamine was not stable during the 2 hour
incubation
at 100 °C. The major degradation product after boiling the pH 6.0
glutamine solution for 1
hour was pyroglutamic acid. After boiling the glutamine solution for 2 hours,
pyroglutamic
acid was still the major degradation product, but glutamic acid was also
detected.
N-acetyl-L-glutamine was much more stable than glutamine. The major
degradation
product was tentatively identified by retention time as N-acetyl-glutamic
acid; this
identification was confirmed by mass spectrometry (MS) and nuclear magnetic
resonance
spectrometry (NMR). The second largest peak, as identified by MS and NMR, was
2, 6-
26
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dioxopiperidinylacetamide. In the N-acetyl-L-glutamine solutions, pyroglutamic
acid
was detected only in the 2 hour sample, and only at the very low level of 0.2
area percent.
TABLE 2: Aqueous Solution of Glutamine and N-acetyl-L-glutamine heated at 100
°C
Glutamine N-acetyl-L-glutamine
solution solution
(height (area
%) %)
Time GLNi GLU' PGA' NAQ'' 2,6-DPA' PGA' NAE"
(min.)
0 100.0 -- none detected99.7 -- -- 0.3
30 90.2 -- 9.8 98.1 0.6 -- 1.2
60 80.0 -- 20.0 96.9 1.3 -- 1.8
120 53.6 10.6 35.8 93.4 2.7 0.2 3.7
1 glutamine, Z glutamate, 3 pyroglutamic acid, 4 N-acetyl-L-glutamine,' 2,6-
dioxopiperidinylacetamide
6 N-acetyl-L-glutamic acid
Aqueous N-acetyl-L-glutamine and Glutamine Stability at Various pH Values
In order to test the stability of N-acetyl-L-glutamine in aqueous solutions at
various pH
values, the following procedure was followed. Aqueous solutions of N-acetyl-L-
glutamine
were prepared in 1 pH unit increments from pH 2.0 to 8Ø The pH of the
solutions was
adjusted with either hydrochloric acid or sodium hydroxide, as needed, just
prior to final
dilution (final concentration = 1 mg/mL or 5.3 mM N-acetyl-L-glutamine). A
single solution
of glutamine was not pH adjusted (measured pH-.= 6.0) and was prepared at 1
mg/mL or 6.8
mM glutamine. All solutions were sterile-filtered (Millipore MillexC~-GS, 25
mm, 0.22
micrometer pore size, sterile) into autosampler vials and capped for storage
at ambient
temperature (17-25 °C). N-acetyl-L-glutamine samples were assessed by
HPLC at various
time points, from 1 to 180 days. The glutamine sample was assessed by HPLC at
similar time
points, from 1 to 45 days.
The stability of N-acetyl-L-glutamine was found to be pH dependent. Results
are
reported in Figures 1 and 2. At all pH values, N-acetyl-L-glutamine showed no
degradation
through 7 days. At pH 5.0 to 8.0, N-acetyl-L-glutamine was stable over 6
months; greater than
99.6% of N-acetyl-L-glutamine remained. The only consistently detected
degradation product
was N-acetyl-glutamic acid at less than 0.5% through six months. At pH 4.0, by
six months,
each of N-acetyl-glutamic acid and 2, 6-dioxopiperidinylacetamide was detected
with 97.9%
N-acetyl-L-glutamine remaining. At pH 3.0, N-acetyl-L-glutamine remained at >
95%
27
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WO 2004/032653 PCT/US2002/032172
through 90 days, dropping to 94.2% at 4 months and 90.4% at 6 months. N-acetyl-
glutamic acid and 2,6-dioxopiperidinylacetamide were detected at approximately
equal levels
in the pH 3.0 samples starting at about 0.15% at 15 days, increasing to about
1% at 30 days
and about 5% at 6 months. At 6 months, pyroglutamic acid was detected at 0.5%.
At pH 2.0,
N-acetyl-L-glutamine~was 97.0% at 15 days, but decreased to only 55.7% at 6
months. N-
acetyl-glutamic acid was the major degradation product in the pH 2.0 sample,
at 2.5% in the
day sample and 37.2% in the,6 months sample. 2, 6-dioxopiperidinyl acetamide
increased
from 0.5% at 15 days to 4.9% at 6 months. The pH 2.0 N-acetyl-L-glutamine
sample was the
only sample that showed increasing values for pyroglutamic acid: 0.2% at 30
days to 2.2% at
10 6 months.
In the glutamine solution (pH 6.0), pyroglutamic acid was found in the sample
after 3
days at room temperature at 0.2%. After 45 days, it was found at 3.3% and
glutamine was at
96.7%. Results from HPLC analysis are reported as height percent in Table 3.
15 TABLE 3: Stability of Glutamine in pH 6.0 Aqueous Solution At 1 mg/mL and
Ambient Temperature
Analyte 2 days 3 days 7 days 15 days 30 days 45 days
GLN' 100.0 99.8 99.5 99.0 98.1 96.7
PGA' none detected0.2 0.5 1.0 1.9 3.3
glutamine, Z pyroglutamic acid
N-acetyl-L-glutamine and Glutamine Stability in Liquid Nutritional Type
Products
In order to test the stability of N-acetyl-L-glutamine in a matrix similar to
that found in
liquid nutritional type products, the following procedure was followed. Three
study products
were formulated, one containing N-acetyl-L-glutamine ( N-acetyl-L-glutamine
was obtained
from Ajinomoto), one containing glutamine (obtained from Ajinomoto) (at
theoretical
concentrations of 16.5 mg/mL and 12.8 mg/mL, respectively, and replacing part
of the protein
on a weight basis), and a control (Optimental~, Ross Products Division, Abbott
Laboratories).
The product containing N-acetyl-L-glutamine was made according to the
procedure set forth
above in Example 1. The product containing glutamine was made in a similar
manner, except
glutamine (7.79 kg) was substituted for N-acetyl-L-glutamine. The products
were assessed for
degradation before and after a retort sterilization process, which is typical
for liquid nutritional
28
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WO 2004/032653 PCT/US2002/032172
processing (here, 128 °C for 5 minutes). The products were stored at
room temperature
(20-22 °C) and assessed for evidence of degradation at 1, 2 and 3
months. Glutamine, N-
acetyl-L-glutamine and pyroglutamic acid (if present) were quantified at each
process and time
point.
In order to analyze by HPLC for glutamine, N-acetyl-L-glutamine and
pyroglutamic
acid, samples were filtered as follows. A 5.0 mL aliquot was transferred to a
50 mL
volumetric flask. Twenty drops of 1 M hydrochloric acid was added and the
sample was
diluted to volume with deionized water. An aliquot was filtered through a 0.45
micron filter
(Millipore, Millex~-HV, 25 mm). The samples were analyzed by HPLC as described
above
(Heat Stability section).
The total amount of pyroglutamic acid present in the protein formula,
including both
free pyroglutamic acid and N-terminal pyroglutamic acid, can be determined by
the following
method. Initially, samples were prepared as a water solution to a
concentration of
approximately 18 g total protein/L. A 20 microliter aliquot of the prepared
sample material
was placed in a 1.5 mL screw cap vial, and 980 microliters of a freshly
prepared enzyme
solution (0.05 M Tris, 0.005 M dithiothreitol, 0.001 M disodium
ethylenediaminetetraacetic
acid (EDTA), pH 8.0, containing 11 units of pyroglutamate aminopeptidase/mL)
was added.
The vial was tightly capped, and incubated at room temperature (21-24
°C) for 24 hours. The
solution was then processed through a C-18 SPE cartridge as detailed below.
For free
pyroglutamic acid determination, the initial sample solution was diluted to a
total protein
content of 2-3 g/L in deionized water, and processed through a C-18 SPE
cartridge.
C-18 SPE (Solid Phase Extraction) cartridges (100 mg/1mL size) were obtained
from
Burdick & Jackson, Muskegon, MI. SPE cartridges were prepared for use with 2 x
5 volumes
of methanol, and then rinsed with 2 x 5 volumes of deionized water. The 1 mL
sample is then
slowly applied, and flow-through material collected in a 1 gram screw cap
vial. Elution was
completed by applying 2 x 500 microliters of deionized water, collecting pass
through volume
in the same vial. The eluate was mixed, and then an aliquot filtered through a
0.45 micrometer
filter prior to HPLC analysis (25 mm, 0.45 micrometer filters were obtained
from Gelman,
Ann Arbor, MI). The HPLC system used had the following parameters: pump model
G1312A, autosampler model ~G1313A, thermostatted column compartment model
G1316A,
diode array detector model G1315A, and peak integrator/data processor model
G2170AA, all
29
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WO 2004/032653 PCT/US2002/032172
obtained from Agilent Technologies, Palo Alto, CA. Column: 6.5 x 150 mm ION-
310, 8
micrometer from Interaction Chromatography, San Jose, CA. The system was pre-
equilibrated
in mobile phase (5 mN HZSOø) at 40 °C at 0.3 mL/min. prior to use.
For analysis a 10 microliter aliquot of sample or standard was injected, and
the column
was eluted with mobile phase at 0.3 mL/min. and 40 °C. Eluting
materials were detected by
UV absorption at 210 nm and 220 nm. The run time was 45 min.
Unknown sample concentrations were determined by comparison to standards.
Three
aqueous solutions of pyroglutamic acid are usually sufficient as standards,
i.e., 10, 20, and 40
mg/L (pyroglutamic acid obtained from Fluka, Milwaukee, WI).
N-acetyl-L-glutamine in the liquid nutritional type product showed no
degradation
during sterilization or after 3 months room temperature storage. Results are
reported in Table
4. A small peak corresponding to N-acetyl-glutamic acid was detected at all
time points, but
remained at approximately the same level indicating no measurable degradation
to N-acetyl-
glutamic acid.
In the glutamine supplemented product, glutamine was reduced to about 1/3 the
original concentration by the sterilization process; and by 2 months no
glutamine was detected.
In this product, pyroglutamic acid was detected at a concentration consistent
with complete
conversion of glutamine.
TABLE 4.: Comparison of Stability of N-acetyl-L-glutamine and glutamine in
Liquid
Nutritional Type Products During Processing and over 3 Months of Storage at
Room
Temperature.
Product with Product with
N-acetyl-L-glutamineglutamine
Analyte N-acetyl-L-glutamineGlutamine pyroglutamic acid
(mmol/L product)(mmol/L product)(mmol/L product)
Theoretical 87.7 87.6 --
Pre-Sterilization92.5 97.8 27.1 *
Post-Sterilization89.8 34.2 77.5
1 month 89.8 13.7 92.9*
2 months 94.1 trace 79. 8
3 months 89.3 none detected 80.6**
* calculated with response factor from glutamine standard. Proper standard was
not available until later in the
experiment.
** calculated with response factor from pyroglutamic acid standard.
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EXAMPLE 3
Glutamine and N-Acetyl-L-Glutamine Bioavailability
Studies were conducted to determine the proportion of bioavailable N-acetyl-L-
glutamine in comparison to glutamine in pig models. The intestinal loop model
employs a
section of isolated intestine to evaluate the absorption and metabolism of N-
acetyl-L-
glutamine and glutamine. The feeding model evaluated the absorption of N-
acetyl-L-
glutamine and glutamine when fed in a typical diet.
Intestinal Loop Model
Twenty-two domestic pigs weighing 15-20 kg were acclimated to lab conditions
over 4 days.
The pigs were fed a standard pig diet, which followed energetic requirements
for these animals
(Nutrient Requiremev~ts of Swif~e, 9th, 1998, Subcommittee on Swine Nutrition,
National
Research Council) and water ad libitum. Animals were randomly assigned into
group C [6
pigs, receiving a glucosaline solution (Braun cat No 622647), 5% glucose, 0.9%
NaCI], group
G [8 pigs, receiving the same glucosaline solution fortified with 8 g/1 of
Gln, (Sigma cat No G-
3126)], and group N [8 pigs, receiving the same glucosaline solution fortified
with 10 g/1 of
NAQ, (Sigma cat No A-9125)]. Before surgery, animals were fasted 15 h. The day
of
experiment, animals were weighed and anaesthetized using Stresnil~ and
penthotal. The
anaesthetized pigs were opened by abdominal medium sagital incision.
Approximately 1
meter of proximal jejunum, about 1 meter from the ligament of Treitz, was,
after clamping
both ends and inserting a proximal fistual, filled with 125 mL of study
solution at 50-75
mL/min. Intestinal infused solution samples were taken by puncture of infused
intestine at 0,
15, 30, 60, 90, 120, 150 and 180 minutes. Samples were frozen in liquid
nitrogen and
maintained at -80 °C until analysis. Portal vein blood samples were
taken by portal vein
puncture at 0, 15, 30, 60, 90, 120, 150 and 180 minutes in tubes with
anticoagulant. Samples
were maintained at 4 °C until centrifugation at 1500 x g for 15 minutes
for plasma and red
blood cell separation. Plasma was frozen at -20 °C until analysis.
Jugular vein blood samples
were taken by puncture at 0, 60, 120 and 180 minutes in tubes with
anticoagulant and plasma
obtained and stored as for portal blood vein. After 3 hours, pigs were
sacrificed and mucosa
samples were obtained from 25 cm of infused intestine segment. The segment was
rinsed
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WO 2004/032653 PCT/US2002/032172
thoroughly with ice-cold saline solution, opened lengthwise and blotted dry.
Mucosa
were removed by scraping the entire luminal surface with a glass coverslip,
then frozen in
liquid nitrogen and~stored at -80 °C.
The analysis for N-acetyl-L-glutamine was conducted as follows. For intestinal
infused solution samples and plasma samples, aliquots were diluted 1:10 (w/v)
with 0.05%
perchloroacetic acid (PCA) solution in water. For mucosa samples, 0.2 mg of
wet mucosa
sample was homogenized with 5 mL of 0.05% PCA solution in water. After
centrifugation
(15,000 x g, 3 minutes, ambient temperature), samples were filtered through
0.45 micrometer
filter and injected into an HPLC chromatographic system consisting of a 2690
Separation
Module, PDA detector and a LichroCart 250-4 cartridge (Purospher RP18 e, 250 x
4 mm, 5
micrometers). The mobile phase consisted of a phosphate buffer 0.1 M at pH
2.7, at a flow
rate of 1 mL/minute. The detection and quantification of N-acetyl-L-glutamine
was monitored
at 210 nm.
The analysis for glutamine and glutamate was conducted as follows. Intestinal
infused
solution samples and plasma samples were prepared as for N-acetyl-L-glutamine
analysis
(described above) with the exception that samples were diluted 1:400 (w/v)
with 0.05% PCA
solution in water. After samples were filtered through 0.45 micrometer filter.
20 microliters
of the mixture was derivatized following the AccQ-Tag method (Waters Corp.),
and diluted to
1 mL with water. Briefly, the sample was buffered with a borate solution and
derivatized with
20 microliters of reactive. After 1 minute the sample was diluted to 1 mL and
injected into the
HPLC system, consisting of a 2690 Separation Module, fluorescence detector and
a SupelcoSil
LC-18 column (250 x 4 mm, 3 micrometers). Mobile phase consisted of a
phosphonate buffer
0.1 M at pH 7.5, with 0.25% triethylamine and 9% acetonitrile, at a flow rate
of 1 mLlminute.
The detection and quantification of glutamate and glutamine was accomplished
using an
excitation wavelength of 250 nm and monitoring emission at 395 nm.
Glucose was analyzed using a well-established coupled enzyme assay. Briefly,
sample
glucose is phosphorylated using hexokinase and ATP (adenosine triphosphate),
and the
resulting glucose-6-phosphate is converted to 6-phosphogluconate using glucose-
6-phosphate
dehydrogenase. During the later reaction, NAD (nicotinamide adenine
dinucleotide) is
converted to NADH (the reduced form of NAD), resulting in increased absorbance
at 340 nm,
which is proportional to~the glucose concentration in the original sample.
This assay can be
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CA 02501540 2005-04-07
WO 2004/032653 PCT/US2002/032172
purchased as a clinical chemistry kit from Sigma Chemical Company, St. Louis,
MO,
(current catalog number 16-20).
Results
Glutamine or N-acetyl-L-glutamine remaining in the intestinal lumen versus
time after
introduction of the infused solution. The remaining percentage of glutamine or
N-acetyl-L-
glutamine in intestinal contents of pigs infused with solutions containing
equivalent amounts
of glutamine or N-acetyl-L-glutamine was similar during the first 90 minutes.
There were
statistically significant differences between groups at 120 and 180 minutes.
There were no
significant differences between glutamine or N-acetyl-L-glutamine at tli2
(approximately 45
minutes). Figure 3 illustrates graphically the amount of analyte (glutamine or
N-acetyl-L-
glutamine) remaining in the intestinal lumen versus time after introduction of
the analyte. The
analyte remaining is expressed as a percentage of the analyte present at time
zero.
Glucose remaining in the intestinal lumen versus time after introduction of
the
infused solution There were no significant differences between C and G groups
at any time.
There were no significant differences between the C and N except at 15-
minutes. G and N
groups tended to be different from time 120 minutes, although penalizing by
the Bonferroni's
correction the only significant difference was at 180 minutes. Figure 4
illustrates graphically
the amount of glucose remaining in the intestinal lumen versus time after
introduction of the
solutions. Glucose remaining is expressed as a percentage of the amount
present at time zero.
Glutamine in portal blood after introduction of the test solution into the
intestinal
loop. When results were expressed as percentages of the initial concentration,
there were
significant differences between control (C) and glutamine (G) and between C
and N-acetyl-L-
glutamine (N) groups (at 90 and 150 minutes, C vs. G; and at 90, 120, 150 and
180 minutes, C
vs. N). There were no significant differences between G and N. When results
were expressed
as absolute values, there were no significant differences between groups
except at 120
minutes, between C and N. Taken together, G and N tend to be different from C
from 120
minutes to the end of the experiment. Figure 5 illustrates graphically the
amount of glutamine
in the portal blood (in mcg/mL) versus time after introduction of the test
solution into the
intestinal loop.
There were no significant differences between groups for glucose in portal
blood and
between groups for glutamine or glucose in peripheral blood. There were only
negligible (parts
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WO 2004/032653 PCT/US2002/032172
- per - million) levels of intact N-acetyl-L-glutamine detected in either
portal or
peripheral blood at any time point during the experiment.
Glutamic Acid (GLU) and Glutamine (GLN) in jejunum mucosa
There were higher glutamate concentrations in groups N and C than in group G,
and,
while both N and G groups showed higher glutamine in the mucosa, group G was
substantially
higher than group N. However, the sum glutamine + glutamate concentration were
similar in
groups G and N, suggesting that delivery of glutamine carbon skeleton to
mucosal metabolic
systems is comparable using these two diets. Intact N-acetyl-L-glutamine could
not be
detected in mucosa samples. Figure 6 illustrates graphically the amount of
glutamine and
glutamate (and their sum) in the jejunum mucosa immediately following
completion of the
experiment (expressed in mcg/gram wet mucosa).
In summary, N-acetyl-L-glutamine shows a similar bioavailability to glucose
and very
slightly lower than glutamine. N-acetyl-L-glutamine seems to be very similax
to glutamine in
utilization after absorption. After being absorbed, N-acetyl-L-glutamine is
quickly hydrolyzed
by enterocyte acylase, entering in the normal glutamine metabolism, and
achieving glutamine
+ glutamate concentration in mucosa as high as that achieved by an equivalent
glutamine diet.
Excess glutamine is excreted to the portal vein, where glutamine concentration
is similar to
that found after an equivalent dose of dietaxy glutamine. N-acetyl-L-glutamine
concentration
in portal vein plasma is only a few ppm, suggesting minimal intact absorption
to the
bloodstream. The high rate of absorption of N-acetyl-L-glutamine as well as a
similar
metabolism to glutamine suggested that both nutrients could have the same
biological behavior
under catabolic stages of the organism.
Feeding Pig Model
Fifteen pigs, 15-20 kg in weight were provided by a certified farm. The pigs
were
acclimated to the laboratory for 2 days. A standard pig diet and water was
provided ad
libitum. After acclimation, the pigs were randomly assigned into group C [5
pigs, receiving a
standard pig diet plus 3 g/kg of Cr203, (Merck cat No 1.02483)], group G [5
pigs, receiving
diet C plus 8 g/kg of Gln, (Ajimoto)], and group N [5 pigs, receiving diet C
plus 10.5 g/kg of
N-acetyl-L-glutamine, (Flamma)]. During the experimental phase of the study,
each group
received 1000 grams of their respective diet per day per animal, fed in 3
portions and water
was provided ad libitum. This experimental phase of feeding lasted 5 days.
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WO 2004/032653 PCT/US2002/032172
On the day of experiment, animals were weighed and received the standard diet
intake (333 g diet per animal) at 7:00 a.m. Three hours after feeding, animals
were weighed,
sedated and bled through jugular vein puncture. Animals were quickly opened by
abdominal
medium sagital incision and the content of the duodenum, medium jejunum (about
2 meters
from the ligament of Treitz) and ileum (30 cm from the ileocecal valve) were
taken, frozen in
liquid nitrogen, lyophylized, and stored at -80 °C until analysis.
Samples of liver and kidney
were removed, dissected of visible fat and connective tissue, quickly frozen
in liquid nitrogen
and stored at -80 °C until analysis. Samples of intestinal mucosa were
obtained as described
for the isolated intestinal loop experiment, and stored as described above
prior to analysis.
Intestinal content was analyzed for glutamine, N-acetyl-L-glutamine and
chromium
(III) oxide. For analysis of N-acetyl-L-glutamine, the lyophilized samples of
intestinal content
were dissolved 1:20 (w/v) with 0.05% PCA in water followed by HPLC analysis ~s
described
in the Intestinal Loop model above.
For analysis of glutamine, the lyophilized intestinal content was treated and
analyzed
as described in the intestinal loop model above.
Chromium was incorporated into the diets to provide a correction factor to
reflect
content per kg of original diet. For analysis of chromium (III) oxide the
following procedure
was utilized. A representative lyophilized intestinal content sample was
weighed into a nickel
crucible and placed in a muffle furnace. Temperature was raised to 500
°C and maintained for
a further 2 hours. After cooling, a fusion rilixture (Na2CO3 K2C03 KNO3,
10:10:4 w/w/w) was
added at about ten times the weight of sample ash and mixed thoroughly. An
extra amount of
fusion mixture was added to form a thin layer on top and fused for 30 minutes
over an open
flame using a gas burner until a clear melt was obtained. The crucible was
removed from the
burner, allowed to cool, and the melt was extracted thoroughly by washing the
walls with
about 20 mL of water and then heated gently on the hot plate for about 30
minutes. When the
crust was thoroughly loosened, the crucible was rinsed four times with water,
and all washings
were added to a 100 mL volumetric flask water, and diluted to volume. The
absorbance at 372
nm against demineralized water as a blank was determined. The absorbance
readings were
converted to mg of Cr203 by employing the equation of a standard curve
prepared by
analyzing 0, 50, 100, 200 and 500 microliters of a standard chromium solution
(2.9034 g of
K2Cr207/L, which is equivalent to 1.5 g/L of Cr203~.
CA 02501540 2005-04-07
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Analysis for acylase was conducted according to the following procedure. 200
mg
of wet mucosa, liver or kidney was homogenized into 5 mL of cold water and
centrifuged at
400 x g for 5 minutes at 5 °C. 100 microliters of an N-acetyl-L-
glutamine solution (5 g/L,
sigma catalog no. A-9125), were mixed with 100 microliters of mucosa
homogenate and
incubated during .1 hour at 37 °C. A blank was done using 100
microliters of mucosa and 100
microliters of water. An enzyme calibration curve was constructed (acylase I,
E.C. 3.5.1.14,
Sigma catalog no. 8376), using from 0.5 IU acylase /mL to 100 IU acylase/mL,
and incubating
with N-acetyl-L-glutamine as above. Free glutamine (released by enzyme
activity) was
determined as described the intestinal loop model above. For each sample, the
acylase activity
was determined by comparison to the standard response curve for the enzyme,
and the value
corrected by appropriate dilution factors.
Results
Absorption data are presented in Table 5 below. Samples from the duodenum
contained insufficient levels of chromium (II) oxide to allow quantitation.
The analytical
results could not be corrected to reflect content per kg of original diet. The
medial jejunum
contained essentially identical levels of glutamine (in the case of diet G)
and N-acetyl-L-
glutamine (in the case of diet N), suggesting similar adsorption in the
duodenum and proximal
jejunum. However, these diets also contained intact protein, and digestion of
that protein
could also produce significant free glutamine, as indicated by the analysis
result for the control
diet. This suggests that the free glutamine content of the original diet is
almost completely
absorbed prior to the medial jejunum. Analysis of the contents of the distal
ilium suggest that,
while absorption of free glutamine can continue between the medial jejunum and
the distal
ilium, absorption of N-acetyl-glutamine is not observed. However, overall
absorption data
indicate absorption of approximately 77% of the high level of administered N-
acetyl-L-
glutamine in this model.
TABLE 5: Adsorption of N-acetyl-L-glutamine and Glutamine as a Component of
Diet in
Pigs.
Glutamine DietN-acetyl-L-glutamine Control Diet
Diet (C)
Duodenum N/D * * N/D NlD
Medium Jejunum10.1 + 1.9 10.3 + 2.4 8.8 0.7
Distal Ileum 1.2 + 0.6 12.8 + 2.1 2.1 + 0.7
36
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WO 2004/032653 PCT/US2002/032172
* For Glutamine and Control diets, data are glutamine (mmole/kg original
diet). For N-acetyl-L-glutamine
diet, data are for N-acetyl-L-glutamine (mmole / kg original diet). Original
diets are (Glutarnine = 54.8
mmole glutamine / kg diet, N-acetyl-L-glutamine = 55.8 mmole N-acetyl-L-
glutamine/kg diet).
* * N/D = Not determined. Chromium (II) oxide values were below quantitation
limits for the assay, and corrected
values could not be generated.
Acylase activity in intestinal mucosa, liver and kidney - Acylase activity was
measured
in several tissues of interest (in view of likely nutritional importance) in
the control pigs.
Acylase activity was found in all tissues tested, including jejunal mucosa,
liver and kidney.
Levels determined were 948 + 300 IU/g wet tissue (17.3 + 7.0 IU/mg protein) in
the jejunal
mucosa, 12,770 + 1110 IU/g wet tissue (159 + 30 IU/mg protein) in liver and
19,630 + 3020
IU/g wet tissue (302 + 47 IU/mg protein) in the kidney.
In summary, N-acetyl-L-glutamine was absorbed mainly in the duodenum and upper-
jejunum, where at least 77% of the dose was adsorbed. There were two main
differences
between N-acetyl-L-glutamine and glutamine: an earlier N-acetyl-L-glutamine
uptake
saturation and a lower ileal absorption.
EXAMPLE 4
Effects of N-Acetyl-L-Glutamine on Intestinal Damage Caused by Malnutrition
A study was conducted to evaluate the potential effects of N-acetyl-L-
glutamine versus
free glutamine on intestinal damage caused by protein-energy malnutrition in
pigs. In this
study, 5-week-old domestic pigs, were provided by a certified farm. The pigs
were randomly
assigned to one of two groups. In one group 3 pigs were freely fed with ENSURE
PLUS~
(Ross Products Division, Abbott Laboratories) for 30 days. In the second
group, 9 pigs were
also fed with ENSURE PLUS, but at only 20% of the daily intake of the first
group. This
second group was divided into 3 subgroups with six pigs each to receive a
daily supplement of
either calcium caseinate, glutamine or N-acetyl-L-glutamine. Daily average
energy and
protein supplied to the control group ranged from 3300 kcal, 138 g protein at
the beginning of
the study to 4500 kcal, 187 g protein at the end of the study. In the second
group,
supplements of caseinate, glutamine and N-acetyl-L-glutamine provided an
additional 1.32
grams nitrogen equivalents per day (basically, 6.89 grams L-glutamine, or 8.87
grams N-
acetyl-L-glutamine or 8.42 grams caseinate protein are supplemented per day).
After 30 days,
all pigs were deprived of food for 16 hours. The animals were then weighed,
sedated,
anesthetized and sacrificed through terminal bleeding by jugular puncture.
37
CA 02501540 2005-04-07
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The entire small intestine was quickly removed. The 60cm segment of the small
intestine from the ligament of Treitz was considered proximal jejunum. A Scm
long segment
from the ligament of Treitz was selected for histological analysis of jejunum.
The 60 cm
length closest to the ileo-cecal valve was considered the. distal ileum. A 5
cm segment from the
ileo-cecal valve was selected for histological analysis of the ileum. The
intestine segments
were rinsed thoroughly with ice-cold saline solution, opened length-wise and
blotted dry. The
mucosa was scraped off using a glass slide onto a cold Petri dish, weighed,
immediately frozen
under liquid nitrogen and stored at -80 °C until biochemical analysis.
Jejunal and ileal mucosa were homogenized in 10 mM phosphate buffer (pH 7.4)
using
a mechanical Potter homogenizer, for protein and DNA assays. For the
determination of the
enzymatic markers of injury, functionality and antioxidant defense system, the
mucosal
homogenates were centrifuged at 3000 g for 10 min. and the resulting
supernatants were used
for enzymatic assays. For the determination of total glutathione, the mucosa
was homogenized
in 5% trichloroacetic acid and centrifuged at 8000 g for 5 min.
Biochemical analysis and immunological analysis were performed on the
specimens.
Concentrations of intestinal mucosa protein and DNA were determined using the
Bradford
method (Analytical Biochemistry, Volume 72, pages 248 - 254, 1976) and the
method of
Labarca and Paigen (Analytical Biochemistry, Volume 102 (2), pages 344 - 352,
1980),
respectively. The degree of intestinal damage caused by malnutrition was
evaluated by
measuring alkaline phosphatase activity using the method of Goldstein (R.
Goldstein, T. Klein,
S. Freier and J. Menczel. American Journal of Clinical Nutrition 24: 1224 -
1231, 1970).
The defensive system against oxidative damage was evaluated by measuring the
activities of glutathione reductase (GR), glutathione transferase (GT) and
glutathione
peroxidase (GPOX) as well as by the concentration of the non-protein
sulfhydryl groups
(mostly reduced glutathione (GSH)). Glutathione reductase activity was
evaluated by the
method of Carlberg and Mannervik (I. Carlberg and B. Mannervik, Methods in
Enzymology,
Volume 113, pp 484-490, 1985). Glutathione transferase activity was measured
using the
method of Habig, et al. (W.H. Habig, M.J. Pabst and W.B. Jakoby, Journal of
Biological
Chemistry. 294: 7130 - 7139, 1984). Glutathione peroxidase activity was
assayed by the
method of Flohe and Gunzler (L. Flohe and W.A. Gunzler, Methods in Enzymology,
Volume
105, pp 114-121, 1984), and the non-protein sulfhydryl content (reported as
reduced
38
CA 02501540 2005-04-07
WO 2004/032653 PCT/US2002/032172
glutathione equivalents) was determined by the method of Anderson (M. E.
Anderson,
Methods in Enzymology, 133: 548 - 554, 1985).
Intestinal lymphocytes were isolated following the procedure of Gautreaux, et
al.
(M.D. Gautreaux, E.A. Deitch and R.D. Berg, Infection and Immunity 62(7): 2874
- 2884, .
1994) modified as detailed below. Two small intestine segments from jejunum
and from
ileum respectively, were isolated and the luminal content was flushed with
phosphate-buffer
saline (PBS, Sigma St. Louis, MO, USA). The visible Peyer's patches were
excised, and the
intestine was opened longitudinally and cut into small pieces. To isolate the
small intestinal
epithelium those pieces were incubated for 30 min at 37 °C in 25 ml of
Hanks Balanced Salt
Solution (HBSS; Sigma, St. Louis, MO, USA) with 5 mM dithiotreitol (DTT; Roche
Molecular Biochemicals, Indianapolis, IN, USA), 2mM EDTA (Sigma, St. Louis,
MO, USA)
and 25 mM Tris buffer (Sigma, St. Louis, Mo, USA) in a shaking water bath (120
strokes per
min); the supernatant was decanted, fresh HBSS-DTT-EDTA-Tris was added, and
the
incubation procedure was repeated. The supernatants containing the epithelial
cells from two
incubations were pooled, and the cells were washed by centrifugation with rpmi
1640 culture
medium containing 5% (v/v) heat-inactivated fetal calf serum (Sigma, St.
Louis, MO, USA),
mM HEPES (Sigma, St. Louis, MO, USA), 2 mM L-glutamine, 500 U penicillin and
100
~,g/ml streptomycin (Sigma, St. Louis, MO, USA)(complete medium). Lamina
propria
lymphocytes (LPL) were liberated from the remaining sediment by placing the
intestinal
20 debris in 40 ml of complete medium with collagenase 0.05 U/ml, dispase 0.30
U/ml (Sigma,
St. Louis, MO, USA) and DNase I 500 U/ml (Roche Molecular Biochemicals,
Indianapolis,
IN, USA) for 120 min in a 37°C shaking water bath at 120 strokes per
min. The excised
Peyer's patches were placed in complete medium and dissected with a couple of
scalpels. The
cleaned Peyer's patches were then collagenase treated (reduced incubation time
to 60 min.) as
described above for LPL isolation to liberate Peyer's patch lymphocytes (PPL).
Each of the cell types isolated from the epithelium, the lamina propria and
Peyer's
patches were subjected to discontinuous PercollTM (Sigma, St. Louis, MO, USA)
density
gradient centrifugation to enrich for lymphocytes. The commercial PercollTM
solution was
diluted 9:10 with 9% NaCI yielding an isotonic PercollTM solution that was
diluted with
complete medium to obtain 3 solutions differing in percent PercollTM
concentration (75%, 40%
and 30%), which were used in decreasing order. The cells were resuspended in 4
ml of
39
CA 02501540 2005-04-07
WO 2004/032653 PCT/US2002/032172
complete medium and were placed over the 30% fraction. After centrifugation at
650 g
for 20 min, the interfaces between the 75 and 40% layers were removed and the
cells were
washed by centrifugation in 25 ml of complete medium. The cells were then
resuspended in 4
ml of 40% PercollTM and centrifugated at 650 g. The cell pellets, enriched for
lymphocytes
(IEL, LPL and~PPL), were collected and washed by centrifugation with PBS.
The isolated lymphocytes were stained with monoclonal antibodies quantitated
by flow
cytometry as follows: O~e hundred pl of each lymphocyte preparation (2x106
cel/ml) were
placed in 3-ml tubes with different concentration of monoclonal antibodies
(Anti CD 1 FITC,
Anti CD3E FITC, Anti CD4a PE, Anti CDBa PE, Anti CDl lb/Mac-1 APC, Anti CD21
APC),
and were incubated for 30 min. in dark at 4°C. The cells were washed
with PBS, pelleted by
centrifugation (500 g, 5 min.), and resuspended in 350 ~1 PBS.
Fluorescence-activated cell sorter (FACS) analysis of cell preparations was
carried out
on a FACScaliburTM flow cytometer (Becton Dickinson). Nonspecific fluorescence
was
determined through 3 controls (for fluorescein isothyocyanate - FITC,
phycoerythrin - PE and
allophycocyanin - APC) prepared for each cell preparation.
Histological analysis of small intestine samples was performed by electron
transmission microscopy. Samples of jejunum and ileum were fixed in 30g/L
glutaraldehyde in
0.1 mol/L sodium cacodylate buffer, pH 7.3, and postfixed in 15 g/L osmium
tetroxide.
Samples were then dehydrated in acetone and embedded in Epon 812 resin.
Ultrathin sections
were double-stained with uranyl acetate and lead citrate, and examined under a
Zeiss 902
transmission electron microscope (Zeiss, Oberkochen, Germany).
Biochemical Results
Reduction of dietary intake to 20% of control resulted in a complete failure
to grow.
Malnourished pigs lost an average of 2 - 3 kg of total weight, while control
pigs gained 18 kg
during the 30 day trial. Liver weight and the weight per length of both
jejunal and deal
mucosa were also severely reduced as consequence of malnutrition (Table 6).
TABLE 6: Liver and small intestinal weights of control and protein-energy
malnourished
pigs.
Weight Mucosa
/ Length Intestine
(g/cm)
Liver Weight Jejunum Ileum-
(g)
Control Pigs 731.4 _+ 26.5 0.092 0.008 0.070 0.007
Malnourished Pigs237.9 + 9.9 * 0.035 0.005 * 0.025 0.006 *
* Significant difference vs. control group (p<0.05).
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WO 2004/032653 PCT/US2002/032172
The amounts of DNA and protein per length of mucosa were significantly lower
(2 to 3
fold) in malnourished pigs compared with controls (data not shown). However,
the
protein/DNA ratio was not affected by PEM in any intestinal segment. These
results suggest
that the overall process of protein and DNA synthesis in the small intestine
of malnourished
pigs is impaired. The intestinal contents of protein (jejunum and ileum) and
DNA (ileum)
tended to be higher in the malnourished pigs that consumed NAQ supplement than
in those
that consumed caseinate or glutamine. These results suggest that NAQ partially
preserves the
protein and DNA synthesis process during the malnutrition period.
Alkaline phosphatase segmental activity, as marker of intestinal injury, was
significantly lower (2 to 3 fold) in malnourished pigs than in controls in
jejunal segment (data
not shown). In the ileal segment, alkaline phosphatase activity was less
affected by the
malnutrition process. In addition, malnourished pigs that consumed the
glutamine or NAQ
supplements tended to have higher AP activity in jejunum than those that
consumed caseinate
supplement.
Glutathione is the central component of the whole antioxidant defense system.
It is an
effective free radical scavenger and is also involved in a range of other
metabolic functions,
including the maintenance of protein sulfliydryl groups in the reduced state,
cofactor for GT
and GPX, amino acid transport, and protein and DNA synthesis. The total
glutathione
concentration was significantly reduced in both small intestinal segments of
the malnourished
pigs in comparison to the control group. However, the amount of GSH in the
intestinal
mucosa of malnourished pigs that consumed NAQ tended to be slightly higher
than in those
that consumed the caseinate or glutamine supplements, though this difference
did not reach
significance.
Glutathione transferase and glutathione reductase enzymatic activities,
responsible of
aldehyde detoxification and of glutathione reduction, respectively, were found
reduced (again,
2 to 3 fold) in the small intestine as a consequence of malnutrition.
Depression in the
glutathione transferase activity could aggravate the intestinal dysfunction by
accumulation of
aldehydes, epoxides and other products containing electrophilic centers within
the mucosa.
This activity looked to be less affected by the malnutrition process in the
pigs that consumed
the N-acetyl-L-glutamine supplement. The activity of glutathione reductase and
of glutathione
peroxidase were also reduced 2 to 3 fold by malnutrition in both small
intestinal segments.
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WO 2004/032653 PCT/US2002/032172
Glutathione reductase is involved in glutathione regeneration from its
oxidized form, and
glutathione peroxidase oxidizes two reduced glutathione molecules to detoxify
peroxides. A
tendency of reduced glutathione to be higher in the intestinal mucosa of pigs
fed with the N-
acetyl-L-glutamine supplement was associated with a. tendency of glutathione
peroxidase
activity to be higher in the same group.
In summary, the deleterious effects of malnutrition on the antioxidant defense
system
appeared less marked in the intestine of animals that consumed the N-acetyl-L-
glutamine
supplement than in the animals that consumed the caseinate or glutamine
supplements.
Immunological Results
There was a decrease in the total number of small intestine peyer's patch
lymphocytes
as a result of malnutrition. In ileum, the total number of peyer's patch
lymphocytes was
significantly lower in caseinate- and glutamine-supplemented pigs than in the
N-acetyl-
glutamine-supplemented or the control groups. In jejunum, there was also a
tendency of the
total number of peyer's patch lymphocytes to be higher in N-acetyl-L-glutamine-
than in
caseinate- or glutamine-supplemented groups. On the other hand, the total
number of jejunum
intra-epithelial lymphocytes was significantly higher in all malnourished
groups compared to
the control group. No differences were found in the number of lymphocytes in
the lamina
proprial of small intestine for any experimental group.
In all malnourished groups the number of peyer's patch lymphocytes expressing
B cell
markers (CD 1 and CD21 ) were lower than in healthy group, being especially
significant in the
case of GD 1+ lymphocytes. The reduction in the number of CD21+ peyer's patch
lymphocytes compared to control group in ileum was significantly different in
the caseinate-
and glutamine- supplemented groups, but not in N-acetyl-L-glutamine
supplemented group. In
jejunum, there was the same tendency but did not reach statistical
significance. The reduction
in the number of CD1 lb+ peyer's patch lymphocytes in jejunum and ileum also
showed a
tendency to be lower in N-acetyl-L-glutamine-supplemented than in caseinate-
or glutamine-
supplemented groups.
The number of T cells (CD3+ cells) in jejunum and ileum peyer's patch
lymphocytes
decreased with malnutrition. The decrease was due to both helper (CD4+) and
citotoxic
(CD8+) T cells. However, there was a general tendency of this decrease of T
cells in PPL to be
lower in the N-acetyl-L-glutamine-supplemented than in the caseinate- or
glutamine-
42
CA 02501540 2005-04-07
WO 2004/032653 PCT/US2002/032172
supplemented groups. In some cases, such as in CD4+ and CD8+ cells in ileum,
significant differences were detected between control and caseinate- or
glutamine-
supplemented groups, but not between the control and N-acetyl-L-glutamine-
supplemented
groups.
As noted above, malnutrition promoted an increase in the total number of intra-
epithelial lymphocytes in jejunum. This increase was detected in both
populations, B cells
(CD21+) and T cells (CD3+). In B cells, the number of CD1+ lymphocytes in the
N-acetyl-L-
glutamine supplemented group was significantly higher than in the rest of the
groups. In T
cells, T cytotoxic subpopulations (CD8+) were significantly higher in all the
malnourished
groups than in the control group. However, the T helper (GD4+) subpopulation
was
significantly higher in glutamine- and N-acetyl-L-glutamine-supplemented
groups (but not in
caseinate-supplemented group) than in the control group. This indicates a
selective effect of
glutamine and N-acetyl-L-glutamine on the T helper (CD4+) subpopulation. No
significant
differences were detected for any of the lymphocyte subpopulations in ileum
intra-epithelial
lymphocytes.
There were no substantial important changes in lamina propria lymphocytes due
to
malnutrition. There was a reduction of the number of CD21+ cells (B cells) in
the caseinate-
supplemented group compared to the control group that was not detected in
either the
glutamine- or N-acetyl-L-glutamine-supplemented groups. In addition, the N-
acetyl-L-
glutamine-supplemented group, but not the glutamine-supplemented group was
significantly
different from the caseinate-supplemented group.
In summary, the N-acetyl-L-glutamine-supplemented group performed better than
the
glutamine or caseinate supplemented groups, showing statistically significant
differences, to
reduce small intestine immunological changes promoted by malnutrition,
especially in total
cell number and B and T helper subpopulations.
Histological Results
Electron transmission micrographs of jejunum enterocytes from healthy and
malnourished pigs
are shown in Figure 7 In control pigs (A, B panels), jejunum enterocytes
showed regular
microvilli, narrow intercellular spaces, regular nucleus and Globet cells
containing high levels
of mucin. Intestinal mucosa of malnourished pigs that consumed ENSURE PLUS
formula
supplemented with caseinate (C, D panels) showed severe atrophy and loss of
microvilli,
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CA 02501540 2005-04-07
WO 2004/032653 PCT/US2002/032172
opening of intercellular spaces, irregular nucleus and clear cytoplasmic zones
with
multivesicular bodies. Cellular desquamation and extrusion of material into
the intestinal
lumen were also evident in this group. Jejunal mucosa of pigs fed during the
malnutrition
period with ENSURE PLUS formula supplemented with glutamine (E, F panels)
displayed
~ shortened microvilli, expanded intercellular spaces, and irregular lobulated
nucleus. Abundant
intraepithelial lymphocyte infiltration was also found in the jejunal mucosa
of malnourished
glutamine pigs. The, jejunal mucosa of pigs that consumed ENSURE PLUS formula
supplemented with NAQ (G, H panels) was less affected by protein-energy
malnutrition. In
this group, the jejunum enterocytes showed microvilli size, nucleus shape and
intercellular
spaces close to the jejunum enterocytes of control pigs. Intraepithelial
lymphocyte infiltration
was scarce in the jejunum mucosa of malnourished NAQ group in comparison to
malnourished
caseinate and glutamine groups. Figure 8 shows the electron transmission
micrographs of
ileum enterocytes from healthy and malnourished pigs. In control pigs (A, B
panels), ileum
enterocytes showed regularly distributed microvilli, intercellular spaces with
no visible
expansion, homogenous and dense cytoplasm and Globet cells with huge amount of
secreted
granules. Ileal mucosa of malnourished pigs that consumed ENSURE PLUS formula
supplemented with caseinate (C, D panels) showed loss of microvilli, expansion
of some
intercellular spaces, clear cytoplasmic zones with multivesicular bodies and
cells in process of
extrusion. In this group, wide lymphocytes infiltration was also evident in
the ileal epithelium.
Ileal mucosa of pigs fed during the malnutrition period with ENSURE PLUS
formula
supplemented with glutamine (E, F panels) displayed loss of microvilli, wide
intercellular
spaces, and intense lymphocytes infiltration. As well as with jejunal mucosa,
the deal mucosa
of pigs that consumed ENSURE PLUS formula supplemented with NAQ (G, H panels)
was
less affected by protein-energy malnutrition. It showed low alteration of
apical microvilli,
intercellular spaces without visible expansion, scarce lymphocyte infiltration
in the apical part
of intestinal epithelium and abundant Goblet cells with high amount of
secreted granules.
Conclusions
Under normal physiological conditions, there is a steady state balance between
the
production of oxygen-derived free radicals and their destruction by the
cellular antioxidant
system. In the present study, the intestinal balance was upset by protein-
energy malnutrition,
44
CA 02501540 2005-04-07
WO 2004/032653 PCT/US2002/032172
leading to a decrease in reduced glutathione and in the enzymatic antioxidant
defense
system. In addition, intestinal immune response was severely impaired by
protein-energy
malnutrition.
Although no clear effect of glutamine was detected on the prevention of
biochemical
and immunological changes induced by malnutrition in the small intestine,
probably due to the
fact that malnutrition was especially severe, there was a positive effect of N-
acetyl-L-
glutamine to reduce the severity of these changes.
This study suggests that N-acetyl-L-glutamine has a positive effect on the
cells of the
small intestine, even beyond that of glutamine. Additionally, electron
transmission
micrographs of enterocyte cytoplasm from healthy and malnourished pigs shown
in Figures 7
and 8 show that N-acetyl-L-glutamine is more effective than glutamine at
preventing the overt
signs of inflammation in the epithelial lining of the gastrointestinal tract.
EXAMPLE 5
Testing toxicity of glutamine enriched preparation in the context of Celiac
Disease using organ cultures of untreated celiac patients
The aim of this study was to evaluate the potential toxicity of several of the
gluten-free
glutamine-enriched products with peptic-tryptic preparations of gluten, known
to induce
mucosal modifications in celiac diseases. Glutamine rich or glutamine-modified
products, not
derived from gluten, were also studied in order to evaluate whether these
latter products could
have induced damage to the mucosa of celiac patients.
The study was performed in a blind fashion. For this study a organ culture
model of
celiac, which is a widely used and validated to explore gluten toxicity and
mucosal damage in
celiac disease was utilized. For the purpose of this project small intestine
bioptic fragments of
patients with untreated celiac disease were used. Biopsies of untreated celiac
patients are ideal
to monitor aspects of mucosal damage including induction of epithelial
apoptosis and increase
of mucosal inflammation.
Twelve adult patients, who were diagnosed as potential untreated celiac
patients, were
enrolled. Their disease was confirmed by clinical signs, the presence of high
titres of anti-
tissue transglutaminase and histological analysis of small intestine biopsies.
CA 02501540 2005-04-07
WO 2004/032653 PCT/US2002/032172
Description of the organ culture end point
Organ cultures were performed using standard methods described in Maiuri, L.
et al,
Gastf~oenter~ology 110, 1368-1378. (1996). Briefly a small biopsy fragment
(around lmm x
lmm) was put on stainless steel mesh and cultured.in medium supplemented with
the different
compounds for a period of 24 hours. The standard positive and negative
controls used in
studies involving celiac patients and in vitro challenge of biopsies were
utilized. The positive
control was 1 mg/ml of gliadin peptic-tryptic digest. The negative control was
the medium
alone. All the compounds to be tested were used at the final concentration of
40 ug/ml. The
compounds/products tested were N-alanyl-glutamine; N-acetyl-glutamine;
Stresson° from
Nutricia, Boca Raton, Florida (P 1 ); Reconvan~ from Fresenius I~abi, Runcorn,
Cheshire,UK
(P2); Nutricomp Immuri from B.Braun, Bethleham, PA (P3); Glutasorb~ from
Hormel Health
Labs, Plymouth, MN (P4); Impact~ from Novartis, White Plains, NY (PS);
Optimental~ plus
NAQ from Ross Products, Columbus, Ohio (P6); Optimental°° plus
hydrolyzed wheat gluten
from Ross Products, Columbus, Ohio (P7). After 24 hours the cultures were
stopped and the
biopsy fragments were oriented and embedded in O.C.T. compound (Tissue Tek,
Miles
Laboratories, Elkhart, IN, USA), snap frozen in isopenthane cooled in liquid
nitrogen, stored
at -70°C until cryosectioning. Five microns sections were then prepared
using a cryostate.
Immunohistochemistry
For this study the following markers were used: TUNEL to monitor epithelial
~ apoptosis and CD25 to monitor sub-epithelial inflammation. The following
reagents were
used and with following procedure.
Detection of DNA fragmentation
DNA fragmentation of the tissue sections were assayed as described in Maiuri,
L. et al.
DNA fragmentation is a feature of cystic fibrosis epithelial cells: a disease
with inappropriate
apoptosis? FEBS Letter 408, 225-31 (1997) and Maiuri, L. et al. FAS engagement
drives
apoptosis of enterocytes of celiac patients. Gut 48, 418-24 (2001) by terminal
deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling
(TUNEL).
Detection of CD25+ cells
Antigen detection on frozen tissue sections was performed by
immunohistochemistry
as described in Maiuri, L. et al. Blockage of T-cell costimulation inhibits T-
cell action in
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WO 2004/032653 PCT/US2002/032172
Celiac disease. Gastroenterology 115, 564-72. (199g) with mAbs anti- CD25
(Dako 1:30)
by alkaline phosphatase staining technique according to the method previously
described in
the same reference. At least 5 slides for each sample were blindly evaluated.
Specificity
control experiments were performed using mouse IgG or IgM against
inappropriate blood
group antigens, and simultaneously analyzing different cultured samples
belonging to the same
individual.
Morphometric anal
The total number of TUNEL+ enterocytes was referred as percentage of
enterocytes.
The total number of CD25+ cells in the lamina propria was determined within a
standard reference area of 1 mm2 . The counts were performed at a microscope
with a
calibrated ocular graticule aligned parallel to the muscolaris mucosae and
independently analysed by two observers; the results were compared afterwards.
Results
As shown in Figure 9 the compounds induced different patterns of epithelial
damage as defined by TUNEL and CD25 upregulation in the sub-epithelial
compartment.
Some preparations induced both strong increase of CD25 expression in the
subepithelial
compartment as well as induction of epithelial apoptosis. These compounds thus
behaved as
the positive control (peptic-tryptic gluten preparation used at 1 mg/ml).
Other compounds
induced some selective modification of the epithelial apoptosis or of the
induction of CD25 in
the subepithelial compartment. Others did not differ from the pattern of
apoptosis or CD25
induction observed in cultures only exposed to medium alone (negative
control). In Figure 10
a and b shows examples of the induction of epithelial apoptosis using compound
N-acetyl-
glutamine or the product Impact, which container glutamine (p5). Figure 11 a
and b describes
the pattern of CD25 induction by the same compounds.
Conclusion
The outcome of this study indicates that many of the tested compounds produced
significant modifications in the small intestine of untreated celiac patients.
Some
were able to induce epithelial apoptosis and increased subepithelial
inflammation, while
others were more efficient in inducing mucosal inflammation. The compounds
that induced both or one of the markers are considered toxic for the mucosa
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WO 2004/032653 PCT/US2002/032172
of celiac patients. Some of the compounds did not induce any effect, in
particular the
compound N-acetyl-L-glutamine. This compound also had a clear trophic effect
on the
biopsies of untreated celiac patients. This trophism was defined by a
generalized
improvement of the mucosa in particular of the epithelia, which were
significantly
ameliorated compared to the other samples.
This study provides clear indication that most of the compounds tested produce
substantial changes,in the mucosa of untreated celiac patients. The most
intriguing, as
unexpected, aspect of the trophic activity of the N-acetyl-L-glutamine. In the
first instance
this product appears to improve the overall condition of the mucosa even
compared to
medium alone.
Particular embodiments have been described above that fall within the scope of
the
invention as set forth in the claims. These embodiments are not intended to
limit the scope of
the invention to the specific forms disclosed. The invention is intended to
cover all
modifications and alternative forms falling within the spirit and scope of the
invention.
48
