U.S. patent application number 11/679517 was filed with the patent office on 2008-01-24 for cellular uptake of bioactive agents.
This patent application is currently assigned to Aesgen, Inc.. Invention is credited to Robert G. II Petit,, Edward C. Shinal.
Application Number | 20080021106 11/679517 |
Document ID | / |
Family ID | 22463417 |
Filed Date | 2008-01-24 |
United States Patent
Application |
20080021106 |
Kind Code |
A1 |
Petit,; Robert G. II ; et
al. |
January 24, 2008 |
CELLULAR UPTAKE OF BIOACTIVE AGENTS
Abstract
Provided is a composition and a method for increasing cellular
uptake of bioactive agents, particularly those compounds termed
"small molecules" into the cells of mammalian tissue, such as the
epithelial cells of the mucosa.
Inventors: |
Petit,; Robert G. II;
(Newtown, PA) ; Shinal; Edward C.; (Pennington,
NJ) |
Correspondence
Address: |
Schwegman, Lundberg, Woessner & Kluth, P.A.
P.O. Box 2938
Minneapolis
MN
55402
US
|
Assignee: |
Aesgen, Inc.
|
Family ID: |
22463417 |
Appl. No.: |
11/679517 |
Filed: |
February 27, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11255771 |
Oct 21, 2005 |
|
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11679517 |
Feb 27, 2007 |
|
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10796261 |
Mar 9, 2004 |
7041651 |
|
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11255771 |
Oct 21, 2005 |
|
|
|
09993465 |
Nov 14, 2001 |
6734170 |
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10796261 |
Mar 9, 2004 |
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PCT/US00/13260 |
May 15, 2000 |
|
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09993465 |
Nov 14, 2001 |
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60134442 |
May 17, 1999 |
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Current U.S.
Class: |
514/563 |
Current CPC
Class: |
A61K 31/197 20130101;
A61P 3/02 20180101; A61P 1/04 20180101; A61P 1/02 20180101; A61K
47/26 20130101; A61K 9/0014 20130101; A61K 31/198 20130101; A23L
33/175 20160801; A61P 17/02 20180101; A61K 9/0095 20130101; A61P
29/00 20180101; A61P 31/12 20180101 |
Class at
Publication: |
514/563 |
International
Class: |
A61K 31/197 20060101
A61K031/197; A61P 29/00 20060101 A61P029/00 |
Claims
1. A solid composition consisting essentially of a dry mixture of
an amount of glutamine effective to treat stomatitis and mucositis
and about 15-50 w % of at least one carbohydrate, wherein the
carbohydrate is present in an amount that is effective to enhance
the cellular uptake of glutamine from aqueous solution.
2. (canceled)
3. (canceled)
4. The composition of claim 1, wherein the amount of the at least
one carbohydrate is about 30-50 w %.
5. The composition of claim 1, wherein the at least one
carbohydrate comprises a monosaccaride, a disaccharide, or a
combination thereof.
6. The composition of claim 5, wherein the at least one
carbohydrate comprises sucrose.
7. The composition of claim 5, wherein the at least one
carbohydrate further comprises a sugar alcohol or polyol.
8. The composition of claim 7, wherein the sugar alcohol is
sorbitol, mannitol or xylitol.
9. The composition of claim 5 or 7, wherein the carbohydrate
includes glycerin.
10. The composition of claim 1, further including an effective
amount of buffer or buffering agent.
11. The composition of claim 10, wherein the buffering agent is
monosodium phosphate anhydrous.
12. The composition of claim 5 or 7, further including an effective
amount of modified cellulose.
13. The composition of claim 12, wherein the modified cellulose is
microcrystalline cellulose.
14. The composition of claim 1, further including a stabilizer,
emulsifying agent, preservative, defoamant, flavoring, or a
combination thereof.
15. The composition of claim 14, wherein the emulsifying agent
includes xanthan gum, carrageenan, or a combination thereof.
16. The composition of claim 14, wherein the preservative includes
methylparaben, potassium sorbate, or a combination thereof.
17. The composition of claim 14, wherein the defoament includes
simethicone.
18. The composition of claim 1, 4 or 5, wherein glutamine is the
sole amino acid in the composition.
19. The composition of claim 1, 4, or 5, wherein the glutamine is
about 58 w % of the composition.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/255,771, filed Oct. 21, 2005, which
application is a continuation of U.S. patent application Ser. No.
10/796,261, filed Mar. 9, 2004, which is a divisional of U.S.
patent application Ser. No. 09/993,465, filed Nov. 14, 2001, now
U.S. Pat. No. 6,734,170, which is a continuation under 37 CFR
.sctn. 1.53(b) of PCT Application Serial No. PCT/US00/13260, filed
May 15, 2000 and published as WO 00/69470 on Nov. 23, 2000, which
claimed priority from provisional U.S. Patent Application No.
60/134,442, filed May 17, 1999, which applications and publications
are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Absorption of biomolecules, such as amino acids and
proteins, is critical to cellular function. About 75 percent of the
solids in the mammalian body are proteins, including enzymes,
polypeptides such as cytokines, nucleoproteins, transport proteins,
and structural proteins. The principal functional constituents of
these proteins, amino acids, polypeptides and isolated amino acids,
are also important for cellular metabolic functions. The amino acid
glutamine, for example, serves important functions in metabolism,
including transport of carbon and nitrogen between tissues. It is a
precursor for hepatic and renal gluconeogenesis, as well as urea
synthesis in the liver and ammonia production in the kidney. A
number of cell types, particularly the cells of the intestinal
mucosa, also utilize large amounts of glutamine as their major
source of respiratory fuel.
[0003] The effectiveness of amino acid supplementation for
treatment of a variety of physiologic disorders has been
demonstrated. D-serine supplementation, for example, augments the
beneficial effects of antipsychotics for the treatment of
schizophrenia. (Tsai, G., et al., Biol. Psychiatry (1998) 44(11):
1081-1089.) L-tryptophan or 5-hydroxytryptophan supplementation has
been shown to improve symptoms of depression, anxiety, insomnia and
pain in patients with fibromyalgia. (Juhl, J. H., Altern. Med. Rev.
(1998) 3(5): 367-375.) Dietary supplementation with 8 essential and
9 nonessential amino acids provided improved health, tone, and mood
in dialysis patients, in whom protein malnutrition is a common
problem. (Mastroiacovo, P., et al., Clin. Ther. (1993) 15(4):
698-704.) Nutritional supplementation with aspartic acid has been
suggested for the treatment of Canavan disease, a rare recessive
autosomal genetic disorder generally resulting in death within
several years of onset. (Baslow, M. H., et al., J. Mol. Neurosci.
(1997) 9(2): 109-125.) L-lysine has also been demonstrated to have
therapeutic use for lesions associated with herpes simplex virus
type 1 (HSV-1). (Ayala, E. And D. Krokorian, J. Med. Virol. (1989)
28(1): 16-20.)
[0004] Glutamine supplementation has been shown to provide numerous
benefits, including stimulation of certain cells of the immune
system and general promotion of cellular growth. Depletion of
glutamine results in atrophy of epithelial tissue, with associated
bacterial translocation. Clinical supplementation of glutamine
reduces epithelial atrophy and accelerates recovery.
[0005] Dietary glutamine supplementation has been proposed for the
treatment of patients recovering from surgery or suffering from
sepsis, inflammation, burns, or trauma. Topical administration,
usually in the form of a "swish and swallow" solution for oral use
to repair the damaged epithelial tissue of mouth or esophageal
sores, can be effective in many patients who have undergone bone
marrow transplantation or chemotherapy. (Skubitz, et al., J. Lab.
Clin. Med. (1996) 127(2): 223-8; Anderson, et al., Bone Marrow
Transplant (1998) 22(4): 339-44.)
[0006] Formulations for the administration of amino acids,
particularly glutamine, are described in U.S. provisional patent
application No. 60/134,442 filed May 17, 1999 and incorporated by
reference herein.
[0007] The effectiveness of amino acid supplementation has been
limited in some individuals due to aging or disease. Effective
supplementation with certain amino acids is further limited to
varying degrees by the low aqueous solubility and limited cellular
uptake of some amino acids. Glutamine, for example, exhibits a low
solubility in water (48 g/l at 30.degree. C., 26 g/l at 18.degree.
C., 18 g/l at 0.degree. C.; The Merck Index, 12th Edition) and a
low chemical stability in aqueous solution (11 days at
22-24.degree. C.). (Cardona, P., Nutr. Hosp. (1998) 13(1):
8-20.).
[0008] Transport of small molecules into various cell types is
controlled by alternate transport systems, making it more difficult
to devise methods for increasing cellular uptake into particular
cell types. Despite the need for methods to enhance the uptake of
amino acids and other small molecules, methods for increasing
initial direct absorption of amino acids, peptides and other
compounds into cells such as epithelial cells, the type of cells
initially responsible for initial uptake of many bioactive
compounds, has not been described.
[0009] Therefore, a continuing need exists for methods to increase
cellular uptake of bioactive compounds into mammalian cells.
SUMMARY OF THE INVENTION
[0010] The invention provides a composition and a method for
increasing cellular uptake of bioactive agents, particularly those
compounds termed "small molecules" into the cells of mammalian
tissue, such as the epithelial cells of the mucosa. The composition
is a solution dispersion or suspension comprising an aqueous
vehicle and an effective amount of a bioactive compound, in
combination with an amount of carbohydrate effective to reduce the
absolute solubility of the bioactive agent in the aqueous vehicle,
so as to achieve increased transport (absorption) of the bioactive
agent into the target cells. The transport (absorption) is
increased over the amount that would enter the cells under
physiological conditions, i.e., under homeostatic conditions, when
the cells are contacted with the agent dissolved or suspended in
water or in a physiological salt solution. Preferably, the
transport (absorption) is increased by a factor of at least about
100-2000 times that is obtainable by a saturated aqueous solution
of the active agent. It is believed that the carbohydrate(s) act by
reducing the amount of free/available water in the composition,
which induces increased transport into mammalian cells, in vitro or
in vivo.
[0011] The carbohydrate carrier can comprise a monosaccharide, such
as glucose, a disaccharide, such as sucrose, or a combination of
monosaccharides and disaccharides. The carbohydrate carrier can
also comprise a sugar alcohol such as mannitol, sorbitol or
xylitol. The carbohydrate carrier can also comprise a
polysaccharide such as high fructose corn syrup or corn syrup
solids, wherein the corn syrup or corn syrup solids, hydrous or
anhydrous, constitute a solution phase for the active agent(s). The
carrier can be combined with water, or with a mixture of water with
pharmaceutically acceptable alkanols, alkylene glycols or polyols
such as glycerol, to form a solution. Preferably the organic
solvents constitute a minor proportion of the aqueous phase,
preferably .ltoreq.5-10 vol-%.
[0012] The solution can be a true solution or a flowable "solid
solution." It can be administered by a variety of means for the
administration of liquids, including toothpaste, chewing gum, hard
or soft gelatin capsules, suppositories, or other liquid dosage
forms such as topically applied lotions, drinks, such as a shake,
an enema, or mouthwash.
[0013] Administration of the composition of the invention can
provide treatment for a variety of physiologic disorders
ameliorated by enhancement of absorption of bioactive agents into
damaged or intact tissues, especially disorders affecting the
endothelial cells and fibroblasts of epithelial tissue. Such
physiologic disorders involving damaged tissue, include, for
example, lesions of the oral and esophageal mucosa following
radiation or chemotherapy in patients treated for cancer or in whom
bone marrow transplant is performed, gastric and peptic ulcers,
burns, major and minor trauma wounds, viral lesions, inflammatory
bowel disorder, Crohn's disease, Sjoren's syndrome, xerostoma, and
cryptosporidiosis.
[0014] A pharmaceutical dosage composition is also provided,
consisting of either bulk-packaged or individually-packaged
pre-mixed dry or liquid formulations of a therapeutically effective
dose of amino acid in admixture with an amount of carbohydrate
carrier effective to achieve increased absorption of the amino acid
into epithelial cells. Kits can also be provided comprising,
separately packaged in one container, dry formulation(s) and
pre-measured aqueous vehicle(s).
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 and FIG. 2 are graphs illustrating the increased
amino acid uptake achieved using a composition and method of the
invention. The amino acid glutamine was administered to CaCo cells
in combination with an effective amount of carbohydrate carrier
(7:1 ratio carbohydrate carrier to amino acid)(Aesgen-14), with
amino acid administered as a saturated solution without additional
components (L-Glut Sat Sol) as a control. As indicated by the
figure legend and the graph, intracellular glutamine concentration
was increased significantly in cells treated with a combination of
amino acid and carbohydrate carrier, as compared to that achieved
by glutamine administration alone. Incubation time in seconds is
indicated on the X axis, with cellular glutamine uptake on the Y
axis.
[0016] FIG. 3 depicts the relative effect of vehicle on L-glutamine
cellular uptake.
[0017] FIG. 4 depicts the relative effect of vehicle on
glycylsarcosine cellular uptake.
[0018] FIG. 5 depicts the relative effect of vehicle on
L-asparagine cellular uptake.
[0019] FIG. 6 depicts the relative effect of vehicle on acyclovir
cellular uptake.
[0020] FIG. 7 depicts the relative effect of vehicle on L-glutamine
cellular uptake (from half saturation).
[0021] FIG. 8 depicts the CaCo-2 permeability of L-glutamine.
[0022] FIG. 9 depicts the CaCo-2 permeability of
glycylsarcosine.
[0023] FIG. 10 depicts the CaCo-2 permeability of L-asparagine.
[0024] FIG. 11 depicts the CaCo-2 permeability of acyclovir.
[0025] FIG. 12 depicts the CaCo-2 permeability of L-glutamine (from
half saturation).
[0026] FIG. 13 depicts the effect of Aesgen-14 on L-glutamine
uptake into human fibroblasts (right boxes) vs. saturated
L-glutamine (left boxes).
[0027] FIG. 14 depicts the effect of Aesgen-14 on L-glutamine
uptake into human umbilical and endothelial cells.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The inventors have discovered a new composition that
increases the cellular uptake of bioactive agents into mammalian
cells in vitro or in vivo. Using the composition and method of the
invention, increased gastrointestinal epithelial cell uptake of the
amino acid glutamine by a factor of over 150.times. within ten
seconds after administration has been demonstrated. The present
invention also provides a method for treating patients suffering
from a number of pathophysiological conditions, using the
composition to increase cellular uptake of bioactive agents in
therapeutic amounts.
[0029] As used herein, the term "bioactive agent" refers to a
molecule that exerts a therapeutic or nutritive effect on a mammal
following absorption of an effective amount of the molecule by the
target cells.
[0030] As used herein, the term "effective amount" refers to an
amount that causes a detectable biological change in a target cell
population, and preferably an amount that accomplishes a
therapeutic effect, i.e., reduces at least one symptom of a
pathology or disease afflicting said mammal.
[0031] As used herein, "amino acid" includes, for example, alanine,
arginine, aspartic acid, asparagine, cysteine, glutamic acid,
glutamine, glycine, histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, proline, serine, threonine, tryptophan,
tyrosine, valine, citrulline, g-aminobutyric acid, hydroxyproline,
and ornithine, as well as dipeptides such as glutamyl glutamate and
tripeptides such as glutathione. (See Remington's Pharmaceutical
Sciences (19th ed.) at pages 389-392.) The composition and method
are particularly useful, however, for increasing absorption of
those amino acids which exhibit limited aqueous solubility and/or
poor cellular uptake, such as glutamine. Limited aqueous
solubility, as used herein, is defined as a solubility of less than
about 5 grams amino acid in 100 ml water at 22-25.degree. C.
[0032] The present solutions can also enhance the in vitro or in
vivo cellular absorption of a wide variety of bioactive agents,
preferably in therapeutic amounts, particularly of the entities
generally referred to as "small molecules."
[0033] As used herein, the term "small molecule" includes single
molecular entities such as amino acids, steroids, cytokines,
hormones, hormonal regulators, enzymes, vitamins and the like that
generally have a molecular weight of less than 30 kD, preferably
less than 25 kD, most preferably less than 10 kD, i.e., a molecular
weight of .ltoreq.5000 daltons.
[0034] As used herein, the term "oligopeptide" is a peptide
comprised of 2 to 20 amino acids.
[0035] Enhanced absorption of bioactive agents into the skin or
intact mucosal tissue of the gut can also be used to administer
bioactive agents having an effect on organs or tissues remote from
the site of administration. Such agents can include small molecules
such as enzymes (enzyme deficiencies), short chain fatty acids
(IBD), pamidronate (osteoporosis), pyruvate (kidney failure),
interferons (immunoregulation), TGF-.beta. (atherosclerosis),
hormones (prostate, breast and other cancers), steroids
(testosterone), and chemotherapeutic agents (taxol, TMX and the
like).
[0036] Other small molecules that may be potentiated using the
present method include antiviral drugs and antibiotics such as
those agents that ligate to binding sites or receptors on the
exterior surface of the cell membrane. These antiviral agents may
include analogs of the viral binding amino acid sequences or
analogs of receptor groups, which inactivate the binding sequence
of the virus, or toxins that are attached to receptor ligands which
are used as a lethal agent to kill the infected cell, or agents
that slow viral replication by inhibiting reverse transcriptase.
(See Remington's Pharmaceutical Sciences (19th ed.) at pages
1237-1241.)
[0037] Analogs of nucleosides, nucleotides or nucreosides may be
used as antiviral agents as well. Additional antiviral agents
include macrophages activated by muramyl tripeptides or other
ligands on liposomes; antiseptics; astringents; and
B-propiolactone.
[0038] Specific antiviral agents include acyclovir, acyclovir
sodium, amantadine hydrochloride, cytarabine, idoxuridine,
ribavirin, rifampin, suramin, trifluridine, vidarabine, zidovudine
(AZT or ZDY), HPA-23, abacavir (Ziagen.RTM.), and any of the
interferons and any combination thereof. Additional antiviral
agents include rCD4-ricin A chain complex; AL-721 which is a
combination of tumor necrosis factor and gamma-interferon;
ampligen, which is poly-IC12U; ansamycin (Rifabutin);
(E)-5-(2-bromovinyl-2'-doxyuridine)(BVDU); butylated
hydroxytoluene; castanospermine; dextran sulfate; dideoxycitidine
(DDC); dideoxyadenosine; dideoxyinosine (DDI); foscarnet;
dihydromethylpyridinylcarbonyloxyazidodide-oxythymidine;
2'-fluoro-2'-deoxy-5-iodo-ara C (FlAC) and its uridine analog
(FlAU); ganciclovir
(9-[2-hydroxy-1-(hydroxymethyl)ethoxymethyl]guanine (DPHG);
Peptide-T; phosphonoformate(foscarnet sodium); rimantadine
hydrochloride; and any combination thereof.
[0039] Other bioactive proteins that may be potentiated using the
present method include the group of proteins that are generally
referred to as nerve growth factors. These include nerve growth
factor itself (NGF), Brain-Derived neurotrophic factor (BDNF),
neutrotrophin-3 (NT-3) and ciliary neurotropic factor (CNTF). NGF
(total dose infused i.v.=1 ug) has been reported to ameliorate
cholinergic neuron atrophy and spatial memory impairment in aged
rats by W. Fischer et al., Nature, 329, 65 (1987). Recombinant
human beta NGF has been produced which has potent in vitro and in
vivo neurotropic activity. See J. Barrett et al., Exp. Neurol.,
110, 11 (1990). Therefore, exogenous administration of neuronal
growth factors may be helpful to treat pathological disorders
involving degenerative processes, including Alzheimer's disease or
diabetic associated polyneuropathy.
[0040] The present method can also be used to deliver insulin.
Since it has been demonstrated that there is a widespread
distribution of insulin receptors in brain, insulin is likely to
also have important functions in the central nervous system. It is
suggested that insulin may function as a neurotrophic factor and
neuromodulator by D. G. Baskin et al., Trends Neurol., 11, 107
(1988) and D. G. Baskin et al., Ann. Rev. Physiol., 49, 335
(1987).
[0041] Another class of proteins are the neuroreceptors or soluble
peptides isolated therefrom. These include receptors for
neurotransmitters (epinephrine, norepinephrine, dopamine,
serotonin, GABA, glycine, glutamate, and the like); neuropeptides
(.beta.-endorphin, enkephalins, somatostatin, neurotensin,
angiotensin vasoactive intestinal peptide, and the like); and
neurohormones (luteinizing hormone releasing hormone,
thyrotrophin-releasing hormone, substance P, and the like).
[0042] High molecular weight bioactive agents can also be employed
in the present method and compositions, including nucleic acids
such as DNA and RNA, i.e., linearized or plasmid DNA. The DNA can
encode "sense" or antisense RNA to block an undesirable cellular
function. The DNA can encode polypeptides such as hormones, and
cytokines in amounts effective to accomplish "gene therapy," i.e.,
the correction of metabolic diseases and defects.
[0043] "Carbohydrate," as used herein, includes those sugars known
as monosaccharides and disaccharides, polyols, hydroxy analogs or
sugar alcohols, such as, for example, xylitol, sorbitol, and
mannitol, and their polymers, such as dextrins, high fructose corn
syrup, and corn syrup solids. It is well known in the art that
certain mono- and disaccharides form sugar alcohols, or hydroxy
analogs. Certain of these hydroxy analogs, particularly sorbitol
and xylitol, have proven to provide the benefit of a sugar taste
without the cariogenic properties of the mono- and disaccharides
from which they are derived.
[0044] It is believed that the carbohydrate, or mixture thereof, as
used in the present invention, acts at least in part by reducing
the free water available to solubilize the bioactive agent(s),
thereby promoting absorption of the amino acids into the cytosol of
the target cells. Preferably, there will be a major proportion by
weight of carbohydrate in the final composition, e.g., greater than
80-90 weight percent. In some cases the composition can be
essentially free of added water, i.e., can be a "solid solution,"
the carbohydrate acting as a "solvent" for the active ingredient.
Such "solid solutions" can be flowable, semisolid or even solid.
The ratio of carbohydrate to active agent can be approximately
1.5:1 w/w to 20:1 w/w in a dry preparation, and preferably 4:1 w/v
to 15:1 w/v in final aqueous solution, most preferably greater than
7:1 w/v, achieved either by constitution of the preparation with
aqueous solvent or by delivery into the aqueous environment of the
extracellular fluids surrounding the target tissue.
[0045] "Cell," as used herein, includes any cell that can be
contacted by the present composition in accord with the present
method, such as epithelial cells, endothelial cells, skin cells,
fibroblasts or neuronal cells. More specifically, cells in which
the composition and method of the present invention have been
demonstrated to increase absorption of the amino acid glutamine are
gastrointestinal epithelial cells, including cells of the mouth,
throat, esophagus, stomach, intestines, colon and rectum,
endothelial cells and fibroblasts.
[0046] "Constitution with aqueous solvent," as used herein,
includes constitution with water, physiological salt solutions or
buffers, fruit juice or other liquid which contains a high
percentage of water, or with extracellular fluids surrounding the
tissue to which the composition is applied, such as saliva, mucous,
gastric fluids, spinal fluid, and the like.
Formulation of a Composition for Increasing Solubility and
Absorption of an Amino Acid
[0047] In accord with the present invention, at least one bioactive
agent is combined with a carbohydrate in the presence of water, so
as to form an aqueous solution. The carbohydrate can be a
monosaccharide, including, for example, allose, altrose, arabinose,
dihydroxyacetone, erythrose, erythrulose, fructose, galactose,
glucose, glyceraldehyde, gulose, lyxose, idose, mannose, psicose,
ribose, ribulose, sorbitol, tagatose, threose, xylose, xylulose,
and their respective hydroxy analogs, such as sorbitol from
sorbose, mannitol from mannose, and xylitol from xylose.
Alternatively, the carbohydrate can be a disaccharide, such as
maltose or sucrose, or both, or their polymers, such as dextrins,
maltodextrins, and high fructose corn syrup products. The
carbohydrate carrier can also be composed of any combination of
monosaccharides, disaccharides, or both. For many applications, the
hydroxy analog of the sugar is preferable, particularly where a
noncariogenic sugar is needed. Examples of hydroxy analogs include
the sugar alcohols, xylitol, sorbitol, and mannitol.
[0048] Carbohydrate concentration, measured as weight/volume, in
the solid composition is preferably 20% to 99%. At a certain
concentration, the carbohydrate will complex and reduce the amount
of free water available as a solute for the active agent, so that
the transport of the active agent into the target cell is
significantly increased.
[0049] A preferred embodiment of the composition provides a mixture
of solids including about 5-50% w/w glutamine (most preferably
L-glutamine), about 15-50% w/w carbohydrate carriers, including a
disaccharide (most preferably sucrose), a sugar alcohol or polyol
(most preferably sorbitol), and glycerin, an effective amount of
buffer, or buffering compound (most preferably anhydrous monobasic
sodium phosphate), about 1-5% w/w modified cellulose (most
preferably Avicel.RTM. Cellulose Gel), with the remainder
optionally comprising stabilizers and emulsifying agents (xanthan
gum, carrageenan), preservatives (methylparaben, potassium
sorbate), a defoamant (simethicone), and flavoring.
[0050] A more preferred embodiment provides approximately 5-15% w/w
glutamine, 30-50% w/w carbohydrate carriers, including a
disaccharide (most preferably sucrose), a sugar alcohol or polyol
(most preferably sorbitol), and glycerin, with the remainder of dry
solids comprising an effective amount of a buffer, or buffering
compound (most preferably anhydrous monobasic sodium phosphate),
modified cellulose (most preferably Avicel.RTM. Cellulose Gel), and
optionally comprising stabilizers and emulsifiers (xanthan gum,
carrageenan), preservatives (methylparaben, potassium sorbate),
defoamants (simethicone), and flavoring.
[0051] A preferred liquid composition provides 5-25% w/v
L-glutamine, 20-40% w/v carbohydrate carrier, including a
disaccharide, a sugar alcohol, and glycerin, 5-10% w/v citric acid,
and an effective amount of buffer (preferably 0.4-0.8% sodium
phosphate), with optional stabilizers, preservatives, emulsifiers
and flavorings.
[0052] Use of a carbohydrate carrier in the composition can
increase the cellular absorption of the amino acid by at least ten
times over direct administration of the amino acid in water. For
example, a preferred aqueous composition of 38% w/v L-glutamine,
30% w/v sucrose, and 2.8% w/v sorbitol produced a 360-fold increase
in glutamine uptake by CaCo cells (an epithelial mucosa cell line)
over that obtained by use of an aqueous glutamine solution
alone.
[0053] Excipients can also be added to the composition, provided
that the necessary concentration of carbohydrate carrier is
maintained. These can include a sweetener/solvent, such as
glycerin; emulsifying and stabilizing agents, such as cellulose gel
(for example, Avicel.RTM. Microcrystalline Cellulose Gel (FMC
Corp., Philadelphia, Pa.)), xanthan gum or carrageenan;
preservatives and stabilizers, such as citric acid, and
methylparaben; a defoamant/base ingredient, such as simethicone;
flavoring, or other ingredients which improve the stability and
administration of the composition.
Delivery of an Increased Concentration of an Active Agent
[0054] The invention provides a method of delivery of increased
concentrations of active agent to target cells in vivo or in vitro
by a number of alternate routes. For example, the active agent can
be mixed with a carbohydrate and water, and optionally gelling or
thickening agents. The mixture can be administered as a solution,
gel, or suspension. Where desired, undissolved materials can be
removed by allowing the mixture to stand to allow undissolved
particles to settle out, or can be centrifuged to isolate the
supernatant. The supernatant solution can then be parenterally, or
orally applied to target tissue, as by intravenous injection of
infusion.
[0055] Application of the preparation can include, but is not
limited to, topical administration by swabbing directly on a wound
resulting from, for example, burn, trauma, or viral infection,
e.g., in ointment, gel or liquid form, including administration by
transdermal patches. The preparation can be applied to oral, nasal,
and esophageal lesions by oral rinse, a gel, or an ingestible
drink. For either oral rinse or ingestible drink, the carbohydrate
carrier can be chosen from among a number of monosaccharides,
disaccharides, or a combination of both, or from their polymers,
such as dextrins, maltodextrins, and high fructose corn syrup
products. Preferred carbohydrate carriers include sucrose, sorbitol
and high fructose corn syrup products. Either a suspension or a
drink can be provided as a dry mixture of carbohydrate carrier and
an effective amount of amino acid, for reconstitution with water,
juice, or other liquid. Bulk packaging of the dry mixture or
packets containing single applications can be provided to a
patient, health care provider, or any individual for whom the
delivery of an increased concentration of active agent is desired.
Premixed liquid bulk or unit dosage forms can also be employed.
[0056] Application of the composition having a relatively low
concentration of free water can also be accomplished by providing a
lozenge or a form of candy or other medicated confection, such as a
common lollipop, which utilizes a suitable carbohydrate carrier,
such as sucrose or sorbitol, and a gelling or thickening agent, as
needed. Chewing gum can also be used to deliver the carbohydrate
carrier, such as sucrose, xylitol, sorbitol, or corn syrup solids,
and amino acid. In a preferred form, the chewing gum can
incorporate a central pocket of flavored syrup, composed of the
appropriate mixture of carbohydrate carrier, such as xylose,
sorbitol, or sucrose, and an effective amount of the amino acid.
Formulations for preparation of chewing gum with a soft core
portion are described in U.S. Pat. No. 4,352,823 (Cherukuri, et
al., Oct. 5, 1982) and U.S. Pat. No. 4,352,825 (Cherukuri, et al.,
Oct. 5, 1982). Alternatively, a solid solution of a biologically
active agent can be used in the preparation of chewing gum,
lozenges, or a candy form such as a lollipop. Such solid solutions
can be formed from comelts, coprecipitates, or by mechanical
activation of the carbohydrate carrier and the biologically active
agent.
[0057] A toothpaste can also be formed to incorporate a
carbohydrate carrier and active agent. Microencapsulation of
ingredients in toothpaste compositions has been described in U.S.
Pat. No. 4,348,378 (Kosti, Sep. 7, 1982), U.S. Pat. No. 4.071,614
(Grimm, Jan. 31, 1978), and U.S. Pat. No. 3, 957,964 (Grimm, May
18, 1976), which describe the addition of encapsulated flavorings
and anti-plaque ingredients to standard toothpaste
preparations.
[0058] The composition of the present invention can also be
delivered by suppository to epithelial tissues of the colon and
rectum. Methods of preparation of suppository formulations are
known in the art. One such method has been described in U.S. Pat.
No. 4,439, 194 (Harwood, et al., Mar. 27, 1984), which describes a
water and drug delivery system for suppository use. An enema
preparation can also be formed of a carbohydrate carrier and an
amino acid, incorporating a sufficient amount of water to form an
aqueous solution. A solid solution of the biologically active agent
in the carbohydrate carrier can also be administered in a
suppository or enema, drawing the aqueous component from the colon
or rectum.
[0059] When delivery to the stomach is preferred, a filled capsule
can be used. One such method has been described in U.S. Pat.
5,569,466 (Tanner, et al., Oct. 29, 1996), which describes the
preparation of fill compositions for soft elastic gelatin capsules.
Enteric coated capsules or tablets, or enteric coated
microparticles can be employed to deliver the compositions to the
upper or lower intestines.
[0060] The composition can be delivered in ice cream formulations,
as well as frozen confections such as the common popsicle. Frozen
formulations can be especially effective for the treatment of oral
and esophageal ulcers, since they can combine, for example, both
the beneficial effects of glutamine, as well as the soothing
effects of the cold mixture.
[0061] The composition of the present invention has been shown to
improve solubility and cellular absorption of a dietary amino acid,
glutamine, into human gastrointestinal epithelial cells, as
illustrated in the following example.
EXAMPLE 1
Evaluation of Cellular Uptake of Glutamine in Combination With
Sucrose and Sorbitol
[0062] 1. Materials and Methods
[0063] Distilled, deionized water (107 ml) was added to 207 grams
of a mixture of sucrose, sorbitol, and glutamine with excipients
(Aesgen-14) as listed in Table 1. TABLE-US-00001 TABLE 1 Aesgen-14
(AES-14) L-glutamine 240.0 Kg 57.94 w %* 50.00% w/v** Sucrose 144.0
Kg 34.77 w % 30.00% w/v Crystalline Sorbitol 13.44 Kg 3.24 w %
2.80% w/v Glycerin 14.0 Kg 2.92 w % 2.52% w/v Sodium Phosphate 2.6
Kg 0.63 w % 0.54% w/v Monobasic (Anhydrous) Avicel Cellulose 874.0
g 0.18 w % 0.17% w/v Gel Type CL-611 Citric Acid (Anhydrous) 280.0
g 0.07 w % 0.06% w/v Xanthan Gum 230.0 g 0.05 w % 0.04% w/v
Carrageenan 230.0 g 0.05 w % 0.04% w/v Artificial Flavor 230.0 g
0.05 w % 0.04% w/v Methylparaben 207.0 g 0.04 w % 0.04% w/v
Potassium Sorbate Powder 180.0 g 0.04 w % 0.04% w/v 30% Simethicone
Emulsion 115.0 g 0.02 w % 0.02% w/v *Weight percents are expressed
as percent of total weight of dry ingredients for reconstitution
with water in a 240 ml bottle. **Weight/volume percents are
expressed as percent of total volume in aqueous mixture.
[0064] As a control, 200 milliliters of distilled, deionized water
was added to 50 grams of L-glutamine (Ajinomoto, Raleigh N.C.) and
mixed by agitation. Both samples were allowed to stand for 1 day at
room temperature. The supernatant was decanted from the residue and
used for the cellular uptake determination.
[0065] On Day 1, cells from a human gastrointestinal epithelial
cell line (CaCo) were plated at a density of 0.5.times.10.sup.6
cells per well in a 6-well tissue culture dish. On Day 2, culture
media was replaced with either normal growth medium or medium
deficient in L-glutamine.
[0066] On Day 3, cells cultured in both normal growth medium
("normal") and L-glutamine deficient growth medium ("starved") were
evaluated for comparison of glutamine uptake using the Aesgen-14
solution in parallel with the L-glutamine solution, according to
the following protocol: Two milliliters of test material (either
Aesgen-14 or L-glutamine solution) was added to the appropriate
wells, then incubated at 37.degree. C. At time points 0, 10, 20,
40, and 60 seconds the test material was aspirated and the cells
washed three times (3.times.) with chilled (4.degree. C.) phosphate
buffered saline (PBS), followed by the addition of 1.0 ml of
perchloric acid. Cells were harvested by scraping, then aspiration
by pipet into a 1.7 ml tube.
[0067] The harvested cells were sonicated for 10 seconds, and 500
.mu.l of sonicated cells were transferred into a 1.7 ml tube. The
perchloric acid was neutralized by the addition of 130 .mu.l of 2M
KHCO.sub.3, and the resulting mixture was frozen overnight at
-80.degree. C.
[0068] Upon thawing, the sample was centrifuged for 10 minutes at
14,000 rpm and the supernatants were transferred to new 1.7 ml
tubes and frozen at -80.degree. C. The resulting clarified samples
were thawed and diluted 1:3 with deionized water. Fifty microliters
were withdrawn, added to 10 microliters complete o-phthaldialdehyde
(Sigma P-0532), and mixed by agitation. After incubation for two
minutes at room temperature, a 20 .mu.l sample was injected on a
Hypersil.RTM. C18 Elite 5 .mu.m HPLC column using 70:30
acetonitrile:water as the mobile phase. Glutamine levels, measured
as .mu.g/ml, were detected at 340 nm.
[0069] 2. Results
[0070] Results are shown in Table 2 as .mu.g/ml mean cellular
glutamine uptake: TABLE-US-00002 TABLE 2 Incubation Time (Seconds)
0 10 20 40 60 Normal cells + 1.00 1568.55 900.60 1185.88 1765.13
Aesgen 14 Normal cells + 3.53 10.30 2.48 3.23 4.85 L-glutamine
Starved cells + 0.00 613.10 672.93 1213.40 1053.85 Aesgen 14
Starved cells + 1.33 1.43 1.49 2.23 49.96 L-glutamine
[0071] As summarized above, glutamine uptake is significantly
increased in both normal cells (363.times.) and in starved cells
(21.times.) in cells treated with Aesgen-14 as compared to cells
treated with aqueous L-glutamine alone.
EXAMPLE 2
Effect of AES-14 on Drug Uptake and Permeability
[0072] The cellular uptake and permeability enhancing effect of a
pharmaceutical vehicle on four model drugs (L-glutamine,
L-asparagine, glycylsarcosine, acyclovir, along with half
saturation L-glutamine) across Caco-2 cell monolayers were measured
in this experiment. Uptake and permeability of each compound was
measured in the apical-to-basolateral direction, with and without
vehicle.
Methods
[0073] Materials. Two amino acids (L-glutamine, L-asparagine), a
dipeptide (glycylsarcosine), and a therapeutic agent (and
acyclovir) with low permeability were studied. Each compound was
tritiated. .sup.14C-mannitol was used as an evaluation of
monolayer/cell integrity (i.e. as a low uptake/permeability
marker).
[0074] Uptake and Permeability Assessments. Compound cellular
uptake into and permeability across Caco-2 monolayers was measured.
Caco-2 monolayers were grown using a recently developed, rapid
culture system, that requires 4 days rather than 21 days. Lentz et
al., (2000), Int. J. Pharm., 200(1): 41-51.
[0075] Uptake and permeability studies were conducted in duplicate
at 37.degree. C. and 50 oscillations per min across Caco-2
monolayers in either (a) blank AES-14 (i.e., AES-14 without
L-glutamine) or (b) Hank's balanced salt solution (HBSS) containing
10 mM HEPES buffer (solution pH=6.8). HBSS was used when no
pharmaceutical vehicle was present for each of the four compounds.
Blank AES-114 was the matrix for L-asparagine, glycylsarcosine,
acyclovir, and "half-saturation" L-glutamine studies when a vehicle
effect is considered. AES-14, which contains L-glutamine, was
studied for L-glutamine. Monolayer integrity was monitored using
.sup.14C-mannitol permeability. Mannitol uptake was also
studied.
[0076] Uptake and permeability studies were conducted using
Transwell.RTM. inserts in the apical to basolateral direction, at
intervals of 10 sec., 60 sec., and 5 min. Donor solution included a
nine saturated systems (except half strength L-glutamine) were the
source solutions for the uptake/permeability studies. Saturated
solutions were obtained by utilizing 5.4 g L-glutamine/100 ml, 1 g
L-asparagine/10 ml, 2 g glycylsarcosine/10 ml, and 16 mg
acyclovir/10 ml system concentrations (Kristol, (1999), J. Pharm.
Sci., 88: 109-110), wherein excess solid solute was present to
assure saturation:
[0077] Saturated solution of L-glutamine in HBSS (5.4 g/100 ml)
[0078] Saturated solution of L-asparagine in HBSS (1 g/10 ml)
[0079] Saturated solution of glycylsarcosine in HBSS (2 g/10
ml)
[0080] Saturated solution of acyclovir (16 mg/10 ml) in HBSS
AES-14
[0081] Saturated solution of L-asparagine in blank AES-14(1 g/10
ml)
[0082] Saturated solution of glycylsarcosine in blank AES-14 (16
mg/10 ml)
[0083] 2.3 g/100 ml L-glutamine in blank AES-14 (i.e.
half-saturated L-glutamine)
[0084] .sup.14C-mannitol and .sup.3H-drugs were quantified by
liquid scintillation counting. For uptake studies, at designated
time points (10 sec, 60 sec, and 5 min), the donor solution was
aspirated off. The cell monolayer was washed twice with ice cold
HBSS to remove any residual binding and then dissolved in 1 ml of
the cell solubilizing agent, Solvable.RTM.. The cell lysate (0.5
ml) was added to 5 ml scintillation cocktail (Econosafe.RTM.) and
counted on liquid scintillation counter (Beckman LS5801, Columbia,
Md.). For permeability studies, 0.5 ml of received solution was
added to 5 ml scintillation cocktail (Econosafe.RTM.) and counted
on liquid scintillation counter.
[0085] Since saturated solutions of unknown concentration of drugs
were used, absolute uptake could not be calculated. Hence, the
vehicle effect on uptake is considered below (FIG. 3-7) in terms of
the relative drug uptake into cell monolayer from vehicle vs
non-vehicle (i.e., ratio of uptake, after normalized for slight
differences in radiolabel tracer).
[0086] Permeability (3) in each experiment was calculated (FIG.
8-12) using eq 1: P = d M / d t A C d ##EQU1## where P is
permeability, dM/dt is rate of drug mass accumulation (i.e.,
radioactivity) in receiver compartment, A is area, and C.sub.d is
donor drug concentration (i.e., radioactivity). Polli et al.,
(1998), Pharm. Res., 15: 47-52. Permeability is an absolute measure
(units of cm/Sec or velocity) and can be determined even though the
absolute drug concentrations were not known. Results
[0087] Uptake. In FIG. 3-7, the relative effect of vehicle on
L-glutamine, glycylsarcosine, L-asparagine, acyclovir, and
L-glutamine (half-strength) uptake into cells is shown. If uptake
(normalized for slight differences in donor radiolabel) were
identical from each vehicle and HBSS, the relative uptake would be
1.0. For all four drugs and half-strength L-glutamine, the relative
uptake exceeded 1.0. In FIG. 3-6, for L-glutamine, L-asparagine,
glycylsarcosine, and acyclovir, vehicle enhanced cellular drug
uptake about four-fold. To perhaps a lesser extent, vehicle
enhanced half-strength L-glutamine (FIG. 7).
[0088] In Table 2 below, vehicle had no effect on mannitol relative
uptake. These mannitol studies, which were performed simultaneously
with those in FIG. 3-7, indicated the vehicle effect differentiates
mannitol from the other compounds, in terms of uptake enhancement.
Thus, the uptake of the saccharides per se is apparently not
increased, and the term "biologically active agent" can be read to
exclude the saccharides present in the solution, dispersion, or
gel. TABLE-US-00003 TABLE 2 Relative Effect of Vehicle on Mannitol
Cellular Uptake L-glutamine Glycyl- (half- Time L-glutamine
sarcosine L-argine Acyclovir strength) (sec) study study study
study study 5 0.52 0.83 1.65 1.24 1.20 60 0.80 1.51 0.77 0.85 0.57
300 0.63 1.06 0.43 0.43 0.30
[0089] Permeability. In FIG. 8-12, the relative effect of Aesgen-14
vehicle on L-glutamine, glycylsarcosine, L-asparagine, acyclovir,
and L-glutamine (half-strength) permeability is shown. Unlike the
uptake data presented above, which shows the relative vehicle
effect on uptake (i.e., the ratio of uptake with vehicle vs without
vehicle), permeability is an absolute measurement, and is
calculated for each formulation (no vehicle and with vehicle).
Since two-fold variation in permeability is within typical
experimental variation, these results indicate that vehicle had no
effect on permeability. Similarly, vehicle had no effect on
mannitol permeability (Table 3).
[0090] In FIG. 14 the effect of Aesgen-14 vehicle on L-glutamine
uptake into human fibroblasts (right boxes) vs. uptake of saturated
L-glutamine (left boxes). FIG. 14 depicts the effect of vehicle on
L-glutamine absorption into human endothelial cells. On the chart,
the effect of saturated L-glutamine alone was not visible.
[0091] It should be noted that 5 min. represents a very brief time
frame for traditional Caco-2 permeability studies. It is unlikely
that steady-state is achieved after 5 min., reducing the
probability of observing any possible vehicle effect.
SUMMARY
[0092] L-glutamine, L-asparagine, glycylsarcosine, and acyclovir
represent two amino acids, a peptide, and an anti-viral agent, each
with poor passive membrane penetration properties under normal
physiological conditions. Hence, enhancement of their cellular
uptake and membrane permeability is advantageous, from a drug
delivery perspective. For saturated solutions of L-glutamine,
L-asparagine, glycylsarcosine, and acyclovir, vehicle AES-14
enhanced their cellular drug uptake about four-fold. This
enhancement of drug uptake into cells occurred immediately (i.e.,
<<1 min), and was sustained over the time period studies (5
min.). To perhaps a lesser extent, vehicle enhanced half-saturated
L-glutamine. Vehicle had no effect to mannitol uptake. Regarding
permeability over a very brief 5 min. period, vehicle had no effect
for any compound. TABLE-US-00004 TABLE 3 Caco-2 Permeability of
Mannitol Mannitol Permeability Mannitol Permeability Study without
Vehicle (cm/sec) with Vehicle (cm/sec.) L-glutamine 3.80 .times.
10.sup.-9 9.16 .times. 10.sup.-7 Glycylsarcosine Below LOQ 1.48
.times. 10.sup.-6 L-asparigine 3.80 .times. 10.sup.-6 9.48 .times.
10.sup.-7 Acyclovir 1.14 .times. 10.sup.-6 1.46 .times. 10.sup.-6
L-glutamine (half- 1.49 .times. 10.sup.-6 Below LOQ strength)
Method for Treating Mammalian Subjects by Enhancing Amino Acid
Absorption
[0093] The composition of the present invention, and its various
methods of delivery, can be used in a method for treating a variety
of mammalian, especially human, physiologic disorders. The method
is most effective for treatment of disorders involving epithelial
tissue, particularly gastrointestinal epithelium (including
oropharynx, esophagus, stomach, intestines and colon).
[0094] The method provides the previously described composition, a
combination of therapeutically effective dosage of a selected amino
acid, or a combination of amino acids, with an effective amount of
carbohydrate carrier(s) which increase(s) aqueous solubility and
cellular absorption of the amino acid or amino acids for
administration to the epithelial tissue of the patient.
[0095] The invention is particularly useful for delivery of
therapeutic levels of amino acids which exhibit limited aqueous
solubility, such as the dietary amino acids tryptophan, tyrosine,
glutamine, aspartic acid, asparagine, glutamic acid, histidine,
isoleucine, leucine, methionine, and phenylalanine. Both D- and
L-amino acids, as well as amino acids such as citrulline,
g-aminobutyric acid, hydroxyproline, and ornithine, for example,
can be delivered by the method to increase cellular absorption.
[0096] Carbohydrate carriers useful for the composition
administered in the method of the invention can be chosen from
among the sugars, either monosaccharide or disaccharide, including,
for example, D-allose, D-altrose, D-arabinose, D-erythrose,
D-erythrulose, D-fructose, D-galactose, D-glucose,
D-glyceraldehyde, D-gulose, D-lyxose, D-idose, D-mannose,
D-psicose, D-ribose, D-ribulose, D-sorbose, D-tagatose, D-talose,
D-threose, D-xylose, D-xylulose, maltose, lactose, and sucrose. In
some patients or physiological conditions, as, for example, when it
is important to choose a carbohydrate carrier which will not
promote tooth decay or cause a sudden increase in blood glucose
levels, it may be preferable to choose a polyol, or sugar alcohol,
such as, for example, sorbitol, erythritol, maltitol, mannitol, or
xylitol.
[0097] For children, particularly, a sugar alcohol may be a
preferable carrier, and can produce added benefit beyond the
desired therapeutic effect on the target tissue. For example,
xylitol reduces the growth of Streptococcus pneumoniae and has been
shown to have a preventive effect against acute otitis media when
incorporated into chewing gum for children. (Uhari, M., et al.,
Brit. Med. J. (1996) 313(7066): 1180-1184.) Use of xylitol as a
carbohydrate carrier for glutamine in a chewing gum formulation
used to treat damaged oral or esophageal epithelial tissue after
chemotherapy or bone marrow transplant can, therefore, also provide
a protective benefit against a pathogenic organism.
[0098] The method comprises identification of physiologic disorders
for which amino acid supplementation is indicated. More
particularly, it provides a method for delivering increased
intracellular amino acid supplementation to patients who exhibit
symptoms of a physiologic disorder for which amino acid
supplementation may be of therapeutic value. Numerous physiologic
disorders, or diseases, have been linked, for example, to defective
amino acid metabolism or defective absorption. In many situations,
it is desirable to deliver large intracellular concentrations of an
amino acid. In most situations, it is also preferable to do so by
administering a limited dose of the selected amino acid or amino
acids. This has not previously been possible, however, since many
amino acids exhibit limited aqueous solubility and intracellular
absorption--and must therefore be administered in large doses to
achieve a desired effect. Physiological conditions for which amino
acids supplementation has been indicated, and for which the method
of the present invention is therefore beneficial for increasing
intracellular delivery of amino acid supplements, are described
below. These examples are not intended to limit the use of the
method described herein, but are presented as examples of the wide
variety of physiologic disorders for which the method of the
present invention will be useful.
Enhancing Amino Acid Absorption for the Treatment of Children and
Adults with Short Bowel Syndrome
[0099] Short bowel syndrome is associated with surgical resection
of the large intestine, and results in decreased surface area for
absorption. The tissue of the bowel is often irritated, with
accompanying symptoms such as cramping and diarrhea. An amino-acid
based complete infant formula has been demonstrated to be effective
in improving feeding tolerance, eliminating the need for parenteral
nutrition, and improving intestinal function in children with
severe short bowel syndrome. (Bines, J., et al., J. Pediatr.
Gastroenterol. Nutr. (1998) 26(2): 123-128.) The present invention
provides a method for increasing absorption of amino acids,
particularly those amino acids which exhibit limited aqueous
solubility and cellular uptake (e.g., tryptophan, tyrosine,
glutamine, aspartic acid, asparagine, glutamic acid, histidine,
isoleucine, leucine, methionine, and phenylalanine), in both
children and adults with short bowel syndrome. When used for the
treatment of patients with short bowel syndrome, the combination of
therapeutically effective concentrations of amino acids and an
effective amount of carbohydrate carrier provide increased levels
of cellular uptake of amino acids into the intestinal epithelium,
thereby providing a greater benefit to the patient and decreasing
the amounts of amino acids that must be administered in order to
achieve satisfactory therapeutic levels.
[0100] The combination of amino acids and carbohydrate carrier can
be administered by a variety of pharmaceutically acceptable routes,
including tablets, caplets, or capsules coated for delivery to the
intestines or colon, as well as enema solutions or suspensions.
Therapeutic dosages can be determined by the patient's physician,
taking into consideration the age, size, and nutritional status of
the patient.
Enhancing Amino Acid Absorption in Dialysis Patients
[0101] Dialysis patients commonly exhibit malnutrition. However,
supplementation with a mixture of 8 essential and 9 nonessential
amino acids has been shown to improve both health and mood of
dialysis patients. (Mastroiacovo, P., et al., Clin. Ther. (1993)
15(4): 698-704.) In the method of the present invention, a
combination of amino acids, in therapeutically effective amounts,
is combined with an effective amount of a carbohydrate carrier to
enhance solubility and cellular uptake of the amino acids, thereby
increasing the therapeutic effect of amino acid supplementation and
decreasing the dosage of amino acid required to achieve therapeutic
effect.
[0102] A preferred mode of administration for dialysis patients is
an enteric coated capsule, caplet, tablet, or coated bead
containing a therapeutically effective amount of each of a variety
of amino acids in combination with an effective amount of a
carbohydrate carrier, such as sucrose or a polyol such as xylitol
or sorbitol. For administration to diabetic patients, the preferred
carbohydrate carrier is a polyol.
Enhanced Absorption of Glutamine for the Treatment of Wounds
[0103] Glutamine is precursor for the synthesis of nucleotides. It
is both an activator of protein synthesis, and an inhibitor of
protein degradation. It is an activator of glycogen synthesis, as
well as a metabolic substrate for rapidly dividing cells. It is
also an energy source for epithelial cells. Treatment of wounds,
whether superficial or non-superficial, with the composition
described for enhancing amino acid absorption, increases the
absorption of glutamine into epithelial tissues, promoting more
rapid wound healing. In addition to promoting wound healing by
increasing glutamine absorption, however, the method provides a
treatment which protects the wound from infection with pathogenic
organisms. Filling infected wounds with sugar has been a practice
for centuries. Honey has long been known to have antibacterial
properties, due, in part, to the hypertonic sugar concentration.
(Basson, N. et al., J. Dent. Assoc. S. Afr. (1994) 49(7): 339-341;
Jeddar, A., et al., S. Afr. Med. J. (1985) 67(7): 257-258; Willix,
D., et al., J. Appl. Bacteriol. (1992) 73(5): 388-394.)
[0104] A combination of sugar and povidone-iodine has been
effective in promoting rapid healing, reducing bacterial
contamination, and filling of defects with granulation tissue when
used to treat patients for wounds, burns, and ulcers. (Knutson, R.,
et al., South Med. J. (1981) 74(11: 1329-1335.) However, while
adding to the antibacterial properties of the hypertonic sugar
environment, povidone-iodine kills white blood cells.
[0105] Combining glutamine with a carbohydrate carrier, therefore,
provides a dual benefit for wound care: the increased glutamine
absorbed by the epithelial cells provides an energy source for the
epithelial cells, promoting cell division and healing, while also
providing an energy source for the white blood cells needed to
protect the underlying tissues from bacterial invasion, and the
carbohydrate carrier protects the surface of the wound from
bacterial contamination by providing an environment in which the
high osmotic pressure and low water availability prevents microbial
growth.
[0106] For wound care, the combination of a therapeutically
effective amount of glutamine and a carbohydrate carrier,
preferably sucrose or honey, is applied topically as a semi-solid
formulation of a high concentration of sugar mixed with water and
glutamine. Alternately, the combination is provided as a thick
syrup for topical application to the affected area. Another
alternative method of application is to provide the formulation as
a solid to be applied to the wound area, drawing its aqueous
fraction from the wound environment. Such a preparation, if
provided in powdered or crystalline form, can be easily placed in a
first-aid kit or other emergency care kit for wound treatment.
[0107] The combination can be especially effective for the
treatment of burns, where the primary goals of treatment are
protection of the tissue from infection and rapid regeneration of
new tissue.
Enhancing Glutamine Absorption for the Treatment of Mucositis and
Stomatitis
[0108] Mucositis is an inflammatory reaction, characterized by
burn-like lesions or ulcerative lesions of the epithelial tissue of
the gastrointestinal tract from mouth to anus. It may result from
exposure to either ionizing radiation or chemotherapeutic agents.
Stomatitis is any inflammatory reaction affecting the oral mucosa,
with or without accompanying ulceration. Mucositis, particularly,
is often further complicated by infection of the ulcerative
tissue.
[0109] Studies have previously shown that oral application of
glutamine solutions can improve the symptoms accompanying mucositis
in some bone marrow transplant patients and chemotherapy patients.
(Skubitz, K., and P. Anderson, J. Lab. Clin. Med. (1996) 127(2):
223-228; Anderson, P., et al., Bone Marrow Transplant (1998) 22(4):
339-344; Anderson, P., et al., Cancer (1998) 83(7): 1433-1439; U.S.
Pat. No. 5,545,668 (Skubitz, et al., Aug. 13, 1996); and U.S. Pat.
No. 5,438,075 (Skubitz, et al., Aug. 1, 1995.) Using the
composition and method described herein, increased and effective
intracellular glutamine concentrations can be delivered to
epithelial tissues of the gastrointestinal system for the treatment
of mucositis or stomatitis without increasing the absolute
glutamine dosage.
[0110] In the method of the invention, the composition can be
provided, for example, as a mouthwash, swish and swallow
preparation, lozenge, or hard candy for treatment of oral
ulcerations. For esophageal ulcers, a drink, including a sugared
drink, a milkshake, or a frozen slurry can be used. Biodegradable
inserts can also be used to treat the mouth and throat. Children,
as well as adults, with mucositis or stomatitis can be treated
using any of these preparations, but may prefer a preparation of
carbohydrate, glutamine, and flavorings delivered as a popsicle or
in combination with sherbet, an ice, or ice cream. These methods of
delivery provide the added benefit of soothing cold on the
ulcerative tissue. A chewing gum preparation, preferably a chewing
gum with a semi-solid or liquid center, can also be used for the
treatment of oral and esophageal ulcers.
[0111] For gastric ulcer therapy, tablets, capsules, capsules, or
coated beads containing the carbohydrate/glutamine composition can
be administered. For intestinal ulcerations, coated tablets,
caplets, capsules, or coated beads can be administered for either
enteric or colonic delivery. Methods for providing enteric coatings
or coatings for colonic delivery are known in the art and have been
described previously herein.
Enhancement of Glutamine Absorption for the Treatment of
Cryptosporidiosis
[0112] Cryptosporidium parvum is a leading cause of persistent
diarrhea in developing countries. Due to its resistance to
chlorine, it has also become a threat in some United States water
supplies. Cryptosporidiosis is particularly problematic in AIDS
patients, the elderly, and the very young, in whom it causes a
severe, life-threatening diarrhea. Cryptosporidium parvum infects
the intestinal tissue, but does not infect beyond the most
superficial surface of the intestinal epithelium. In a piglet
model, approximately two-thirds of the intestinal villus surface
area was damaged during Cryptosporidia infection. In the remaining
epithelial tissue, increased glutamine metabolism is associated
with a sodium-hydrogen exchange coupled to a chloride transport
mechanism. Because of its direct association with the chloride
transport mechanism, glutamine can be particularly therapeutic for
repair of tissue damaged by Cryptosporidium infection. (Guerrant,
R., Emerging Infectious Diseases (1997) 3(1): 51-57.) Infected
tissue has lost much of the absorptive surface area, however, and
the method of the present invention, by treating the patient with
the composition of carbohydrate carrier and a therapeutic dose of
glutamine, enhances glutamine uptake in the remaining cells to
compensate for the decreased absorptive surface area.
[0113] The composition can be administered using a coated capsule,
tablet, or caplet for intestinal delivery. Alternately, the
composition can be infused or administered as an enema solution to
coat the intestinal lining with the glutamine/carbohydrate carrier
and enhance glutamine absorption into the remaining intestinal
epithelial cells.
[0114] The method can also be useful as a factor in disease
prevention, since glutamine is known to provide a primary energy
source for white blood cells, which migrate among the cells of the
intestinal lining and are responsible for destruction of pathogenic
organisms such as C. parvum. Enhancement of glutamine absorption
into the epithelial and white blood cells by the method of the
present invention therefore provides a method for improving the
immune response while maintaining the structural integrity of the
epithelial lining of the intestine. For patients at risk for
Cryptosporidium infection, enteric-coated capsules can be
administered to maintain epithelial cell integrity and improve the
immune response.
Enhancement of Glutamine Absorption to Improve Post-Surgical Wound
Healing in the Gastrointestinal Tract
[0115] Following surgical resection within the oral cavity, the
intestine, or bowel, epithelial tissue damage can be treated by the
method of the present invention to increase tissue integrity and
promote wound healing. Following oral surgery, a swish and swallow
preparation, mouthwash, lozenge, candy, or chewing gum preparation
containing the composition of the present invention can be provided
to the patient to allow easy administration of a therapeutically
effective dose of glutamine in combination with a carbohydrate
carrier. Particularly in patients who have undergone oral surgery,
non-cariogenic carbohydrate carriers are preferred. Such sugar
carriers include, for example, maltitol, lactitol, sorbitol, and
xylitol. The most preferable polyol carbohydrate carrier for
incorporation into the composition is xylitol.
[0116] Following intestinal surgery, the composition can be
administered in the form of a coated tablet, caplet, capsule, or
coated bead. The tablet, caplet, capsule, or coated bead can be
coated with an organic solvent, such as, for example, cellulose
acetate phthalate, cellulose acetate trimellitate, cellulose
acetate succinate, hydroxypropyl methyl cellulose phthalate,
hydroxypropyl methyl cellulose acetate succinate, and carboxy
methyl ethyl cellulose, for enteric delivery. A tablet, caplet, or
capsule can be coated with an acrylic-based resin to dissolve at
higher pH (pH 7) to provide delivery to the distal ileum and colon.
Alternatively, delivery of the glutamine/carbohydrate carrier
composition can be provided in the form of a suppository, using a
base such as cocoa butter or other glyceride, or as a rectal tablet
without a conventional suppository base. Such compositions for
suppository use have been described by Mizuno, et al., in U.S. Pat.
No. 4,462, 984, and Harwood, et al., in U.S. Pat. No.
4,439,194.
[0117] For treatment of diabetic patients, xylitol is the preferred
carbohydrate carrier, as sorbitol is not absorbed in the intestine
and could cause added intestinal discomfort.
Enhancement of Glutamine Absorption for Treatment of Low Birth
Weight Infants
[0118] Neu, et al., have reported that very-low-birth-weight
neonates who receive enteral glutamine supplementation have an
increased survival rate. (J. Pediatrics, (1997) 131(5): 691-699.)
The method of the present invention provides increased therapeutic
intracellular glutamine dosages with decreased actual glutamine
administration. In low-birth-weight neonates, particularly,
achievement of the desired effect with smaller doses of nutrient
can be essential.
[0119] For delivery of the composition, an enteral feeding tube is
preferred. Any one of a number of carbohydrate carriers can be
chosen, although sucrose and high fructose corn syrup are
preferred. The therapeutic dosage of glutamine can be determined by
the individual physician, using standard means of dosage
calculation, bearing in mind that glutamine absorption is enhanced
by combination with the carbohydrate carrier to levels of at least
ten times higher than that achieved by administration of glutamine
alone. Excipients can be added to the feeding formula, including
flavorings and stabilizers. Added nutrients can also be included,
including vitamins, amino acids, and recommended nutrients such as
lactoferrin.
Enhancement of Glutamine Absorption to Treat Dermatological Lesions
of Viral and Bacterial Origin
[0120] A number of viral illnesses can be recognized by epithelial
lesions. Among these are, for example, herpetic lesions around the
mouth, the lesions associated with impetigo, and the painful
lesions known as shingles, characteristic of varicella-zoster
virus. The method of the present invention can be used to treat
such lesions by topically applying the glutamine/carbohydrate
carrier composition to the affected area. The glutamine component
of the composition aids in healing by providing energy to the
epithelial cells, while the sugar provides antibacterial properties
to protect the damaged or infected tissue from further
infection.
[0121] For topical application, a lotion or cream is preferred,
incorporating glutamine, a carbohydrate carrier, and excipients
such as stabilizing agents, gelling agents, or thickening
agents.
Enhancement of Glutamine Absorption to Treat Patients Infected with
Human Immunodeficiency Virus
[0122] Gastrointestinal lymphoid tissue harbors more than 90% of
the total lymphocytes in the body. Studies have shown that the
gastrointestinal epithelium contains a large population of
CD34.sup.+ CD4-progenitors. (Mattapallil, J., et al., J. Virol.
(1999) 73(5): 4518-4523.) The gastrointestinal tract has also been
demonstrated to be a major site of CD4.sup.+ T cell depletion and
viral replication in simian immunodeficiency virus infection. Other
studies have shown that glutamine enhances production of T
lymphocyte-derived cytokines. (Yaqoob, P. and P. Calder, Cytokine
(1998) 10(10): 790-794.) Enhancing glutamine absorption into the
intestinal mucosa by the method of the present invention therefore
can provide a therapeutic benefit to HIV-infected patients,
particularly those patients who are in the early stages of
infection. Enhancement of the cytokine response to the viral
infection can contribute to viral destruction by the immune system
at the site of significant viral replication.
[0123] The glutamine/carbohydrate carrier composition can be
administered in the form of an enteric-coated tablet, caplet,
capsule, or coated bead. Suitable sugar carriers will preferably
include, for example, sucrose, glucose, high fructose corn syrup,
and xylitol.
[0124] Daily administration of recommended dietary levels of
glutamine is preferred, since administration of this quantity of
glutamine by the method of the present invention can result in an
increased delivery of glutamine to the intestinal epithelium by a
factor of, for example, 10-30.times.. Therefore, administration of
more moderate amounts can produce an even greater intracellular
concentration of glutamine than has been previously been achieved
by administration of higher dosages of glutamine alone.
Enhancement of Glutamine Absorption for Cancer Therapy
[0125] Glutamine supplementation can be beneficial for cancer
therapy for both its direct and indirect results. Glutamine
supplementation has been shown to increase glutathione release from
the gut in Fisher-344 rats. (Cao, Y., et al., J. Parenter. Enteral
Nutr. (1998) 22(4): 224-227.) When given in conjunction with either
radiation or chemotherapy, glutamine has been demonstrated to
increase selectivity of either therapy for tumor cells. (Klimberg,
V. and J. McClellan, Am. J. Surg. (1996) 172(5): 418-424.) In one
study, tumor growth in rats receiving glutamine, either by gavage
or as a food additive, decreased by 40% within three weeks. (Fahr,
M., et al., J. Parenter. Enteral Nutr. (1994) 18(6): 471-476.) In a
separate study, tumor volume loss in rats receiving methotrexate
was nearly doubled when glutamine was added to the diet. (Klimberg,
V., et al., J. Parenter. Enteral Nutr. (1992) 16 (6 Suppl):
83S-87S.) Decreased tumor growth in glutamine-supplemented rats has
been correlated with greater natural killer cell activity,
presumably due to glutathione-mediated suppression of prostaglandin
E2 (PGE2) synthesis. (Klimberg, V., et al., J. Surg. Res. (1996)
63(1): 293-297.)
[0126] By providing normal cells with an energy source and a means
to accomplish cellular repair, glutamine supplementation has also
been indirectly associated with increased tolerance to
chemotherapeutic agents.
[0127] The composition and method of the present invention provide
increased glutamine absorption into gastrointestinal epithelial
cells. Once absorbed into these cells, more glutamine is made
available to circulate to other tissues of the body. Enhancement of
absorption of glutamine also provides a means to increase
glutathione production in the intestine. Cancer therapy can
therefore consist of, or be enhanced by, daily administration of
glutamine in admixture with an amount of carbohydrate carrier, such
as, for example, sucrose, glucose, xylose, xylitol, high fructose
corn syrup or corn syrup solids effective to increase glutamine
absorption into the gastrointestinal epithelium. The composition
and method can be used for both human and veterinary cancer
therapy.
[0128] Daily doses of glutamine will be determined by the
individual patient's physician, taking into consideration factors
which are known by those of skill in the art to affect dosage
calculation, such as, for example, body size and age. Recommended
daily doses of glutamine for cancer therapy are preferably at least
at the maximum dietary intake of 3-4 grams per day, although lower
doses can be administered, since the composition and method of the
present invention increase glutamine absorption by at least a
factor of ten, and more preferably, 100.
Other Uses for a Method for Increased Amino Acid Absorption
[0129] Although the method for treating physiological disorders in
patients has been described primarily in terms of administration of
glutamine, the invention is not intended to be limited to a method
of administering enhanced levels of glutamine alone. For example,
D-serine has been demonstrated to be therapeutic for the treatment
of schizophrenia when administered in conjunction with
antipsychotic medications. (Tsai, G., et al., Biol. Psychiatry
(1998) 44(11): 1081-1089.) Enhanced absorption of D-serine into the
intestinal epithelia after oral administration, can, therefore,
provide a method for increasing available D-serine for systemic
circulation. Canavan disease, an autosomal genetic disorder, is
proposed to benefit from supplementation of dietary aspartic acid.
(Baslow, M. And T. Resnik, J. Mol. Neurosci. (1997) 9(2): 109-125.)
Early detection of the disease, therefore, can be accompanied by
aspartic acid supplementation by the method of the present
invention to enhance uptake of aspartic acid, an amino acid with an
aqueous solubility of only 0.778 g/100 g at 25.degree. C., to
protect against the progressive degeneration of the brain which is
characteristic of the disease.
[0130] These are only two examples of a number of physiologic
conditions which can be therapeutically treated using enhanced
amino acid absorption provided by the method of the present
invention. As amino acids are identified as having therapeutic
value, dietary supplementation can be further enhanced by providing
the amino acid supplement in combination with a carbohydrate
carrier as described by the method of the invention.
Veterinary Use for Enhanced Amino Acid Absorption into Epithelial
Cells
[0131] The early-weaned pig develops intestinal atrophy, and
glutamine supplementation has been proposed to prevent intestinal
epithelial damage and provide a benefit in swine production. (Wu,
et al., J. Nutr. (1996) 126 (10): 2578-84.) The composition and
method of the present invention can be used to enhance amino acid
absorption into those epithelial tissue cells, thereby decreasing
costs associated with amino acid supplementation. The composition
and method are also useful for veterinary treatment of dogs and
other mammals in whom chemotherapy has been initiated. For example,
doxorubicin, associated with gastrointestinal ulcers in human
chemotherapy patients, is the recommended treatment for a number of
other mammalian cancers, including canine hemangiosarcoma. The
composition and method of the present invention provide enhanced
amino acid absorption into the damaged epithelium of the mammalian
subject, as well as increasing systemically available amino acid by
increasing absorption into the gastrointestinal epithelium.
Stable Glutamine Preparations for Administration to a Patient
[0132] The present invention also describes a composition for
providing glutamine to a patient in a form which has improved
aqueous solubility and stability. In one form, the composition can
be provided as a granulated or powdered drink mix, contained in
bulk packaging or packaged as individual doses. Before
administration, the preparation can be constituted with water,
juice, or other liquid to provide for easy administration and
increase the absorption of glutamine into the epithelial tissue.
Glutamine can also be provided in stable form with the sugar
carrier as a solid solution in the form of a candy or lozenge. The
patient can administer the glutamine/carbohydrate carrier
composition by simply placing the candy or lozenge into his mouth
and allowing it to remain there while the surrounding fluids
dissolve it. In this aqueous environment, the carbohydrate can
provide the carrier to facilitate absorption of the glutamine into
the epithelial cells of the oral cavity, the esophagus, and the
stomach.
[0133] Either the granulated/powdered formulation or the solid
solution can also be administered to the environment of the small
intestine or the large intestine by adding an enteric coating or an
acrylic-based resin as previously described for delivery to the
distal ileum or colon.
[0134] In any of these preparations, glutamine has a stable
shelf-life and can be provided to the patient well in advance of
the time of administration. The preparations can be stored in the
clinic or the patient's home for administration as needed.
[0135] The invention is described with reference to various
specific and preferred embodiments and techniques. However, it
should be understood that many variations and modifications may be
made while remaining within its scope.
[0136] All referenced publications, patents and patent documents
are intended to be incorporated by reference, as though
individually incorporated by reference.
* * * * *