U.S. patent application number 13/140353 was filed with the patent office on 2012-01-19 for sustained release of nutrients in vivo.
This patent application is currently assigned to New World Pharmaceuticals, LLC. Invention is credited to Sitaraman Krishnan, Frederick A. Sexton, Venkat Kalyan Vendra.
Application Number | 20120015039 13/140353 |
Document ID | / |
Family ID | 42316829 |
Filed Date | 2012-01-19 |
United States Patent
Application |
20120015039 |
Kind Code |
A1 |
Sexton; Frederick A. ; et
al. |
January 19, 2012 |
SUSTAINED RELEASE OF NUTRIENTS IN VIVO
Abstract
Nutritional compositions delivered in vivo in a time controlled
manner sustainable over long periods of time, provide enhancing
athletic performance, increased hand/eye coordination and
concentration on the task at hand.
Inventors: |
Sexton; Frederick A.;
(Rumson, NJ) ; Krishnan; Sitaraman; (Potsdam,
NY) ; Vendra; Venkat Kalyan; (Louisville,
KY) |
Assignee: |
New World Pharmaceuticals,
LLC
Rumson
NJ
|
Family ID: |
42316829 |
Appl. No.: |
13/140353 |
Filed: |
December 17, 2009 |
PCT Filed: |
December 17, 2009 |
PCT NO: |
PCT/US2009/068608 |
371 Date: |
September 29, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12337022 |
Dec 17, 2008 |
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13140353 |
|
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61014251 |
Dec 17, 2007 |
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Current U.S.
Class: |
424/493 ;
424/489; 424/490; 424/499; 514/23; 514/53; 514/54; 514/58;
514/60 |
Current CPC
Class: |
A23L 29/206 20160801;
A23L 33/125 20160801; A23P 10/30 20160801; A23L 29/272 20160801;
A23P 10/25 20160801; A23L 33/24 20160801; A23L 2/52 20130101; A23L
33/40 20160801; A61P 3/02 20180101; A23L 29/30 20160801; A23V
2002/00 20130101; A61P 5/50 20180101; A23L 5/00 20160801; A61P 3/08
20180101; A23L 33/175 20160801; A23L 33/26 20160801; A23L 33/25
20160801; A23L 33/30 20160801; A23L 33/15 20160801; A23G 4/06
20130101; A23L 33/16 20160801; A23V 2002/00 20130101; A23V 2200/33
20130101; A23V 2250/51086 20130101; A23V 2250/51 20130101; A23V
2250/5026 20130101; A23V 2250/606 20130101; A23V 2250/608 20130101;
A23V 2250/616 20130101; A23V 2250/612 20130101; A23V 2250/5114
20130101; A23V 2002/00 20130101; A23V 2200/33 20130101; A23V
2250/06 20130101; A23V 2250/50 20130101 |
Class at
Publication: |
424/493 ; 514/23;
424/489; 424/499; 514/53; 514/54; 514/58; 514/60; 424/490 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 31/7016 20060101 A61K031/7016; A61K 31/715
20060101 A61K031/715; A61P 3/02 20060101 A61P003/02; A61K 31/716
20060101 A61K031/716; A61K 31/718 20060101 A61K031/718; A61K 9/50
20060101 A61K009/50; A61K 31/70 20060101 A61K031/70; A61K 31/7004
20060101 A61K031/7004 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 17, 2008 |
US |
PCT/US08/87104 |
Claims
1. A composition for in vivo consumption comprising: nutritional
supplements; and, compounds for time controlled and sustained
release of the nutritional supplements in vivo, wherein
carbohydrate release and absorption kinetics of the composition are
different than a composition without compounds for time controlled
and sustained release of the nutritional supplements in vivo.
2. The composition of claim 1, wherein the carbohydrate release and
absorption kinetics comprise a change in blood glucose
concentration.
3. A method for manufacturing particles for time controlled and
sustained release of the nutritional supplements in vivo,
comprising: (a) heating a solution of hydroxypropyl cellulose (HPC)
above a lower critical solution temperature; (b) crosslinking
polymer chains to obtain microparticle hydrogels; and (c) loading
the microparticle hydrogels with one or more carbohydrates, wherein
the particles result in control over a partitioning coefficient and
associated particle release and absorption kinetics.
4. The method of claim 3, wherein the one or more carbohydrates in
(c) is selected from the group consisting of: monosaccharides,
disaccharides, polysaccharides, and combinations thereof.
5. The method of claim 4, wherein the one or more carbohydrates is
selected from the group consisting of: dextrose, fructose,
galactose, sucrose, maltose, lactose, polydextrose, dextrins, corn
syrup solids, starch and combinations thereof.
6. The method of claim 3, wherein the polymer chains are
crosslinked with trisodium trimetaphophate (TSTMP).
7. The composition of claim 1, wherein the composition comprises a
hydrogel.
8. The composition of claim 7, wherein the hydrogel comprises a
polysaccharide.
9. The composition of claim 8, wherein the polysaccharide is
selected from the group consisting of a thermally responsive
polysaccharide, a hydrophobically modified polysaccharide, a pH
responsive polysaccharide, and combinations thereof.
10. The composition of claim 9, wherein the polysaccharide is
hydroxypropyl cellulose.
11. The composition of claim 9, wherein the polysaccharide is
sodium alginate.
12. A composition for in vivo consumption comprising: one or more
carbohydrates; and, compounds for time controlled and sustained
release of the carbohydrates in vivo.
13. The composition of claim 1 or 12, wherein the composition
comprises hydrogel particles.
14. The composition of claim 13, wherein the hydrogel particles
sequester the carbohydrates.
15. The composition of claim 14, wherein the carbohydrates are
released from the hydrogel particles at a rate determined by
diffusion of the carbohydrates inside the hydrogel particles.
16. The composition of claim 13, wherein the hydrogel particles
comprise a polysaccharide.
17. The composition of claim 12, the polysaccharide is selected
from the group consisting of: a thermally responsive
polysaccharide, a hydrophobically modified polysaccharide, a pH
responsive polysaccharide, and combinations thereof.
18. The composition of claim 17, wherein the polysaccharide is
hydroxypropyl cellulose.
19. The composition of claim 17, wherein the polysaccharide is
sodium alginate.
20. The composition of claim 13, wherein the hydrogel particles are
coated with a polymer.
21. The composition of claim 20, wherein the polymer is a
pH-responsive polysaccharide.
22. The composition of claim 1, wherein the composition can sustain
blood glucose concentrations above fasted state levels during rest
for a longer duration than an equal volume of the composition
without compounds for time controlled and sustained release of the
nutritional supplements in vivo.
23. The composition of claim 1, wherein the composition can sustain
blood glucose concentrations above fasted state levels during low-,
moderate-, or high-intensity exercise for a longer duration than an
equal volume of the composition without compounds for time
controlled and sustained release of the nutritional supplements in
vivo.
24. The composition of claim 1, wherein in vivo administration of
the composition results in a lower insulin response than in vivo
administration of the composition without compounds for time
controlled and sustained release of nutritional supplements.
25. The composition of claim 1, wherein in vivo administration of
the composition results in increased utilization of fat stores than
in vivo administration of the composition without compounds for
time controlled and sustained release of nutritional
supplements.
26. The composition of claim 12, wherein the carbohydrate has a
high glycemic index.
27. The composition of claim 1, wherein the nutritional supplements
are selected from the group consisting of: carbohydrates, amino
acids, lipids, electrolytes, and vitamins.
28. The composition of claim 27, wherein the electrolytes are
selected from the group consisting of: sodium, potassium,
magnesium, chloride, calcium, bicarbonate, phosphate, and
sulfate.
29. The method of claim 3, wherein the partitioning coefficient and
associated particle release and absorption kinetics comprise a
diffusional barrier at acidic pH for the one or more carbohydrate
molecules of (c) within the particles.
30. The method of claim 29, wherein the acidic pH is less than pH
3.8.
Description
INCORPORATION BY REFERENCE
[0001] This application claims priority to International
Application No. PCT/US2008/087104, filed Dec. 17, 2008 and to U.S.
Application Serial No. 12/337,022, filed Dec. 17, 2008, both of
which claim the benefit of priority of U.S. provisional patent
application Ser. No. 61/014,251 filed Dec. 17, 2007.
[0002] The foregoing applications, and all documents cited therein
or during their prosecution ("appln cited documents") and all
documents cited or referenced in the appln cited documents, and all
documents cited or referenced herein ("herein cited documents"),
and all documents cited or referenced in herein cited documents,
together with any manufacturer's instructions, descriptions,
product specifications, and product sheets for any products
mentioned herein or in any document incorporated by reference
herein, are hereby incorporated herein by reference, and may be
employed in the practice of the invention.
FIELD OF THE INVENTION
[0003] The invention relates to compositions for increasing
performance of athletes by providing appropriately timed release
and increased absorption of nutrients including carbohydrates,
amino acids, and electrolytes.
BACKGROUND OF THE INVENTION
[0004] Contemporary understanding of sports related nutritional
requirements indicates that carbohydrate consumption plays a key
role improving athlete endurance and performance. With the entrance
of Gatorade.RTM. and other similar sports drinks beginning in the
mid 1960's more attention has been paid to understanding the role
of various nutrients on human performance in athletic events.
[0005] Understanding the body's ability to absorb and process
carbohydrates and other nutrients for maximal performance output
has been studied by many. In 2003, the International Olympic
Committee on Nutrition for Athletes issued a position stating that
a high carbohydrate diet in the days before competition helps
enhance performance, particularly when exercise lasts longer than
about 60 min and that athletes should aim to achieve carbohydrate
intakes that meet the fuel requirements of their training programs
and also adequately replace their carbohydrate stores during
recovery between training sessions and competition. However, this
is a difficult task, especially for athletes in training who need a
sustained supply of carbohydrates and other nutrients without the
drawback of an intake of large amounts or smaller amounts but at
more regular intervals, of food in order to meet the dietary
requirements necessary to maintain enhanced performance.
[0006] There is thus a need in the art to provide a sustained and
controlled supply of carbohydrates and other nutrients to
individuals without the necessity of intake of excess food and
snacks.
[0007] Citation or identification of any document in this
application is not an admission that such document is available as
prior art to the present invention.
SUMMARY OF THE INVENTION
[0008] The inventors have discovered compositions for in vivo
administration, which surprisingly provide an elevated and
prolonged glycemic response compared to conventional food products,
such as GATORADE.RTM.. The present invention is based upon the
premise that athletic performance can be improved by providing
controlled release of nutritional supplements (such as
carbohydrates and electrolytes).
[0009] The present invention relates to a composition comprising
nutritional supplements (such as carbohydrates, amino acids,
vitamins, and/or electrolytes), which when administered to a human
provides a sustained release of the nutritional supplements over an
extended period of time. Preferably, the nutritional supplements
may be delivered in a sustained and extended manner for peak
athletic performance and recovery. For instance, the composition
may release the nutritional supplements so that absorption and
oxidation rates of exogenous carbohydrates are increased during
exercise. According to one embodiment, the composition releases
nutritional supplements such that saturation of the SGLT1 and
GLUT-5 transporters with exogenous carbohydrates is maintained
during exercise.
[0010] In a preferred embodiment, a composition for in vivo
consumption may comprise nutritional supplements; and, compounds
for sustained release of the nutritional supplements in vivo.
Preferably, the nutritional supplements may comprise carbohydrates,
amino acids, vitamins, and electrolytes.
[0011] In another preferred embodiment, the compounds for the
sustained release of the nutritional supplements may comprise one
or more components selected from biodegradable polymers,
bioadhesives, nanoparticles, colloidal suspensions and binders.
[0012] According to one embodiment, the biodegradable polymers and
binders may be selected from poly(lactide)s, poly(glycolide)s,
poly(lactide-co-glycolide)s, poly(lactic acid)s, poly(glycolic
acid)s, poly(lactic acid-co-glycolic acid)s, polycaprolactone,
polycarbonates, polyesteramides, polyanhydrides, poly(amino acids),
polyorthoesters, polyacetyls, polycyanoacrylates, polyetheresters,
poly(dioxanone)s, poly(alkylene alkylate)s, copolymers of
polyethylene glycol and polyorthoester, biodegradable
polyurethanes, hydrogels, blends and copolymers thereof.
[0013] In an advantageous embodiment, the invention encompasses
nanoparticles and microparticles comprising hydrogels.
Advantageously, the hydrogels may comprise modified
polysaccharides. The polysaccharides may comprise modifications
with chemical groups such as carboxylic acid (as in carboxymethyl
cellulose [CAS no. 9000-11-7], starch glycolate [CAS no.
9057-06-1], etc.), acryloyl or methacryloyl (as in starch acrylate
[CAS no. 39316-65-9], hydroxypropyl cellulose acrylate [CAS no.
94187-94-7], hydroxyethyl starch acrylate, etc.), hydroxyalkyl (as
in hydroxypropyl cellulose [CAS no. 9004-64-2], hydroxyethyl starch
[CAS no. 9005-27-0], hydroxyethyl cellulose [CAS no. 9004-62-0],
etc.), and ester (as in starch acetate, starch phosphate, etc.).
The modified starch may also contain grafted fatty acid moieties
(as in starch adipate [CAS no. 39347-22-3], etc.), oligo(alkylene
oxide) grafts (as in hydroxypropyl cellulose, etc.), grafted
hydrophobes such as cholesterol, grafted amphiphiles such as
alkenylsuccinate (as in 1-octenylsuccinic acid anhydride modified
starch, etc.) or side chain derivatizations to introduce aldehydic
or carboxylic groups.
[0014] In another advantageous embodiment, the invention may also
comprise methods of crosslinking of the microparticles and
nanoparticles of the present invention to form hydrogels.
Crosslinking may be performed using free radical initiators such as
persulfate salts, or redox systems involving ascorbic acid, or a
naturally occurring crosslinker such as genipin. Ionic crosslinking
may also be contemplated, such as with anionic polysaccharides such
as gellan.
[0015] In another yet advantageous embodiment, the invention may
also comprise the preparation of hydrogels. In an advantageous
embodiment, a blend of hydrophobically modified polysaccharide such
as, but not limited to, hydroxypropyl cellulose and a carboxy
containing polysaccharide such as, but not limited to, alginate or
carboxymethyl cellulose may be used to prepare the hydrogel
particles of the present invention. In another advantageous
embodiment, nanoparticle suspensions may be synthesized by
self-assembly of chitosan and carboxymethyl cellulose hydrolysate,
wherein the polymers may be hydrolyzed with the enzymes chitosanase
and cellulase, respectively.
[0016] In another embodiment, hydrogels may be prepared from
mixtures of acidic polysaccharides such as, but not limited to,
alginates, and basic polysaccharides such as, but not limited to,
oligosaccharide derivatives of chitosan; a basic polysaccharide
such as, but not limited to, chitosan and anionic polysaccharide
such as, but not limited to, hyaluronic acid; alginate and oxidized
alginate blended with chitosan; grafted agar and sodium alginate
blend with acrylamide; gellan co-crosslinked with scleroglucan;
photocrosslinked modified dextran; starch reacted with glycidyl
methacrylate; or polymerizable saccharide monomers, such as
sucrose, created by reaction of the sugar with epoxy acrylate, or
methacryloyl chloride and acetyl chloride. The nanoparticle and
microparticle hydrogels may be prepared using polymer self-assembly
(temperature, pH, or ionic strength induced precipitation; micelle
formation; colloidal precipitation because of electrostatic
interactions between oppositely charged polymers) or conventional
emulsification-based methods.
[0017] In another preferred embodiment, the nutritional supplements
are released in vivo in a sustained manner and in a concentration
effective to increase exercise duration and cumulative power output
potential by at least about 1% to about 50%, about 50% to about
100%, about 100% to about 500% or about 500% to about 1000% when
compared to a control with no nutritional supplement ingestion pre
and during exercise.
[0018] In another preferred embodiment, the nutritional supplements
are released in vivo in a sustained manner and in a concentration
effective to increase burst energy duration and power output by
about 1% to about 50%, about 50% to about 100%, about 100% to about
500% or about 500% to about 1000% when compared to a control with
no nutritional supplement ingestion before and during exercise.
[0019] In another preferred embodiment, the nutritional supplements
are released in vivo in a sustained manner and in a concentration
effective to increase burst energy duration and power output by
about 1% to about 50%, about 50% to about 100%, about 100% to about
500% or about 500% to about 1000% when compared to a control with
no nutritional supplement ingestion pre and during exercise.
[0020] In yet another preferred embodiment, the nutritional
supplements are released in vivo in a sustained manner and in a
concentration effective to increase the number of effective burst
events by at least about 1% to about 50%, about 50% to about 100%,
about 100% to about 500% or about 500% to about 1000% when compared
to a control with no nutritional supplement ingestion pre and
during exercise.
[0021] In another preferred embodiment, the nutritional supplements
are released in vivo in a sustained manner and in a concentration
effective to decrease the duration of recovery time between burst
events by at least about 1% to about 50%, about 50% to about 100%,
about 100% to about 500% or about 500% to about 1000% when compared
to a control with no nutritional supplement ingestion before and
during exercise.
[0022] In another preferred embodiment, the nutritional supplements
are released in vivo in a sustained manner and in a concentration
effective to increase continuous exercise power output (watts) by
about 1% to about 50%, about 50% to about 100%, about 100% to about
500% or about 500% to about 1000%, advantageously about 50% at
about VO.sub.2 62% for 90 minutes and not less than about 25%
increase in high intensity (burst) exercise, about VO.sub.2 86%,
power output and duration following 90 min of continuous exercise,
and increase the number of effective "burst" periods by about 100%
as compared to performance when utilizing commercially available
performance enhancing drinks.
[0023] In yet another preferred embodiment, the compounds for
sustained release of the nutritional supplements in vivo release
the nutritional supplements in effective amounts and concentrations
over extended periods of time to increase exercise duration and
cumulative power output potential by about 1% to about 50%, about
50% to about 100%, about 100% to about 500% or about 500% to about
1000%, advantageously at least about 10% to about 70%, increase
burst energy duration and power output by at least about 10% to
about 70%, increase the number of effective burst events from about
10% to 100%, decreases the duration of recovery time between burst
events by at least about 10% to about 100%, when compared to a
control with no nutritional supplement ingestion pre and during
exercise.
[0024] In another preferred embodiment; the compounds for sustained
release of the nutritional supplements in vivo release the
nutritional supplements in effective amounts and concentrations
over extended periods of time to decrease duration of recovery time
between burst events by at least about 2%, about 5%, about 10%,
about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,
about 80%, about 90% or about 100% when compared to a control with
no nutritional supplement ingestion pre and during exercise.
[0025] In another preferred embodiment, the compounds for sustained
release of the nutritional supplements in vivo release the
nutritional supplements in effective amounts and concentrations
over extended periods of time to increase continuous exercise power
output (watts) by about 2%, about 5%, about 10%, about 20%, about
30%, about 40%, about 50%, about 60%, about 70%, about 80%, about
90% or about 100%, advantageously about 50% at about VO.sub.2 62%
for 90 minutes and not less than about a 25% increase in high
intensity (Burst) exercise, about VO.sub.2 86%, power output and
duration following 90 minutes of continuous exercise, and increase
the number of effective burst periods by at least about 100% as
compared to performance when utilizing commercially available
performance enhancing drinks.
[0026] In another preferred embodiment, the compositions are
formulated as a beverage, a gum, a food snack, a powder or any
other consumable.
[0027] In another preferred embodiment, a method of improving
athletic performance and endurance comprises administering to an
individual a composition comprising nutritional supplements and,
compounds for sustained release of the nutritional supplements in
vivo, wherein the nutritional supplements comprise carbohydrates,
amino acids, vitamins, and electrolytes; and, consuming the
composition wherein the compounds for sustained release of the
nutritional supplements in vivo release the nutritional supplements
in effective amounts and concentrations over extended periods of
time to increase exercise duration and cumulative power output
potential by at least about 1% to about 50%, about 50% to about
100%, about 100% to about 500% or about 500% to about 1000% about
10% to about 70%, increase burst energy duration and power output
by at least about 10% to about 70%, increase the number of
effective burst events from about 10% to 100%, decreases the
duration of recovery time between burst events by at least about 10
to about 100%, when compared to a control with no nutritional
supplement ingestion before and during exercise.
[0028] In another preferred embodiment, a composition for in vivo
consumption comprises one or more nutritional supplements and
compounds for time controlled and sustained release of the
nutritional supplements in vivo, wherein carbohydrate release and
absorption kinetics of the composition are different than a
composition without compounds for time controlled and sustained
release of the nutritional supplements in vivo. In some
embodiments, the carbohydrate release and absorption kinetics
comprise a change in blood glucose concentration. In a preferred
embodiment, the composition comprises a hydrogel. In preferred
embodiments, the nutritional supplements are selected from the
group consisting of: carbohydrates, amino acids, lipids,
electrolytes, and vitamins. Examples of electrolytes include
sodium, potassium, magnesium, chloride, calcium, bicarbonate,
phosphate, and sulfate.
[0029] In another preferred embodiment, a method for manufacturing
particles for time controlled and sustained release of the
nutritional supplements in vivo, comprises (a) heating a solution
of hydroxypropyl cellulose (HPC) above a lower critical solution
temperature; (b) crosslinking polymer chains to obtain
microparticle hydrogels; and (c) loading the microparticle
hydrogels with one or more carbohydrates, wherein the particles
result in control over a partitioning coefficient and associated
particle release and absorption kinetics. In some embodiments, the
one or more carbohydrates in (c) is selected from the group
consisting of monosaccharides, disaccharides, polysaccharides, and
combinations thereof. Examples of suitable carbohydrates include
dextrose, fructose, galactose, sucrose, maltose, lactose,
polydextrose, dextrins, corn syrup solids, starch and combinations
thereof. In certain embodiments, the polymer chains are crosslinked
with trisodium trimetaphophate (TSTMP). In certain embodiments, the
partitioning coefficient and associated particle release and
absorption kinetics comprise a diffusional barrier at acidic pH for
the one or more carbohydrate molecules of (c) within the particles.
In certain embodiments, the acidic pH is less than pH 3.8.
[0030] In another preferred embodiment, a composition of the
invention comprises a hydrogel, wherein the hydrogel comprises a
polysaccharide. In certain embodiments, the polysaccharide is
selected from the group consisting of a thermally responsive
polysaccharide, a hydrophobically modified polysaccharide, a pH
responsive polysaccharide, and combinations thereof. Examples of
suitable polysaccharides include hydroxypropyl cellulose and sodium
alginate.
[0031] In another preferred embodiment, a composition for in vivo
consumption comprises one or more carbohydrates; and compounds for
time controlled and sustained release of the carbohydrates in vivo.
In a preferred embodiment, the composition comprises hydrogel
particles. In yet another preferred embodiment, the hydrogel
particles sequester the carbohydrates. In a further embodiment, the
carbohydrates are released from the hydrogel particles at a rate
determined by diffusion of the carbohydrates inside the hydrogel
particles. In a preferred embodiment, the hydrogel particles
comprise a polysaccharide. Examples of suitable polysaccharides
include thermally responsive polysaccharides, hydrophobically
modified polysaccharides, and pH responsive polysaccharides, and
combinations thereof. In a particular embodiment, the
polysaccharide is hydroxypropyl cellulose. In another embodiment,
the polysaccharide is sodium alginate. In some embodiments, the
carbohydrate has a high glycemic index.
[0032] In another preferred embodiment, the hydrogel particles
according to the invention are coated with a polymer. In some
embodiments, the polymer is a pH-responsive polysaccharide.
[0033] In another preferred embodiment, a composition according to
the invention can sustain blood glucose concentrations above fasted
state levels during rest for a longer duration than an equal volume
of the composition without compounds for time controlled and
sustained release of the nutritional supplements in vivo.
[0034] In another preferred embodiment, a composition according to
the invention can sustain blood glucose concentrations above fasted
state levels during low-, moderate-, or high-intensity exercise for
a longer duration than an equal volume of the composition without
compounds for time controlled and sustained release of the
nutritional supplements in vivo.
[0035] In another preferred embodiment, in vivo administration of a
composition according to the invention results in a lower insulin
response than in vivo administration of the composition without
compounds for time controlled and sustained release of nutritional
supplements.
[0036] In another preferred embodiment, in vivo administration of a
composition according to the invention results in increased
utilization of fat stores than in vivo administration of the
composition without compounds for time controlled and sustained
release of nutritional supplements.
[0037] It is noted that in this disclosure and particularly in the
claims and/or paragraphs, terms such as "comprises", "comprised",
"comprising" and the like can have the meaning attributed to it in
U.S. Patent law; e.g., they can mean "includes", "included",
"including", and the like; and that terms such as "consisting
essentially of" and "consists essentially of" have the meaning
ascribed to them in U.S. Patent law, e.g., they allow for elements
not explicitly recited, but exclude elements that are found in the
prior art or that affect a basic or novel characteristic of the
invention.
[0038] These and other embodiments are disclosed or are obvious
from and encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] The following detailed description, given by way of example,
but not intended to limit the invention solely to the specific
embodiments described, may best be understood in conjunction with
the accompanying drawings, in which:
[0040] FIG. 1 depicts polysaccharides containing carboxylic acid
groups;
[0041] FIG. 2 depicts an oxidized scleroglucan derivative;
[0042] FIG. 3 depicts Passerini and Ugi multicomponent condensation
reactions;
[0043] FIG. 4 depicts the structure of genipin;
[0044] FIG. 5 depicts the structure of carrageenan;
[0045] FIG. 6 depicts vinyl-functionalized dextran;
[0046] FIG. 7 depicts glycidyl methacrylate adduct of starch
resulting from (a) transesterification, and (b) ring opening of the
epoxy group;
[0047] FIGS. 8A and 8B depict crosslinking of polysaccharides
containing hydroxyl groups. P represents a polysaccharide
segment;
[0048] FIG. 9 depicts a crosslinked hydroxypropyl cellulose
macrogel and a colloidally stable dispersion of hydroxypropyl
cellulose microgel particles in water;
[0049] FIG. 10 depicts crosslinking of hydroxyl containing
polysaccharides using TSTMP in the presence of sodium
hydroxide;
[0050] FIG. 11 depicts synthesis of acrylated hydroxypropyl
cellulose;
[0051] FIG. 12 depicts the chemical structure of hydroxylpropyl
cellulose;
[0052] FIG. 13 depicts chemical crosslinking using TSTMP; pH=11.5;
T=50.degree. C.; 1 h; R represents a polysaccharide chain;
[0053] FIG. 14 depicts enzymatic determination of glucose;
[0054] FIGS. 15A-15C depict (A) number-weighted particle size
distribution in the 4.4% (w/v) HPC dispersion, (B) mass-weighted
particle size distribution in the 4.4% (w/v) HPC dispersion, and
(C) scanning electron microscopy images of a crosslinked HPC
microparticles.
[0055] FIG. 16 depicts kinetics of glucose transport across the HPC
particles and through the membrane of the diffusion cell for: the
HPC dispersion of Example 2.1; and a glucose solution (without HPC
particles) that had the same overall concentration of glucose as
the HPC dispersion (8.7% w/w); pH=7; T=28.degree. C.
[0056] FIG. 17 depicts kinetics of glucose transport across the HPC
particles and through the membrane of the diffusion cell for: the
HPC dispersion of Example 2.9 containing sodium alginate as a
pH-responsive diffusional barrier; and a dispersion prepared by a
1:1 dilution of this dispersion with distilled water; pH=3.8;
T=28.degree. C.
[0057] FIG. 18 depicts kinetics of glucose transport across the HPC
particles and through the membrane of the diffusion cell for: the
HPC dispersion of Example 2.1 and a glucose solution (without HPC
particles) that had the same overall concentration of glucose as
the HPC dispersion (8.7% w/w); pH=7; T=37.degree. C.
[0058] FIG. 19 depicts kinetics of glucose transport across the HPC
particles and through the membrane of the diffusion cell for: the
HPC dispersion of Example 2.4; and a glucose solution (without HPC
particles) that had the same overall concentration of glucose as
the HPC dispersion (8.7% w/w); pH=2; T=37.degree. C.
[0059] FIG. 20 depicts the effect of pH on the kinetics of glucose
transport across the HPC particles and through the membrane of the
diffusion cell for the HPC dispersions of Example 2.12 that
contained sodium alginate as a pH-responsive diffusional barrier;
pH=7, 3.8, and 2; T=28.degree. C.
[0060] FIGS. 21A and 21B depict (A) blood glucose concentration vs.
time after consumption of 380 mL of the dispersion of Example 2.4,
and 380 mL of GATORADE.RTM. control and (B) normalized blood
glucose concentration profiles, relative to the fasted-state blood
glucose concentration. The subject remained seated on a chair
during the experiment.
[0061] FIG. 22 depicts normalized blood glucose concentration vs.
time, after consumption of 380 mL of a dispersion of Example 2.5,
and 380 mL of GATORADE.RTM. control. The subject remained seated on
a chair during the experiment.
[0062] FIG. 23 depicts normalized blood glucose concentration vs.
time, after consumption of two different dispersions of Example
2.5. The first dispersion contained pure dextrose, while the second
dispersion contained a 1:3 mass ratio of dextrose to fructose. The
overall sugar concentration in both the dispersions was the same.
The subject remained seated on a chair during the experiment.
[0063] FIG. 24 depicts normalized blood glucose concentration vs.
time, after consumption of 450 g of the dispersion of Example 2.7,
and 450 g of a 10 wt % dextrose solution in distilled water. The
subject remained seated on a chair during the experiment.
[0064] FIG. 25 depicts normalized blood glucose concentration vs.
time, after consumption of 450 g of the dispersion of Example 2.7:
(a) as a large bolus dose; and (b) in three aliquots, each weighing
150 g. The aliquots were consumed at t=0, 39 and 69 min. The
subject remained seated on a chair during the experiment.
[0065] FIG. 26 depicts normalized blood glucose concentration vs.
time, after consumption of 380 mL of: (a) the dispersion of Example
2.7 in three aliquots, and (b) GATORADES in three aliquots. The
subject remained seated on a chair during the experiment.
[0066] FIG. 27 depicts the effect of the consumption of 380 mL of
the HPC dispersion, of Example 2.4, before a continuous
moderate-intensity exercise (.about.60% {dot over
(V)}.sub.O.sub.2.sub.max) on a treadmill. GATORADE.RTM. with added
dextrose and water were used as positive and negative controls,
respectively.
[0067] FIG. 28 depicts the parameters used in quantitative analysis
of the blood glucose concentration profiles.
DETAILED DESCRIPTION
[0068] While the making and using of the various embodiments of the
present invention are discussed in detail below, it should be
appreciated that the present invention provides many applicable
inventive concepts which can be embodied in a wide variety of
specific contexts. The specific embodiments described herein are
merely illustrative of specific ways to make and use the invention
and do not delimit the scope of the invention or the scope of the
claims appended hereto.
[0069] According to one embodiment, the composition of the present
invention provides sustained release over extended periods of time
of carbohydrates and optionally other nutrients to an individual.
Preferably, carbohydrates are released from the composition in a
manner which maintains maximum carbohydrate absorption and
oxidation rates for a prolonged period of time. Generally, the
maximal carbohydrate absorption rate in an individual is in a range
of about 1.2 to about 1.7 g/min. A peak carbohydrate oxidation rate
of about 1.75 g/min can be achieved, for example, when
carbohydrates are administered as a blend of glucose (1.2 g/min)
and fructose (0.8 g/min). See, e.g., Azevedo, J. L. Jr.; Tietz, E.;
Two-Feathers, T.; Paull, J.; Chapman, K. Lactate, Fructose and
Glucose Oxidation Profiles in Sports Drinks and the Effect on
Exercise Performance. PLoS ONE [Online] 2007, 2, pp e927-e927
(doi:10.1371/journal.pone.0000927), for representative carbohydrate
oxidation profiles.
[0070] The rate and extent of exogenous carbohydrate absorption may
be limited not only by the amount of carbohydrate available but
also by the maximum intestinal transport capacity for glucose and
fructose. Intestinal transport of glucose is mediated by a sodium
dependent glucose transporter (SGLT1), located in the brush-border
membrane. SGLT1 transporters may become saturated at a glucose
ingestion rate of .about.1 g/min. Fructose on the other hand is
absorbed from the intestine by GLUT-5, a sodium-independent
facilitative fructose transporter. Generally, ingestion of a
mixture of carbohydrates that have different transport mechanisms
for absorption in to the blood stream, simultaneously increases
carbohydrate and water absorption.
[0071] Applicants have approximated the number of calories required
based on certain activity levels as indicated in Table 1. The
energy requirement is estimated based on the weight-normalized
Caloric burn rate (kcal per minute of activity per unit body mass
of an individual) and the Caloric content of glucose (.about.4
kcal/g). The energy available from exogenous glucose oxidation is
about 396 kcal. As can be seen from Table 1, this energy is
insufficient to meet the total energy requirement of the listed
activities. The present invention seeks to narrow the Caloric gap
using a controlled-release nutrient formulation.
TABLE-US-00001 TABLE 1 Energy requirement based on activity levels
Running Running Outside Competitive (8.5 min/ (12 min/ construction
soccer mile) mile) work Caloric burn rate 0.0757 0.0871 0.0606
0.0417 (kcal min.sup.-1 lb.sup.-1) Total energy 1022 1176 818 563
required* (kcal) Required glucose 0.0189 0.0218 0.0151 0.0104
oxidation rates.sup..sctn. (g min.sup.-1 lb.sup.-1) Total glucose
256 294 204 141 required* (g) Exogenous glucose 99 99 99 99
oxidized.sup.# (g) Difference (g) 157 195 105 42 Energy obtained
396 396 396 396 from exogenous glucose (kcal) Caloric gap (kcal)
627 780 422 167 *Based on a 150-lb individual and a 90-min
activity. .sup..sctn.Oxidation rate of glucose that is required to
sustain the Caloric burn rate reported in row 1. The values are
estimated using the fact that 1 g of pure carbohydrate yields about
4 kcal of energy. .sup.#Based on an exogenous glucose oxidation
rate of ~1.1 g/min, which is determined by the rate of transport of
glucose (from the intestinal lumen in to the blood stream) across
the intestinal epithelium.
[0072] The term "sustained release" (i.e., extended release and/or
controlled release) are used herein to refer to nutritional
supplement, for example carbohydrates etc., delivery system or
composition that is introduced into the body of a human and that
continuously releases a stream of one or more nutrients over a
predetermined time period and at a level sufficient to achieve a
desired effect throughout the predetermined time period. Reference
to a continuous release stream is intended to encompass release
that occurs as the result of diffusion-limited release of the
component from the matrix, or biodegradation in vivo of the
composition, or a matrix or component thereof, or as the result of
metabolic transformation or dissolution of the added nutrients or
other desired agent(s). Delayed release may be achieved by
entrapping the nutrients within particulate carriers with
mucoadhesive surface characteristics. Adhesion of the
nutrient-loaded particles to intestinal mucosa will increase
retention time of the particles inside the intestinal lumen,
thereby providing continuous release and transport of nutrients
across the epithelium into blood, beyond the normal retention time
of non-adhesive composition inside the gastrointestinal tract.
[0073] In a preferred embodiment, the nutritional supplement
composition is in the form of a solution, suspension, gel capsule,
powder, snack (e.g. a bar), granola form, or tablet. The "delivery"
of nutrients comprises, for example, suspending the nutrients
individually or in combinations in sustained release particulate
microparticles; compounds which bind to the nutrients with
different affinities and the like. According to one embodiment, the
requisite volume for consumption by the individual is about 500 mL
when in liquid form, however, formulations increasing and or
decreasing the concentrations and amounts are contemplated.
[0074] Examples of nutrients include, but not limited to
carbohydrates, proteins, amino acids, vitamins, co-enzymes,
phospholipids, minerals, and electrolytes. Examples of vitamins and
co-enzymes that may be delivered using this invention include but
are not limited to water or fat soluble vitamins such as thiamin,
riboflavin, nicotinic acid, pyridoxine, pantothenic acid, biotin,
flavin, choline, inositol and paraminobenzoic acid, carnitine,
vitamin C, vitamin D and its analogs (such as ergocalciferol,
calcitriol, doxercalciferol, and paricalcitol), vitamin A and the
carotenoids, retinoic acid, vitamin E and vitamin K.
[0075] In a preferred embodiment, the nutritional composition
comprises carbohydrates that are taken up by different receptors,
e.g. SGLT and GLUT receptors. Suitable carbohydrates include, but
are not limited to, mono-, di- and polysaccharides such as glucose,
sucrose, maltose as well as more complex edible carbohydrates such
as maltodextrins. According to one preferred embodiment, the
composition includes a blend of glucose and fructose. The weight
ratio of glucose to fructose preferably ranges from about 1:1 to
about 100:1, about 5:1 to about 95:1, about 10:1 to about 90:1,
about 15:1 to about 85:1, about 20:1 to about 80:1, about 25:1 to
about 75:1, about 30:1 to about 70:1, about 35:1 to about 65:1,
about 40:1 to about 60:1, about 45:1 to about 55:1 or about 50:1.
Preferably, the composition includes from about 0.1 to about 99.9
wt. %, about 1 to about 99 wt. %, about 5 to about 95 wt. %, about
10 to about 90 wt. %, about 15 to about 85 wt. %, about 20 to about
80 wt. %, about 25 to about 75 wt. %, about 30 to about 70 wt. %,
of carbohydrates, about 35 to about 65 wt. %, about 40 to about 60
wt. %, about 45 to about 55 wt. %, or about 50 wt. %, calculated on
a 100% dry matter basis of the composition.
[0076] In another preferred embodiment, the composition includes
amino acids. The amino acids may be in the form of free amino acids
or peptides, and are preferably present in an amount in the range
of from about 0.1 to about 99.9 wt. %, about 1 to about 99 wt. %,
about 5 to about 95 wt. %, about 10 to about 90 wt. %, about 15 to
about 85 wt. %, about 20 to about 80 wt. %, about 25 to about 75
wt. %, about 30 to about 70 wt. %, of carbohydrates, about 35 to
about 65 wt. %, about 40 to about 60 wt. %, about 45 to about 55
wt. %, or about 50 wt. % calculated on a 100% dry matter basis of
the composition.
[0077] The peptide material can be derived from proteins of animal
or plant origin and examples of such proteins are milk proteins,
meat proteins, soy proteins, wheat proteins, pea proteins, rice
proteins and maize proteins. Preferably the protein raw material is
wheat gluten protein or a subfraction thereof such as gliadin. In
the present context, the term "peptide material" is understood to
indicate a protein hydrolysate and may contain all types of
peptides that may vary in length as well as a certain amount of
free amino acids resulting from the hydrolysis. The protein raw
material is hydrolyzed by one or more hydrolytic enzymes. The
hydrolytic enzyme can be of animal, plant, yeast, bacterial or
fungal origin. Preferably enzyme preparations are used which have a
low exo-peptidase activity to minimize the liberation of free amino
acids and to improve taste profiles of the protein hydrolysates.
The preferred hydrolyzed protein material of the present invention
has an average peptide chain length in the range of 1-40 amino acid
residues and more preferably in the range of 1-20 amino acid
residues. The average peptide chain can be determined using the
method as described in WO 96/26266. Further the peptide material is
present in an amount of about 0.1-90 wt. %, calculated on dry
matter basis of the composition.
[0078] Other optional components of the composition according to
the invention are vitamins, minerals, electrolytes, flavors,
antioxidants, components having co-enzyme and antioxidant
properties, lipids including emulsifiers, and proteins for meeting
specific nutritional and/or physiological needs.
[0079] Carbohydrates, such as dextrose, fructose, and the like and
combinations thereof may be present in a composition according to
the invention in any desirable amount, including, for example,
about 1-20 wt. % of the composition, e.g., 1 wt. %, 2 wt. %, 3 wt.
%, 4 wt. %, 5 wt. %, 6 wt. %, 7 wt. %, 8 wt. %, 9 wt. %, 10 wt. %,
11 wt. %, 12 wt. %, 13 wt. %, 14 wt. %, 15 wt. %, 16 wt. %, 17 wt.
%, 18 wt. %, 19 wt. %, or 20 wt. % of the composition. Also 20-25
wt. %, 25-30 wt. %, 30-35 wt. %, 35-40 wt. %, 40-45 wt. %, 45-50
wt. %, and greater than 50 wt. %.
[0080] Other optional components of the composition according to
the invention include preservatives; colorants such as F.D. &
C. dyes and lakes; flavorants; and sweeteners. Suitable
preservatives include, for example, methylparaben, propylparaben,
sodium benzoate, and potassium sorbate and may be present in a
composition according to the invention in an amount such as, for
example, about 0.0001 wt. %, about 0.001 wt. %, about 0.005 wt. %,
about 0.01 wt. %, about 0.02 wt. %, about 0.03 wt. %, about 0.04
wt. %, about 0.05 wt. %, about 0.1 wt. %, about 0.5 wt. %, or
greater than 0.5 wt. % of the composition.
[0081] In another preferred embodiment, the nutritional composition
comprises a binder and/or biodegradable polymer or mixture of
biodegradable polymers with appropriate time release
characteristics and release kinetics. The composition of the
invention may then be formed into microparticles comprising the
appropriate nutritional compositions suitable for providing
efficacious concentrations of the compounds of the invention over a
prolonged period of time without the need for frequent re-dosing.
The composition of the present invention can be incorporated into
the biodegradable polymer or polymer mixture in any suitable manner
known to one of ordinary skill in the art and may form a
homogeneous matrix with the biodegradable polymer.
[0082] In another preferred embodiment, the nutrient supplements
are present in nano suspensions/colloidal particles. The
nanoparticles or colloidal particles (CP) can form a stable
colloidal suspension in water and in a physiological medium. The CP
associate with the nutrients, e.g. carbohydrates, in aqueous media
by a spontaneous mechanism; and the CP release the nutrients in a
physiological medium and, more precisely, in vivo. The release
kinetics depend on the nature of the polymer that is the CP
precursor. A protein, whose nutritional value depends on the
tertiary structure of the molecule may also be delivered by this
method, using biocompatible polymer hosts that will not denature
the protein.
[0083] Thus, by varying the specific structure of the polymers, it
is possible to control the association and release phenomena from
the kinetic and quantitative points of view.
[0084] Another preferred embodiment of the invention concerns the
preparation of: selected particles; and other selected particles
which are structured, submicron and capable of being used
especially for carrying one or more nutrients, these particles
being individualized (discrete) supramolecular arrangements that
are: based on linear amphiphilic polyamino acids having peptide
linkages and comprising at least two different types of hydrophilic
repeating amino acids, and hydrophobic repeating amino acids, the
amino acids of each type being identical to or different from one
another; capable of associating at least one nutrient in colloidal
suspension, in the undissolved state, and releasing it, especially
in vivo in a prolonged and/or delayed manner; and stable in the
aqueous phase at a pH of between 4 and 13, in the absence of
surfactant(s).
[0085] Preferably, the particles are submicron structured particles
capable of being used especially for carrying one or more
nutrients, these particles being discrete supramolecular
arrangements; capable of associating at least one nutrient in
colloidal suspension, in the undissolved state, and releasing it,
especially in vivo, in a prolonged and/or delayed manner; and
stable in the aqueous phase at a pH of between 4 and 13, in the
absence of surfactant(s).
[0086] In another preferred embodiment, the composition can be
formulated to encapsulate the nutritional compositions in
microspheres or microparticles so that it may be admixed or
formulated into any form, such as a powder, a beverage, gum,
nutritional food product, pill and the like.
[0087] A "microsphere" or "microparticle", as defined herein,
includes a particle of a biocompatible solid-phase material having
a diameter of about one millimeter to about one micrometer, or
less, wherein the particle may contain a biologically active agent
and, wherein the solid-phase material sustains the in vivo release
of the nutritional compositions from the microsphere. A microsphere
can have a spherical, non-spherical or irregular shape. The typical
microsphere shape is generally spherical.
[0088] A "biocompatible" material, as defined herein; means that
the material, and any degradation products of the material, is
non-toxic to the recipient and also presents no significant
deleterious or untoward effects on the recipient's body.
[0089] In a preferred embodiment, the microspheres contain a
mixture of nutritional compounds and the microsphere is composed of
a biodegradable material that is released over a certain period of
time. For example, in order to provide an initial burst of
nutrients to provide an immediate reservoir of energy or nutrients
to the individual, the nutritional compounds are formulated as such
and can contain a variety of carbohydrates, amino acids,
electrolytes, vitamins, etc. in differing ratios. The second group
can contain a differing ratio of carbohydrates:amino acids:vitamins
etc., or strictly different or similar carbohydrates that are
released over a longer period of time to maintain a sustainable
release of the nutrients. The formulation of the nutrients in the
microspheres and the timing of release can be varied depending on
the types of activity, the individual, age, weight and nutritional
needs. For example, a marathon runner (sustained nutrition over
long period) would have different nutritional needs to a sprinter
(burst of nutrition).
[0090] In another preferred embodiment, different types of
carbohydrates, e.g. those that are taken up by for example, SGLT
transporters versus GLUT transporters are added in differing ratios
at differing release rates to achieve the results as described
infra.
[0091] In another preferred embodiment, compositions comprise
compounds which dissolve over a period of time in vivo sequentially
in acid, neutral and weak alkaline regions of the gastrointestinal
tract. These compounds include for example, an acidic polymeric
dispersion coating as the first coating to prolong nutrient
supplement release. In this embodiment, the microparticle comprises
as a core a material comprising calcium carbonate, sugar, dextrose
and nonpareil seeds. The first coating is a material which retards
rapid passage of water. The first coating is preferably an aqueous
dispersion of poly(methacrylic acid-co-ethyl acrylate)
(commercially available under the designation Eudragit L30D-55).
The second coat is a latex acrylic polymer. The second coating is
preferably poly(ethyl acrylate-co-methyl
methacrylate-co-2-trimethylammonioethyl methacrylate chloride)
(commercially available under the designation Eudragit RS-30D). The
thickness of the second coating is established to achieve the
desired time-release rate for the drug.
[0092] The time release products are preferably substantially
spherical in configuration. The diameter of the time release drug
products typically ranges between 20 and 650 microns, between 30
and 500 microns or between 40 and 350 microns and is preferably
between about 50 and 250 microns when the products are in a liquid
suspension form. It is a feature of the present invention that the
time release nutrient composition containing products of the
present invention, because of their size, can be suspended in an
aqueous medium, thereby providing a liquid suspension.
[0093] In this embodiment, the nutrient compositions are formulated
as a time release formulation comprising: a core which can be
optional; nutritional supplements bound to the core; a first
coating having limited permeability to water; and a second coating,
which is more permeable to water than the first coating, wherein
the first and second coatings together comprise the time release
components of the nutrient compositions.
[0094] The core will generally have a diameter of about 19 to 57,
about 20 to 56, about 21 to 55, about 22 to 54, about 23 to 53,
about 24 to 52, about 25 to 51, about 26 to 50, about 27 to 49,
about 28 to 48, about 29 to 47, about 30 to 46, or about 31 to 45
microns. The core is generally comprised of an inert ingredient,
preferably a material selected from the group consisting of calcium
carbonate, sugar, dextrose and nonpareil seeds.
[0095] The first coating, which has a limited permeability to water
and which retards rapid passage of acid and water. This first
coating will typically have a diameter of between about 1.30 and
4.60, about 1.40 and 4.50, about 1.50 and 4.40, about 1.60 and
4.30, about 1.70 and 4.20, about 1.80 and 4.10, about 1.90 and
4.00, about 2.00 and 3.90, about 2.10 and 3.80, about 2.20 and
3.70, about 2.30 and 3.60 or about 2.40 and 3.50 microns. The first
coating is preferably an acidic polymeric dispersion coating which
prolongs drug release, more preferably an aqueous dispersion of
poly(methacrylic acid-co-ethyl acrylate). Such a polymer is
commercially available under the name EUDRAGIT L30D-55. The core
and first coating together typically have a diameter of between
about 60 and 77, about 61 and 76, about 62 and 75, about 63 and 74,
about 64 and 75 or about 65 and 74 microns.
[0096] It is appreciated that the first and second coatings
together comprise the time release components of the product of the
present invention. The first and second coatings together effect
time release of the orally administrable drug within an individual
over a maximum period of about 12 hours. It is appreciated by those
skilled in the art that the thickness of the second coating can be
altered to achieve the desired time release rate for the
supplement. That is, the thickness of the second coating can be
increased to achieve a longer period of time release in the body.
The coatings work due to differential porosity. The inner coating
comprised of, for example, poly(methacrylic acid-co-ethyl acrylate)
is sensitive to pH. Nutrient transport across the inner coating is
determined by the porosity and water content of the coating, both
of which are determined by the different pH values within regions
of the gastrointestinal tract. In an acidic environment (in the
stomach), the inner coating becomes relatively hydrophobic and
shrinks, leading to decreased pore size and nutrient permeability.
In contrast, the pH inside the intestinal lumen is higher. The
inner coating becomes relatively hydrophilic due to ionization, and
allows faster release of nutrients from the particle cores. The
outer coating is not pH-responsive, but can be used to control
nutrient permeability by controlling the pore size. The present
invention provides in the first and second coating porosity such
that water entering the time release component will pass through
the second coating more rapidly that through the first coating and
the drug and water exiting the time-release component will pass
through the first coating more slowly than through the second
coating. In the preferred form, passage through each coating is by
mechanical means with the passage through the first coating being
augmented by ionic interaction.
[0097] In another preferred embodiment, one or more of nutrient
supplements are bound or encapsulated by a particle which is stable
in an aqueous environment and are released over an extended period
of time once the supplements have been consumed.
[0098] The composition according to the invention may have the form
of a powder, gum, a beverage or any other food product. A beverage
according to the invention can be prepared by dissolving the
above-defined ingredients in an appropriate amount of water.
Preferably an isotonic drink has been prepared. For drinks;
intended to be used during and after exercise it is recommended to
have a concentration of the composition according to the invention
in the range of about 0.10-60 wt. % calculated on the total weight
of the drink.
[0099] In a preferred embodiment, the formulation has a viscosity
and "mouth-feel" approximately equivalent to water. The viscosity
of the formulation can be quantified using a capillary viscometer,
such as the Ubblehold viscometer, by determining the time required
for the liquid to fall from one fiducial mark to another in a glass
capillary [See, e.g., Pearce, E. M.; Wright, C. E.; Bordoloi, B. K.
Laboratory Experiments in Polymer Synthesis and Characterization;
Pennsylvania State University: University Park, 1982; p. 187]. Room
temperature viscosity of water is about 1 cP, while that of olive
oil is about 80 cP, castor oil about 1000 cP and corn syrup about
1400 cP. The viscosity of fat-free milk is about 30 cP [Vesa, T.
H.; Marteau, P. R.; Briet, F. B. et al. Am. J. Clin. Nutr. 1997,
66, 123-126].
[0100] In another preferred embodiment; the composition comprises
flavoring agents which provide a variety of tastes/aromas that are
pleasant, palatable and impart a feeling of confidence for adults,
adolescents, and child consumers.
[0101] In one preferred embodiment, consumption of the nutrient
compositions increases exercise duration and cumulative power
output potential by at least about 2%, about 5%, about 10%, about
20%, about 30%, about 40%, about 50%, about 60%, about 70%, about
80%, about 90% or about 100% when compared to no supplemental
carbohydrate (i.e., the nutrient compositions) ingestion pre and
during exercise.
[0102] In another preferred embodiment, consumption of the nutrient
compositions increases burst energy duration and power output by at
least about 2%, about 5%, about 10%, about 20%, about 30%, about
40%, about 50%, about 60%, about 70%, about 80%, about 90% or about
100% when compared to no supplemental carbohydrate ingestion pre
and during exercise.
[0103] In another preferred embodiment, consumption of the nutrient
compositions increases the number of effective burst events by at
least about 1% to about 50%, about 50% to about 100%, about 100% to
about 500% or about 500% to about 1000% when compared to no
supplemental carbohydrate (i.e., the nutrient compositions)
ingestion pre and during exercise.
[0104] In another preferred embodiment, consumption of the nutrient
compositions decreases the duration of recovery time between burst
events by at least about 2%, about 5%, about 10%, about 20%, about
30%, about 40%, about 50%, about 60%, about 70%, about 80%, about
90% or about 100% when compared to no supplemental carbohydrate
(i.e., the nutrient compositions) ingestion pre and during
exercise.
[0105] In another preferred embodiment, consumption of the nutrient
compositions results in about 1% to about 50%, about 50% to about
100%, about 100% to about 500% or about 500% to about 1000%
increase in continuous exercise power output (watts) at about
VO.sub.2 62% for 90 minutes and not less than about a 25% increase
in high intensity (Burst) exercise, about VO.sub.2 86%, power
output and duration following 90 minutes of continuous exercise,
and increase the number of effective "burst" periods by >100% as
compared to performance when utilizing as performance enhancing
drinks such as GATORADE, CYTOMAX, or POWERADE.
[0106] The measurement of cumulative power output potential is well
known to one of skill in the art (see, e.g., Byrne, C.; Twist, C.;
Eston, R. Neuromuscular function after exercise-induced muscle
damage: theoretical and applied implications. Sports Med. 2004, 34,
49-69; Hunter, A.; St, C.; Lambert, M. et al. Effects of
supramaximal exercise on the electromyographic signal. Br. J.
Sports Med. 2003, 37, 296-299; and Williams, S. G.; Cooke, G. A.;
Wright, D. J. et al. Peak exercise cardiac power output: A direct
indicator of cardiac function strongly predictive of prognosis in
chronic heart failure. Eur. Heart J. 2001, 22, 1496-1503). In a
particularly advantageous embodiment, the yo-yo-intermittent
recovery test is utilized for measuring cumulative power output
potential (see, e.g., Krustrup, P.; Mohr, M.; Amstrup, T. R. et al.
The yo-yo intermittent recovery test: physiological response,
reliability, and validity. Med. Sci. Sports Exerc. 2003, 35,
697-705).
[0107] In another preferred embodiment, consumption of the nutrient
compositions results in an increased sustainability in
concentration, hand/eye coordination etc. during extended exercise.
Tests of this nature are known in the art and anyone or more can be
utilized. See, for example, U.S. Pat. No. 7,300,365. This would
translate into improved athletic performance but also aid promotion
for non-athletic, academic and or work, performance uses.
[0108] In another preferred embodiment, consumption of the nutrient
compositions results in improved concentration and brain function
as compared to no supplemental carbohydrate (i.e., the nutrient
compositions) ingestion.
[0109] In another preferred embodiment, consumption of the nutrient
compositions is expected to result in improved and stabilized blood
glucose concentration and is therefore a beneficial beverage or
food alternative for individuals with diabetes.
[0110] In another preferred embodiment, the compositions are
packaged for accessibility during longer periods of athletic
exertion (e.g., a marathon) or when immediate energy and associated
supplements are required, the product comprising the nutrients is
in a solid or gel from which is wearable, yet protected, on the
person in a ready to consume form.
[0111] In another preferred embodiment, the nutritional composition
is packaged so that it is easily handled and stored in backpacks,
duffle bags, pockets, etc. Preferably, the packaging is
environmentally friendly.
[0112] In a preferred embodiment the nutritional compositions are
admixed with a biodegradable binder or encapsulated within a
biodegradable microsphere which allows for sustained release of
desired carbohydrates and other nutrients. "Biodegradable", as
defined herein, means the polymer will degrade or erode in vivo to
form smaller chemical species. Degradation can result, for example,
by enzymatic, chemical and/or physical processes. Suitable
biocompatible, biodegradable polymers include, for example,
polysaccharides, poly(lactide)s, poly(glycolide)s,
poly(lactide-co-glycolide)s, poly(lactic acid)s, poly(glycolic
acid)s, poly(lactic acid-co-glycolic acid)s, polycaprolactone,
polycarbonates, polyesteramides, polyanhydrides, poly(amino acids),
polyorthoesters, polyacetyls, polycyanoacrylates, polyetheresters,
poly(dioxanone)s, poly(alkylene alkylate)s, copolymers of
polyethylene glycol and polyorthoester, biodegradable
polyurethanes, hydrogels, blends and copolymers thereof.
[0113] Biocompatible, non-biodegradable polymers suitable for the
methods and compositions of the present invention include
non-biodegradable polymers selected from the group consisting of
polyacrylates, polymethacrylates, polymers of ethylene-vinyl
acetates and other acyl substituted cellulose acetates,
non-degradable polyurethanes, polystyrenes, polyvinyl chloride,
polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonate
polyolefins, polyethylene oxide, hydrogels, blends and copolymers
thereof.
[0114] In another preferred embodiment, hydrogels are used in the
sustained release of the nutritional supplements. Physical
polymeric hydrogels have been widely explored for biomaterials
applications. Examples include hydrogels formed by complexation of
enantiomeric polymer or polypeptide segments and hydrogels with
temperature- or pH-sensitive properties. They attract special
attention for sustained drug delivery because of the mild and
aqueous conditions involved in trapping delicate bioactive agents
such as proteins. For example, in situ formed hydrogels, formed
from thermosensitive block copolymers, have also been proposed as
sustained release matrices for drugs. They have the advantage that
there is no chemical reaction involved in the gel formation. These
copolymer hydrogels are usually designed for macromolecular drugs
such as proteins and hormones. Preferably the polymer is in an
aqueous solution, which forms a hydrogel. For example, suitable
aqueous polymer solutions contain about 1% to about 80%, about 2%
to about 75%, about 3% to about 70%, about 4% to about 65%, about
3% to about 70%, about 4% to about 65%, about 5% to about 60%,
about 6% to about 55%, about 7% to about 50%, about 8% to about
45%, about 9% to about 42% polymer, preferably about 10% to about
40% polymer. Suitable hydrogels can also contain about 1% to about
20%, about 2% to about 19%, about 3% to about 18%, about 4% to
about 17% cyclodextrin (w/w) (based on the weight of total
solution), preferably about 5% to 15% cyclodextrin, to solubilize
nutrients that have limited water solubility. The hydrogel is
typically formed using an aqueous carrier fluid. For example,
typical aqueous solutions contain about 1% to about 80%, about 2%
to about 75%, about 3% to about 70%, about 4% to about 65%, about
3% to about 70%, about 4% to about 65%, about 5% to about 60%,
about 6% to about 55%, about 7% to about 50%, about 8% to about
45%, about 9% to about 42% polymer, preferably about 10% to about
40% polymer.
[0115] The hydrogel composition may also contain a secondary
polymer, which may complex with the nutrient, conjugate the
nutrient, or both. The secondary polymer may suitably be a
polyester, polyurethane, polyamide, polyether, polysaccharide,
poly(amino acid), polypeptide, or a protein. Preferably the
secondary polymer is a di- or mono-functional polymer or polyionic
polymer with poly(ethylene glycol) segments. In the case where
nutritional supplements conjugate or complex to the hydrogels, then
the hydrogel formulations act not only as a matrix but also a
carrier of the nutritional supplements. This means that the
nutritional supplements, e.g. a variety of carbohydrates, are not
only physically entrapped in the hydrogel, but also are complexed
or conjugated to the molecules that form the hydrogel. A secondary
polymer may also be used to alter the properties, such as porosity
and viscosity, of the hydrogel matrix.
[0116] The properties of the hydrogels are tunable by using
different polymer block molecular weights, by adjusting the
cyclodextrin content, and through the use of secondary polymers.
For example, the hydrogel may be adjusted to be a more flexible
hydrogel or a more rigid hydrogel. The hydrogel structure can be
tailored to have variable viscosity and longer or shorter drug
release rates. The degree of hydrophobicity of the
poly(hydroxyalkanoate) can also be selected for a desired sustained
release rate.
[0117] The duration of extended release is dependent on the
molecular weights of the block polymers, particularly the molecular
weight of the hydrophobic poly(hydroxyalkanoate) section (e.g.,
PHB). The release rate may be altered in accordance with the
invention to achieve a desired duration of response by selecting: a
particular poly(hydroxyalkanoate); the stereo-isomeric state of the
selected poly(hydroxyalkanoate); the molecular weight of the
selected poly(hydroxyalkanoate); and the relative quantity of
cyclodextrin used in the hydrogel, to achieve a desired duration
and rate of sustained release. The molecular weight and selection
of the hydrophilic poly(alkylene oxide) also impacts the sustained
release kinetics, but to a lesser extent than the hydrophobic
poly(hydroxyalkanoate) component. Secondary polymers may also be
utilized to change the release kinetics. Hydrogels can provide
sustained release over a period of one or more days by adjustment
of the molecular weights of the block polymers and the copolymer,
as well as the cyclodextrin content within the hydrogel of the
present invention and the potential use of secondary polymers.
[0118] Microencapsulation of components of the nutritional
supplement in biodegradable polymers such as
polylactide-polyglycolide is also contemplated. Depending on the
ratio of component to polymer, and the nature of the particular
polymer employed, the rate of component release may be sustained.
Examples of other biodegradable polymers include poly(orthoester)s
and poly(anhydride)s. The formulations are also prepared by
entrapping the component in liposomes or microemulsions which are
compatible with body tissue.
[0119] Further, the terminal functionalities of a polymer can be
modified. For example, polyesters may be blocked, unblocked or a
blend of blocked and unblocked polymers. A blocked polyester is as
classically defined in the art, specifically having blocked
carboxyl end groups. Generally, the blocking group is derived from
the initiator of the polymerization and is typically an alkyl
group. An unblocked polyester is as classically defined in the art,
specifically having free carboxyl end groups.
[0120] In an advantageous embodiment, blends of polysaccharides are
utilized to synthesize aqueous dispersions of microparticles or
nanonparticles. Advantageously, the polysaccharides are
hydrophobically modified polysaccharides wherein the
polysaccharides form interpenetrating polymer networks. In an
especially advantageous embodiment, the polysaccharides contain
carboxylic acid groups, such as, but not limited to, the
polysaccharides of FIG. 1.
[0121] Without being bound by theory, it is expected that the
carboxy containing hydrogel particles are in a collapsed state in
the acidic environment of the stomach. Hence, the encapsulated
sugar molecules are retained within the particles in the stomach.
The hydrogel particles will achieve an expanded state when they
reach the small intestine (pH 5-7), and will release the
encapsulated sugar at a rate faster than that in the stomach. A key
feature of the proposed polysaccharide hydrogels is their pH
responsiveness. Ideally, the hydrogels should not swell in the
acidic environment of the stomach, but should swell upon entry into
the small intestine and release the encapsulated sugars at a
controlled rate. Advantageously, the carbohydrates of the present
invention are controlled release particles dispersed in an aqueous
medium, but may also be stored in a solid particulate form.
[0122] In a particularly advantageous embodiment, the hydrogels
comprise hydrophobized polysaccharides. Polysaccharides may be
functionalized with hydrophobes such as cholesterol. For example,
polysaccharides such as, but not limited to, pullulan, dextran and
mannan may be partly substituted by various hydrophobic groups such
as, but not limited to, long alkyl chains and cholesterol.
[0123] The nanoparticles or microparticles of the present invention
may comprise modified starch molecules with grafted fatty acid
moieties. The fatty acid may be grafted on to starch using
potassium persulfate, for example, as a catalyst. In another
embodiment, the invention also encompasses surface-modification of
nanoscale starch particles using, for example, stearic acid
chloride (a hydrophobe), poly(ethylene glycol) or methyl ether (a
hydrophilic molecule). In another embodiment, the modified starch
may be an acryloyl-modified starch or an acryloyl-modified
hydroxyethyl starch.
[0124] In an advantageous embodiment of the invention, the
polysaccharide was first derivatized to introduce aldehydic or
carboxylic groups on the side chain. These groups were then
crosslinked to produce more stable three-dimensional networks.
[0125] In an advantageous embodiment, the particles are crosslinked
to form hydrogels. Crosslinking may be performed using free radical
initiators such as persulfate salts, or redox systems involving
ascorbic acid, or a naturally occurring crosslinker such as
genipin. Ionic crosslinking is also explored. Anionic
polysaccharides such as gellan can be used for ionic crosslinking,
instead of chemicals such as borax which may not be desirable in a
food formulation.
[0126] The present invention further relates to the preparation of
hydrogels. In an advantageous embodiment, a blend of
hydrophobically modified polysaccharide such as, but not limited
to, hydroxypropyl cellulose and a carboxy containing polysaccharide
such as, but not limited to, alginate or carboxymethyl cellulose
may be used to prepare the hydrogel particles of the present
invention. Examples of suitable alginates include sodium alginate
polymers (e.g., sodium alginate NF, F-200, SAHMUP and sodium
alginate NF, SAHMUP), which may be present in a composition
according to the invention in an amount of e.g., about 0.01 wt. %
to about 1.0 wt. % of the composition.
[0127] The hydrophobically modified polysaccharide results in
spontaneous particle formation due to phase separation in water,
while the polysaccharide containing carboxylic acid groups imparts
a pH-responsive behavior and will also increase intestinal transit
time. In one embodiment, nanoparticle suspensions may be
synthesized by self-assembly of chitosan and carboxymethyl
cellulose hydrolysates. The polymers are hydrolyzed with the
enzymes chitosanase and cellulase, respectively. Electrostatic
interactions between the carboxylate groups of carboxymethyl
cellulose with the amino groups of chitosan result in spontaneous
formation of nanoparticles by mixing solutions of the two polymers.
Particle size depended on the mixing ratio of the solutions, and
also by the molecular weight of the polymers. It was necessary to
hydrolyze the polymers and lower the molecular weight before mixing
in order to prevent the formation of macroscopic gel.
[0128] In another embodiment, hydrogels may be prepared from
mixtures of acidic polysaccharides such as, but not limited to,
alginates, and basic polysaccharides such as, but not limited to,
oligosaccharide derivatives of chitosan; a basic polysaccharide
such as, but not limited to, chitosan and anionic polysaccharide
such as, but not limited to, hyaluronic acid; alginate and oxidized
alginate blended with chitosan; grafted agar and sodium alginate
blend with acrylamide; gellan co-crosslinked with scleroglucan;
photocrosslinked modified dextran; starch reacted with glycidyl
methacrylate; or polymerizable saccharide monomers, such as
sucrose, created by reaction of the sugar with epoxy acrylate, or
methacryloyl chloride and acetyl chloride.
[0129] Crosslinking of polysaccharides containing hydroxyl groups,
e.g. starch, hydroxyalkyl starch, hydroxyalkyl cellulose, etc., can
be achieved using a variety of reagents including bis-epoxides,
divinyl sulfone, N,N'-carbonyldiimidazole, cyanuric chloride,
terephthaloyl chloride, carbon disulfide, formaldehyde, and
glutaraldehyde as shown in FIGS. 8A and 8B [Park, H.; Park, K.;
Shalaby, W. S. W. Biodegradable Hydrogels for Drug Delivery,
Technomic Publishing Company: Lancaster, Pa., 1993]. Crosslinking
to form macroscopic hydrogels may be readily achieved using these
reagents. Kabra et al. [Kabra, B. G.; Gehrke, S. H.; Spontak, R. J.
Microporous, responsive hydroxypropyl cellulose gels. 1. Synthesis
and microstructure. Macromolecules 1998, 31, 2166-2173] have used
divinyl sulfone crosslinker to prepare macrogels of hydroxypropyl
cellulose. The synthesis of hydrogel nano- or microparticles, on
the other hand, requires careful selection of reaction conditions
to prevent precipitation (due to colloidal instability) or macrogel
formation. The difference between a macrogel and a
colloidally-stable microgel suspension is evident from the
photographs of two crosslinked samples of hydroxypropyl cellulose
polymer, shown in FIG. 9. Both the vials shown in FIG. 9 contain
about 4% (w/v) of hydroxypropyl cellulose in water. The vial
labeled `1` shows a macrogel, while the vial labeled `2` contains a
colloidally stable microgel suspension of hydroxypropyl cellulose.
To prevent macrogel formation and colloidal aggregation, the
polysaccharide concentration has been kept fairly low (below about
1 wt %) in the crosslinking reactions. Cai et al. [Cai, T.; Hu, Z.;
Marquez, M. Synthesis and self-assembly of nearly monodisperse
nanoparticles of a naturally occurring polymer. Langmuir 2004, 20,
7355-7359] have prepared nanoparticles of crosslinked hydroxypropyl
cellulose using divinyl sulfone crosslinker at 0.05 wt % polymer
concentration. The toxicity of divinyl sulfone is of concern in
synthesizing formulations for controlled release of nutrients.
FIGS. 8A and 8B depict crosslinking of polysaccharides containing
hydroxyl groups. P represents a polysaccharide segment.
[0130] The transport of small molecules such as glucose through
polysaccharide hydrogels has been investigated for cell
encapsulation and tissue engineering [McEntee, M.-K. E.; Bhatia, S.
K.; Tao, L.; Roberts, S. C.; Bhatia, S. R. Tunable transport of
glucose through ionically-crosslinked alginate gels: effect of
alginate and calcium concentration. J. Appl. Polym. Sci. 2008, 107,
2956-2962]. Ionically-crosslinked alginate hydrogel beads, with an
average bead diameter of 2 mm, were prepared using alginate and
calcium chlorides. The researchers found a two-step release profile
for glucose over a time range of 20-50 min. It should be noted that
the release rates were measured by suspending the glucose-loaded
spheres in pure water. The large difference in the concentration of
glucose inside the sphere and the suspending fluid (pure water)
resulted in a relatively rapid release of sugar (within about 50
min after suspension).
[0131] Covalent-crosslinking is expected to impart greater
stability (against premature disintegration) to the hydrogel
spheres, in the wide range of pH and ionic strength conditions that
are encountered in the GI tract, than ionically-crosslinked
hydrogels. When trisodium metaphosphate is used as the crosslinking
agent, covalent-crosslinks are formed. The release rate of
nutrients is tuned by controlling the crosslink density of the
microspheres. More importantly, the release rate depends on the
concentration of the nutrients outside the particles, in the
aqueous phase of the suspension. Applicants' dispersions contain a
relatively high sugar concentration in the aqueous phase. Diffusion
of nutrients from the hydrogel microparticles occurs only when the
nutrients get depleted from the aqueous phase. Hence, the particles
act as reservoirs of sugar and supply nutrients within the
intestinal lumen over a time period significantly beyond the
duration reported in the study using ionically-crosslinked alginate
beads (.about.50 min) [McEntee, M.-K. E.; Bhatia, S. K.; Tao, L.;
Roberts, S. C.; Bhatia, S. R. Tunable transport of glucose through
ionically-crosslinked alginate gels: effect of alginate and calcium
concentration. J. Appl. Polym. Sci. 2008, 107, 2956-2962]. In
Applicants' formulations, the nutrients dissolved in the aqueous
phase will be initially absorbed across the intestinal ephithelium.
The microparticles release entrapped nutrients at low rates
initially (because of low concentration gradient), and at a faster
rate when the aqueous phase nutrients are depleted (because of a
greater concentration difference).
[0132] Acceptable molecular weights for polymers used in the
present invention may be determined by a person of ordinary skill
in the art accounting for factors such as the desired polymer
degradation rate, physical properties such as mechanical strength
and rate of dissolution of polymer in solvent. Typically, an
acceptable range of molecular weights is of about 2,000 Daltons to
about 2,000,000 Daltons, about 3,000 Daltons to about 1,900,000
Daltons, about 4,000 Daltons to about 1,800,000 Daltons, about
5,000 Daltons to about 1,700,000 Daltons, about 6,000 Daltons to
about 1,600,000 Daltons, about 7,000 Daltons to about 1,500,000
Daltons, about 8,000 Daltons to about 1,400,000 Daltons, about
9,000 Daltons to about 1,300,000 Daltons, about 10,000 Daltons to
about 1,200,000 Daltons, about 12,000 Daltons to about 1,100,000
Daltons, about 13,000 Daltons to about 1,000,000 Daltons, about
14,000 Daltons to about 900,000 Daltons, about 15,000 Daltons to
about 800,000 Daltons, about 16,000 Daltons to about 700,000
Daltons, about 17,000 Daltons to about 600,000 Daltons, about
18,000 Daltons to about 500,000 Daltons, about 19,000 Daltons to
about 400,000 Daltons, about 20,000 Daltons to about 300,000
Daltons, about 21,000 Daltons to about 200,000 Daltons, about
22,000 Daltons to about 100,000 Daltons, or about 23,000 Daltons to
about 50,000 Daltons. In one embodiment, the polymer is a
biodegradable polymer or copolymer.
[0133] In another preferred embodiment, the nutritional supplements
can be encapsulated in microparticles or microspheres. These
particles optionally comprise surfactants such as a cationic or
anionic surfactant that is entrapped and fixed to the particle
surface. The bioadhesive properties of the microparticles are
attributed to the charged surfactants entrapped on the particle
surface as the hydrophobic ends of the surfactants are embedded in
the solid core and the hydrophilic ends are exposed on the surface
of the microparticles.
[0134] Bioadhesive substances, also denoted mucoadhesive
substances, are generally known to be materials that are capable of
being bound to a biological membrane and retained on that membrane
for an extended period of time. Compared with conventional
controlled release systems, bioadhesive controlled release systems
have the following advantages: i) a bioadhesive controlled release
system localizes a biological active ingredient in a particular
region, thereby improving and enhancing the bioavailability for
active ingredients which may have poor bioavailability by
themselves, ii) a bioadhesive controlled release system leads to a
relatively strong interaction between a bioadhesive substance and a
mucosa, such an interaction contributes to an increasing contact
time between the controlled release system and the tissue in
question and permits localization of the active released from the
controlled release system to a specific site, iii) a bioadhesive
controlled release system prolongs delivery of biological active
ingredients in almost any non-parenteral route, iv) a bioadhesive
controlled release system can be localized on a specific site with
the purpose of local therapy, v) a bioadhesive controlled release
system can be targeted to specific diseased tissues, and vi) a
bioadhesive controlled release system is useful when conventional
approaches are unsuitable, such as for certain biological active
ingredients which are not adequately absorbed.
[0135] The microparticles can also include at least one
co-surfactant. The co-surfactant can be a natural biologically
compatible surfactant or a pharmaceutically acceptable non-natural
surfactant. The co-surfactant assists in maintaining particles
within the desired size range and preventing their aggregation. The
co-surfactant comprises less than about 5%, less than about 4%,
less than about 3%, less than about 2%, preferably less than about
1%, less than about 0.9%, less than about 0.8%, less than about
0.7%, less than about 0.6%, less than about 0.5%, less than about
0.4%, less than about 0.3%, less than about 0.2% and more
preferably less than about 0.1% by weight of the particle.
[0136] The microparticles are preferably formed as an aqueous
continuous phase suspending a colloidal phase of submicron
particles. The aqueous continuous phase of the particle suspension
can contain antioxidants, preservatives, microbicides, buffers,
osmoticants, cryoprotectants, and other known pharmaceutically
useful additives or solutes.
[0137] The microparticles sustain the release rate of nutritional
supplements for an extended period of time. For example, the micro
particles sustain the release of nutritional supplements for a
period between about 1 minute and twelve hours.
[0138] The use of microparticles which provide varying rates of
nutrient release are contemplated. For example, the kinetics of
nutrient-release may be any of the following: (i) a steady-state or
zero-order release rate in which there is a substantially uniform
rate of release throughout; (ii) a first-order release rate in
which the rate of release declines towards zero with time; and
(iii) a delayed release in which the initial rate is slow, but then
increases with time.
[0139] The term "bioadhesion" relates to the attachment of a
material to a biological substrate such as a biological membrane.
The term "mucoadhesive substance" is in accordance with the
generally accepted terminology and is used synonymously with the
term "a bioadhesive substance".
[0140] A cationic surfactant is incorporated on an outer surface of
the microparticle to form a bioadhesive microparticle. The
surfactant is entrapped and fixed to the particle surface and forms
a coating at the interface surrounding the particle core. The
interface surrounding the core is hydrophobic. The cationic
surfactant also stabilizes the outer surface of the hydrophobic
core component of the microparticles, thereby promoting a more
uniform particle size.
[0141] Examples of surface active materials that are capable of
strong bonding to the negatively charged and hydrophilic surfaces
of tissues are preferable for use as cationic charged surfactants.
Suitable surface active materials include straight-chain
alkylammonium compounds, cyclic alkylammonium compounds, petroleum
derived cationics, and polymeric cationic materials.
Cetylpyridinium chloride was found to exhibit strong bioadhesive
properties on biological surfaces, and is a preferred surface
active material. The surfactant is present in a proportion of about
0.01% to about 5%, preferably about 0.05% to about 2%, by weight of
the suspension.
[0142] Straight-chain alkylammonium compounds are cationic surface
active materials in which one or more hydrophobic alkyl groups are
linked to a cationic nitrogen atom. The linkage can also be more
complex as, for example, in
R--C(.dbd.O)--NHCH.sub.2CH.sub.2CH.sub.2N(CH.sub.3).sub.2.
Alternatively, the cationic surface active material can contain
more than one cationic nitrogen atom such as the class of compounds
of R--NHCH.sub.2CH.sub.2CH.sub.2NH.sub.2 and derivatives thereof.
Representative examples of suitable compounds for the cationic
surfactant include, but are not limited to: cetyl trimethylammonium
chloride (CTAB), hexadecyltrimethylammonium bromide (HDTAB),
stearyl dimethylbenzylammonium chloride, lauryl
dimethylbenzylammonium chloride, cetyl dimethylethylammonium
halide, cetyl dimethylbenzylammonium halide, cetyl
trimethylammonium halide, dodecyl ethyldimethylammonium halide,
lauryl trimethylammonium halide, coconut alkyltrimethylammonium
halide, and C8-C20 N,N-dialkyldimethylammonium halide.
[0143] Other suitable compounds for the cationic surfactant
include, but are not limited to, bis(hydrogenated tallow alkyl)
dimethylammonium chloride which is known to adsorb onto the surface
with hydrophobic groups oriented away from it,
2-hydroxydodecyl-2-hydroxyethyl dimethyl ammonium chloride [CAS no.
xx] and
N-octadecyl-N,N',N'-tris-(2-hydroxyethyl)-1,3-diaminopropane
dihydrofluoride [CAS no. 6818-37-7].
[0144] Surface-active quaternary ammonium compounds in which the
nitrogen atom carrying the cationic charge is part of a
heterocyclic ring can be used as the cationic surfactant. Examples
of suitable compounds are laurylpyridinium chloride, bromide
laurylpyridinium, tetradecylpyridinium bromide, and cetylpyridinium
halide where the halide is selected from chloride, bromide or
fluoride.
[0145] Polymeric amines which can be used as the cationic
surfactant comprise a class of polymers containing ionic groups
along the backbone chain and, exhibit properties of both
electrolytes and polymers. These materials contain nitrogen, of
primary, secondary, tertiary or quaternary functionality in their
backbone and may have weight average molecular weights as low as
about 100 or higher than about 100,000. Suitable polymeric amines
useful as a cationic surfactant include, but are not limited to,
polydimeryl polyamine available from General Mills Chemical Co.,
polyamide, polyacrylamides, polydiallyldimethylammonium chloride,
polyhexamethylene biguanide compounds, and also other biguanides,
for example those disclosed in U.S. Pat. Nos. 2,684,924, 2,990,425,
3,183,230, 3,468,898, 4,022,834, 4,053,636 and 4,198,425, herein
incorporated by reference into this application,
1,5-dimethyl-1,5-diazaundecamethylene polymethobromide, such as
"Polybrene" manufactured by Aldrich, polyvinylpyrrolidone and their
derivatives, polypeptides, poly(allylamine) hydrochloride,
polyoxyethylenated amines, and polyethyleneimine, such as "Polymin"
manufactured by BASF.
[0146] Suitable polymeric materials for the cationic surfactant
also include surface active cationic polymers prepared by
converting a fraction of the amino groups to their acyl
derivatives. For example, the polyethyleneimine is first condensed
with less than the stoichiometric quantity of acid halides thus
alkylating some of the amino groups and the remaining amino groups
are then condensed with hydrogen halides such as hydrogen chloride
or, preferably, hydrogen fluoride. The surface activity of these
compounds varies with the number of amino groups which are acylated
and with the chain length of the acylating group RCO. The
condensation reaction can be performed with stearic or oleic acid
chlorides in the presence of a solvent containing metal fluoride,
preferably silver fluoride, in such a manner that metal chloride
formed in the reaction precipitates from the solvent.
[0147] Also suitable, for the purpose of this invention, are
cationic derivatives of polysaccharides such as dextran, starch or
cellulose, for example, diethylaminoethyl cellulose. Examples of
applicable copolymers based on acrylamide and a cationic monomer
are available from Hercules Inc. under the trade name RETEN
including RETEN 220, or from National Adhesives under the trade
name FLOC AID including FLOC AID 305. Other useful acrylamide-based
polyelectrolytes are available from Allied Colloids under the trade
name PERCOL. Further examples of suitable materials are cationic
guar derivatives such as those sold under the trade name JAGUAR by
Celanese-Hall.
[0148] In another preferred embodiment, the microparticles comprise
a hydrophobic core which is preferably formed of a biodegradable
hydrophobic materials having barrier properties. Suitable,
nontoxic, pharmaceutical solid core materials are inert hydrophobic
biocompatible materials with a melting range between about
50.degree. C. and about 120.degree. C., between about 60.degree. C.
and about 110.degree. C., between about 70.degree. C. and about
100.degree. C. or between about 80.degree. C. and about 90.degree.
C. Examples include, but are not limited to, natural, regenerated,
or synthetic waxes including: animal waxes, such as beeswax;
lanolin and shellac wax; vegetable waxes such as carnauba,
candelilla, sugar cane, rice bran, and bayberry wax; mineral waxes
such as petroleum waxes including paraffin and microcrystalline
wax; cholesterol; fatty acid esters such as ethyl stearate,
isopropyl myristate, and isopropyl palmitate; high molecular weight
fatty alcohols such as cetostearyl alcohol, cetyl alcohol, stearyl
alcohol, and oleyl alcohol; solid hydrogenated castor and vegetable
oils; hard paraffins; hard fats; biodegradable polymers such as
polycaprolactone, polyamides, polyanhydrides, polycarbonates,
polyorthoesters, polylactic acids, and copolymers of lactic acid
and glycolic acid; cellulose derivatives and mixtures thereof.
Other hydrophobic compounds which may be used in the present
invention include triglycerides, preferably of food grade purity or
better, which may be produced by synthesis or by isolation from
natural sources. Natural sources may include animal fat or
vegetable oil, such as, soy oil, a source of long chain
triglycerides (LCT). Other suitable triglycerides are composed
predominantly of medium length fatty acids (C10-C18), denoted
medium chain triglycerides (MCT). The fatty acid moieties of such
triglycerides can be unsaturated, monounsaturated or
polyunsaturated. Mixtures of triglycerides having various fatty
acid moieties are also useful for the present invention. The core
can comprise a single hydrophobic compound or a mixture of
hydrophobic compounds. Hydrophobic materials are known to those
skilled in the art and are commercially available, as described in
the list of suitable carrier materials in Martindale, The Extra
Pharmacopoeia, 28.sup.th ed.; The Pharmaceutical Press: London,
1982; pp 1063-1072. Considerations in the selection of the core
material include good barrier properties to the active ingredients
and sensory markers, low toxicity and irritancy, biocompatibility,
stability, and high loading capacity for the active ingredients of
interest.
[0149] An amphiphilic or nonionic co-surfactant can be used in the
microparticles of the present invention to provide improved
stability. Co-surfactants can be formed of natural compounds or
normatural compounds. Examples of natural compounds are
phospholipids and cholates. Examples of normatural compounds
include: polysorbates, which are fatty acid esters of
polyethoxylated sorbitol sold by Unigema surfactants as Tween;
polyethylene glycol esters of fatty acids from sources such as
castor oil; polyethoxylated fatty acid, such as stearic acid;
polyethoxylated isooctylphenol/formaldehyde polymer; poloxamers,
such as, poly(oxyethylene)poly(oxypropylene) block copolymers
available from BASF as Pluronic; polyoxyethylene fatty alcohol
ethers available from ICI surfactants as Brij; polyoxyethylene
nonylphenyl ethers sold by Union Carbide as Triton N;
polyoxyethylene isooctylphenyl ethers sold by Union Carbide as
Triton X; and SDS. Mixtures of surfactant molecules, including
mixtures of surfactants of different chemical types, can be used in
the present invention. Surfactants preferably are suitable for
pharmaceutical administration and compatible with the drug to be
delivered.
[0150] Particularly suitable surfactants include phospholipids,
which are highly biocompatible. Especially preferred phospholipids
are phosphatidylcholines (lecithins), such as soy or egg lecithin.
Other suitable phospholipids include phosphatidylglycerol,
phosphatidylinositol, phosphatidylserine, phosphatidic acid,
cardiolipin, and phosphatidylethanolamine. The phospholipids may be
isolated from natural sources or prepared by synthesis.
Phospholipid surfactants are believed to usually form a single
monolayer coating of the hydrophobic core. The co-surfactant can be
present in an amount less than about 5%, preferably less than about
1%, and more preferably less than about 0.1%, relative to the
weight of hydrophobic core component. In some embodiments, one or
more co-surfactants can be used.
[0151] In another preferred embodiment, the nutritional supplements
comprise compounds which modulate uptake of carbohydrates. In the
gastrointestinal tract, chromium and vanadium (either individually,
or preferably in concert) modulate sugar transport (e.g., glucose
transport) by typically slowing glucose absorption. Slower glucose
absorption slows insulin release and reduces excessive insulin
responses in response to rising blood glucose levels after a meal.
This benefits pancreatic secretion of insulin by reducing both the
glucose load and rate of glucose load over the initial phases of
glucose detection, absorption and metabolism by the body. Reduced
rates of glucose loading reduce the stress on beta cells normally
associated with the insulin response to rising glucose. Moreover,
slower or modulated glucose absorption permits more time for
insulin to stimulate normal sugar metabolic routes either before
glucose loading is complete, or during a slower rate of glucose
loading. Consequently, insulin dependent mechanisms have more time
to prepare for the arrival of sugars from the intestine. This
modulation of glucose absorption improves short-term insulin
modulation in the liver, muscle, and adipose tissue. These effects
in the gastrointestinal tract are, in all likelihood, short-term
responses, and they are not necessarily associated with the
longer-term systemic effects of chromium and vanadium
administration.
[0152] In addition, chromium and vanadium may potentially slow
glucose metabolism by interacting with the intestine, particularly
the epithelium of the intestine responsible for sugar metabolism
(including absorption). One primary mechanism for sugar transport
in the gut is sodium facilitated sugar transport. Such transporters
are located in the lumenal membrane of the epithelium. The
basolateral membrane may also have an additional sugar transporter
that facilitates transport out the cell and into the blood. For net
sugar absorption from the lumen of the gut to the blood, sodium
facilitated sugar transport generally requires a sodium
concentration favorable to the diffusion of sodium into the
epithelium cell from the lumen. This concentration gradient is
largely generated by the active transport of the Na/K ATPase in the
epithelium cells, which generally transports three sodium atoms out
of the cell to the blood side of the epithelium in exchange for two
sodium atoms in the reverse direction.
[0153] Each cycle of the pump requires hydrolysis of one ATP to
transport sodium and potassium against their respective
concentration gradients. The hydrolysis reaction requires a
divalent cation, typically magnesium. In many instances, however
other divalent cations may substitute or enter into the hydrolysis
reaction with varying degrees of catalytic activity or inhibition.
Substitution of trivalent cations for divalent cations in the cycle
generally leads to significant inhibition of the pumping activity
and/or dephosphorylation from the phosphoenzyme intermediate state.
Chromium may thus inhibit the Na/K ATPase activity by substituting
for magnesium and thereby inhibiting, relative to magnesium,
catalytic and transport activity, giving rise to a decreased sodium
gradient across the lumenal membrane. The reduced gradient effects
sugar transport by reducing the thermodynamic and kinetic forces
favoring sugar entry from the gut.
[0154] In addition, during the hydrolysis of ATP in the catalytic
cycle of the Na/K ATPase, a phosphoenzyme intermediate (EP) is
formed between phosphate and an aspartic acid at the active site of
APTase. This covalent EP is transient and is chemically distinct
from phosphorylated proteins associated with kinases and
phosphatases, which have also been shown to be affected by
vanadium. Formation of EP in the catalytic cycle for Na/K ATPase is
inhibited by vanadate present at low concentrations of less than 1
micromolar. Vanadate binds to the active site as a transition state
analog of phosphate in a vanadyl-enzyme, or EV complex, rather than
EP. The EV complex is highly stable, as the kinetics of loss of
vanadate from the EV complex is relatively slow. Vanadate may thus
effectively inhibit the Na/K ATPase by disrupting catalysis,
through the formation of EV, giving rise to a decreased sodium
gradient across the lumenal membrane. Consequently, the reduced
gradient reduces sugar entry from the intestine.
[0155] Chromium and vanadium also operate at the systemic level
after absorption of the two transition metals from the gut. Major
sites of activity include the liver, muscle and adipose tissue.
Vanadium may have particular activity with respect to
phosphorylation systems, including the many phosphorylated proteins
responsible for modulating metabolism. Chromium may also modulate
metabolism at the cellular level. These systemic effects generally
improve the action of insulin and/or metabolic pathways associated
with sugar and/or lipid metabolism.
[0156] In regard to absorption and metabolism of the subject
compositions, and the different components thereof, features of the
alimentary tract may affect how compositions of the present
invention, and methods of using the same, are utilized when
ingested orally. The elements of the alimentary tract, including
the gastrointestinal tract, may affect the dosage required for any
such modality. Such features are well known to those of skill in
the art.
[0157] In another preferred embodiment, the nutritional
compositions are formulated into unit dosage forms such as tablets,
caplets, powder, granules, beads, chewable lozenges, capsules,
liquids, aqueous suspensions or solutions or similar dosage forms,
using conventional equipment and techniques known in the art. Such
formulations typically include a solid, semisolid, or liquid
carrier. Exemplar carriers include lactose, dextrose, sucrose,
sorbitol, mannitol, sutarches, gum acacia, calcium phosphate,
mineral oil, cocoa butter, oil of theobroma, alginates, tragacanth,
gelatin syrup, methyl cellulose, polyoxyethylene sorbitan
monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc,
magnesium stearate, and the like.
[0158] Other formulations suitable for oral administration may be
in the form of capsules, cachets, pills, tablets, lozenges (using a
flavored basis, usually sucrose and acacia or tragacanth), powders,
granules, or as a solution or a suspension in an aqueous or
non-aqueous liquid, or as an oil-in-water or water-in-oil liquid
emulsion, or as an elixir or syrup, or as pastilles (using an inert
base, such as gelatin and glycerin, or sucrose and acacia), each
containing a predetermined amount of a supplement or components
thereof as an active ingredient. A supplement or components thereof
may also be administered as a bolus, electuary, or paste.
[0159] In other formulations, the nutritional supplements are
provided in beverages. The beverages of this invention can be
carbonated beverages e.g., flavored seltzer waters, soft drinks or
mineral drinks, as well as non-carbonated juices, punches and
concentrated forms of these beverages. Beverages, especially juice
and cola beverages, which are carbonated in the manner of soft
drinks, as well as "still" beverages and nectars and full-strength
beverages or beverage concentrates which contain at least about 45%
by weight of juice are also contemplated.
[0160] By way of example, the fruit juices and fruit flavors used
here in include grape, pear, passion fruit, pineapple, banana or
banana puree, apricot, orange, lemon, grapefruit, apple, cranberry,
tomtato, mango, papaya, lime, tangerine, cherry, raspberry, carrot
and mixtures thereof. Additionally, artificial flavors, e.g. cola,
or natural flavors derived from these juices can be used in the
beverages. Chocolate flavors and other non-fruit flavors can also
be used to make beverages containing the vitamin and mineral
supplement. Additionally, milk, obtained from cows or synthetic, is
a contemplated beverage to which the powder compositions of this
invention can be added. The milk may itself include other beverage
components, in particular flavors such as chocolate, coffee, or
strawberry. As used herein, the term "juice product" refers to both
fruit and vegetable juice beverages and fruit and vegetable juice
concentrates which comprise at least about 45% fruit juice.
Vegetable when used herein includes both nonfruit edible plant
parts such as tubers, leaves, rinds, and also if not otherwise
indicated, any grains, nuts, beans, and sprouts which are provided
as juices or beverage flavorings.
[0161] In one preferred embodiment, sport beverages can be
supplemented by the powder compositions of the present invention.
Typical sport beverages contain water, sucrose syrup,
glucose-fructose syrup, and natural or artificial flavors. These
beverages can also contain citric acid, sodium citrate,
monopotassium phosphate, as well as other materials which are
useful in replenishing electrolytes lost during perspiration.
[0162] Tables 2 and 3 depict components of representative sports
and energy drink products.
TABLE-US-00002 TABLE 2 Representative energy drink products. The
`x` indicates that an unspecified amount of the ingredient is
present in the formulation. Monster Energy Red Bull RockStar
Container Size 16 oz 8.3 oz 8, 16, or 24 oz Serving size 8 oz 8.3
oz 8 oz Vitamin B2 1.7 mg 3.4 mg Vitamin B3 20 mg 100 mg 20 mg
Vitamin B5 10 mg Vitamin B6 2 mg 125 mg 2 mg Vitamin B12 6 .mu.g
240 .mu.g 6 .mu.g Sodium 180 mg 200 mg 40 mg Potassium Folic acid
Magnesium Zinc Taurine 1000 mg 1000 mg Penax Ginseng extract 200 mg
25 mg Energy Blend 2500 mg 1350 mg L-Carnitine x 25 mg Glucose x
Caffiene x x 80 mg Guarana x 25 mg Inositol x 25 mg
Glucuronolactone x Maltodextrin x Ginko biloba extr 150 mg Milk
thistle 20 mg Muira pauma extract Catuaba extract Epimedium extract
Yerba mate extract Ingredients Water Carbonated water x x x Sucrose
x x x Fructose Fructose-glucose syrup Glucose x x x Acelsulfame x
Aspartame/Sucralose x x Trehalose Whey-protein isolate Citric acid
x x Phoshoric acid Lactic acid Magnesium carbonate Vitamin E
acetate Salt x Monopotassium phosphate Ester gum Sodium ascorbate
Sodium citrate x x x Flavor x x x Benzoic acid x x Sorbic acid x x
Ascorbic acid x L-Carnitine x x Niacinamide x x x Calcium
pantothenate x x Pantothenic acid Calcium disodium Pyroxidine
hydrochloride x x Magnesium lactate Calcium lactate Gum acacia
Cyanocobalamin x x Potassium citrate Modified food starch Hibiscus
extract Calcium phosphate Gum Arabic Vanocobalamin Red clover
flower extract Magnesium oxide Zinc picolinate Muira pauma extr
Epimedium sagittatum extract Guarana seed extract x Yerba mate
extract Catuaba extract Folic acid Taurine x x Penax Ginseng
extract x Glucuronolactone x x Inositol x x Riboflavin x
Maltodextrin x
TABLE-US-00003 TABLE 3 Representative sports drink products Life
Propel Vitamin Water Function: Fit Water (Goji Alternative Gatorade
Accelerade Water Revive Melon) Energy Container Size 20 oz 20 oz
18.9 oz 20 oz 20 oz 16.9 oz Serving size 8 oz 8 oz 8 oz 8 oz 8 oz 8
oz Vitamin B2 Vitamin B3 20 mg 10 mg Vitamin B5 20 mg Vitamin B6
12.5 mg 12.5 mg 5 mg 1 mg Vitamin B12 75 .mu.g 60 .mu.g 30 .mu.g 3
.mu.g Sodium 110 mg 120 mg 35 mg 20 mg 120 mg Potassium 30 mg 15 mg
140 mg Folic acid 200 .mu.g Magnesium 20 mg Zinc 4.95 mg Taurine
Penax Ginseng extract Energy Blend 300 mg L-Carnitine Glucose
Caffiene x Guarana x Inositol Glucuronolactone Maltodextrin Ginko
biloba extract Milk thistle Muira pauma extract x Catuaba extract x
Epimedium extract x Yerba mate extract x Ingredients Water x x x x
x x Carbonated water Sucrose x x x x x x Fructose x
Fructose-glucose syrup x Glucose Acelsulfame x Aspartame/sucralose
x Trehalose x Whey-protein isolate x Citric acid x x x x x x
Phoshoric acid x Lactic acid x Magnesium carbonate x Vitamin E
acetate x 10% x RDA Salt x x x Ester gum x x x Sodium ascorbate x
Sodium citrate x x x Flavor x x x x x x Benzoic acid Sorbic acid
Ascorbic acid x x x L-Carnitine Niacinamide x x x x Calcium
pantothenate x x Pantothenic acid x Calcium disodium x Pyroxidine
hydrochloride x x x x Magnesium lactate Calcium lactate x Gum
acacia x Cyanocobalamin x x Potassium citrate x Modified food
starch x Hibiscus extract x Calcium phosphate x Gum Arabic x
Vanocobalamin x Red clover flower extract x Magnesium oxide x Zinc
picolinate x Muira pauma extract x Guarana seed extract x Yerba
mate extract x Catuaba extract x Folic acid x Taurine Penax Ginseng
extr. Glucuronolactone Inositol Riboflavin Maltodextrin
[0163] As used herein, the term "juice beverage" refers to a fruit
or vegetable juice product which is in a single-strength,
ready-to-serve, drinkable form. Juice beverages of the present
invention can be of the "full-strength" type which typically
comprise at least about 95% juice. Full strength juice beverages
also include those products of 100% juice such as, for example,
orange, lemon, apple, raspberry, cherry, apricot, pear, grapefruit,
grape, lime, tangerine, carrot, pineapple, melon, mango, papaya,
passion fruit, banana and banana puree, cranberry, tomato, carrot,
cabbage, celery, cucumber, spinach, and various mixtures thereof.
Juice beverages also include extended juice products which are
referred to as "nectars". These extended juice products typically
comprise from about 50% to about 90%, about 55% to about 85%, about
60% to about 80%, about 65% to about 75% juice, preferably, from
about 50% to about 70% juice. Nectars usually have added sugars or
artificial sweeteners or carbohydrate substitutes. As used herein,
the term "citrus juice" refers to fruit juices selected from orange
juice, lemon juice, lime juice, grapefruit juice, tangerine juice
and mixtures thereof.
[0164] As used herein, the term "juice materials" refers to
concentrated fruit or vegetable juice, plus other juice materials
such as juice aroma and flavor volatiles, peel oils, and pulp or
pomace. As used herein, the term "juice concentrate" refers to a
fruit or vegetable juice product which, when diluted with the
appropriate amount of water, forms drinkable juice beverages. Juice
concentrates within the scope of the present invention are
typically formulated to provide drinkable beverages when diluted
with 3 to 5 parts by weight water.
[0165] As used herein the term "beverage concentrate" or "bottling
syrup" refers to a mixture of flavors, water and from about 10% to
about 60%, about 20% to about 50% or about 30% to about 40% sugar
or carbohydrate substitute, e.g., sucrose, dextrose, corn syrup
solids, fructose, dextrins, polydextrose and mixtures thereof.
[0166] The flavor component of the beverages and beverage
concentrates contains flavors selected from fruit flavors,
vegetable flavors, botanical flavors and mixtures thereof. As used
herein, the term "fruit flavor" refers to those flavors derived
from the edible reproductive part of a seed plant, especially one
having a sweet pulp associated with the seed, and "vegetable
flavor" refers to flavors derived from other edible parts of seed
and other plants. Also included within the term "fruit flavor" and
"vegetable flavor" are synthetically prepared flavors made to
simulate fruit or vegetable flavors derived from natural sources.
Particularly preferred fruit flavors are the citrus flavors
including orange, lemon, lime and grapefruit flavors. Besides
citrus flavors, a variety of other fruit flavors can be used such
as apple, grape, cherry, pineapple, mango and papaya flavors and
the like. These fruit flavors can be derived from natural sources
such as juices and flavor oils, or can be synthetically prepared.
As used herein, the term "botanical flavor" refers to flavors
derived from parts of a plant other than the fruit; i.e., derived
from nuts, bark, roots and leaves, and beans such as coffee, cocoa,
and vanilla. Also included within the term "botanical flavor" are
synthetically prepared flavors made to simulate botanical flavors
derived from natural sources. Examples of such flavors include
cola, tea, coffee, chocolate, vanilla, almond, and the like.
Botanical flavors can be derived from natural sources such as
essential oils and extracts, or can be synthetically prepared.
[0167] The flavor component can comprise a blend of various
flavors, e.g. lemon and lime flavors, cola flavors and citrus
flavors to form cola flavors, etc. If desired, juices such as
orange, lemon, lime, apple, grape, carrot, celery, and like juices
can be used in the flavor component. The flavors in the flavor
component are sometimes formed into emulsion droplets which are
then dispersed in the beverage concentrate. Because these droplets
usually have a specific gravity less than that of water and would
therefore form a separate phase, weighting agents (which can also
act as clouding agents) are typically used to keep the emulsion
droplets dispersed in the beverage. Examples of such weighting
agents are brominated vegetable oils (BVO) and rosin esters, in
particular the ester gums. See Green, L. F. Developments in Soft
Drinks Technology; Applied Science Publishers: London, 1978; Vol.
1, pp 87-93, for a further description of the use of weighting and
clouding agents in liquid beverages. Besides weighting agents,
emulsifiers and emulsion stabilizers can be used to stabilize the
emulsion droplets. Examples of such emulsifiers and emulsion
stabilizers include the gums, pectins, celluloses, polysorbates,
sorbitan esters and propylene glycol alginates. See Green, L. F.
supra at p. 92. The particular amount of the flavor component
effective for imparting flavor characteristics to the beverages and
beverage concentrates ("flavor enhancing") can depend upon the
flavor(s) selected, the flavor impression desired, and the form of
the flavor component.
[0168] The flavor component can comprise at least 0.05% by weight
of the beverage composition, and typically from 0.1% to 2% by
weight for carbonated beverages. When juices are used as the
flavor, the flavor component can comprise, on a single-strength
basis, up to 25% fruit juice by weight of the beverage, preferably
from 5% to 15% juice by weight for carbonated beverages.
[0169] Carbon dioxide can be introduced into the water which is
mixed with the beverage syrup or into the drinkable beverage after
dilution to achieve carbonation. The carbonated beverage can be
placed into a container such as a bottle or can and then sealed.
Any conventional carbonation methodology can be used to make the
carbonated beverages of this invention. The amount of carbon
dioxide introduced into the beverage will depend upon the
particular flavor system used and the amount of carbonation
desired. Usually, carbonated beverages of the present invention
contain from 1.0 to 4.5 volumes of carbon dioxide. The preferred
carbonated beverages contain from 2 to about 3.5 volumes of carbon
dioxide.
[0170] The present invention is also particularly suited for the
supplementation of beverages and beverage concentrates, including
water and citrus juices. The beverages can contain from 3% to 100%
juice or from about 0.05% to about 10% of an artificial or natural
flavor, particularly orange juice. The concentrated orange juice,
orange juice aroma and flavor volatiles, pulp and peel oils used in
the method of the present invention can be obtained from standard
orange juice. See Nagy, S.; Shaw, P. E.; Veldhuis, M. K. Citrus
Science and Technology; AVI Publishing: Westport, Conn., 1977; Vol.
2, pp 177-252 for standard processing of oranges, grapefruit and
tangerines. (See also Nelson et al. Fruit and Vegetable Juice
Processing Technology, 3rd ed.; AVI Publishing: Westport, Conn.,
1980; pp. 180-505, for standard processing of noncitrus juices such
as apple, grape, pineapple, etc. to provide sources of juice and
juice materials for noncitrus juice products).
[0171] Juices from different sources are frequently blended to
adjust the sugar to acid ratio of the juice. Different varieties of
oranges can be blended or different juices can be blended to get
the desired flavor and sugar to acid ratio. A sugar to acid ratio
of from about 8:1 to about 20:1 is considered acceptable for fruit
juices. However, preferred sugar to acid ratios are typically from
about 11:1 to about 15:1, especially for citrus juices. Sweeteners
include the sugars normally present in juice products, for example
glucose, sucrose, and fructose. Sugars also include high fructose
corn syrup, invert syrup, sugar alcohols, including sorbitol,
refiners syrup, and mixtures thereof. In addition to sugar,
extended juice beverages of the present invention can contain other
sweeteners. Other suitable sweeteners include saccharin,
cyclamates, acetosulfam, L-aspartyl-L-phenylalanine lower alkyl
ester sweeteners (e.g. aspartame). A preferred sweetener for use in
such extended juice products is aspartame. For single-strength
juice beverages, the sugar content can range from about 2.degree.
to about 16.degree. Brix (16.degree. Brix means the juice contains
about 16% soluble solid, and so on). Typically, the sugar content
of such beverages depends upon the amount of juice contained
herein.
[0172] In solid dosage forms for oral administration (capsules,
tablets, pills, dragees, powders, granules and the like), the
supplement or components thereof is mixed with one or more
pharmaceutically-acceptable carriers, such as sodium citrate or
dicalcium phosphate, and/or any of the following: (1) fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; (2) binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; (3) humectants, such as glycerol; (4)
disintegrating agents, such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate; (5) solution retarding agents, such as paraffin; (6)
absorption accelerators, such as quaternary ammonium compounds; (7)
wetting agents, such as, for example, acetyl alcohol and glycerol
monostearate; (8) absorbents, such as kaolin and bentonite clay;
(9) lubricants, such a talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof; and (10) coloring agents. In the case of capsules, tablets
and pills, the pharmaceutical compositions may also comprise
buffering agents. Solid compositions of a similar type may also be
employed as fillers in soft and hard-filled gelatin capsules using
such excipients as lactose or milk sugars, as well as high
molecular weight polyethylene glycols and the like.
[0173] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the supplement or components thereof moistened with an
inert liquid diluent. Tablets, and other solid dosage forms, such
as dragees, capsules, pills and granules, may optionally be scored
or prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating
art.
[0174] Tablets and other solid dosage forms may also be formulated
so as to provide slow or controlled release of the active
ingredient therein using, for example, hydroxypropylmethyl
cellulose in varying proportions to provide the desired release
profile, other polymer matrices, liposomes and/or microspheres.
They may be sterilized by, for example, filtration through a
bacteria-retaining filter, or by incorporating sterilizing agents
in the form of sterile solid compositions which may be dissolved in
sterile water, or sonic other sterile injectable medium immediately
before use. These compositions may also optionally contain
opacifying agents and may be of a composition that they release the
active ingredient(s) only, or preferentially, in a certain portion
of the gastrointestinal tract, optionally, in a delayed manner.
Examples of embedding compositions which may be used include
polymeric substances and waxes. The active ingredient may also be
in micro-encapsulated form, if appropriate, with one or more of the
above-described nutritional supplements.
[0175] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs. In addition to the supplement or
component, the liquid dosage forms may contain inert diluents
commonly used in the art, such as, for example, water or other
solvents, solubilizing agents and emulsifiers, such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
oils (in particular, cottonseed, groundnut, corn, germ, olive,
castor and sesame oils), glycerol, tetrahydrofuryl alcohol,
polyethylene glycols and fatty acid esters of sorbitan, and
mixtures thereof.
[0176] Besides inert diluents, the oral compositions may also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents. Suspensions, in addition to the supplement or
components thereof, may contain suspending agents as, for example,
ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and
sorbitan esters, microcrystalline cellulose, aluminum
metahydroxide, bentonite, agar-agar and tragacanth, and mixtures
thereof.
[0177] The composition of the invention can be administered as a
capsule or tablet containing a single or divided dose of the
inhibitor. Preferably, the composition is administered as a sterile
solution, suspension, or emulsion, in a single or divided dose.
Tablets may contain carriers such as lactose and corn starch,
and/or lubricating agents such as magnesium stearate. Capsules may
contain diluents including lactose and dried corn starch.
[0178] A tablet may be made by compressing or molding the active
ingredient optionally with one or more accessory ingredients.
Compressed tablets may be prepared by compressing, in a suitable
machine, the active ingredient in a free-flowing form such as a
powder or granules, optionally mixed with a binder, lubricant,
inert diluent, surface active, or dispersing agent. Molded tablets
may be made by molding in a suitable machine, a mixture of the
powdered active ingredient and a suitable carrier moistened with an
inert liquid diluent.
[0179] When preparing dosage form incorporating the compositions of
the invention, the compounds may also be blended with conventional
excipients such as binders, including gelatin, pregelatinized
starch, and the like; lubricants, such as hydrogenated vegetable
oil, sutearic acid, and the like; diluents, such as lactose,
mannose, and sucrose; disintegrants, such as carboxymethylcellulose
and sodium starch glycolate; suspending agents, such as povidone,
polyvinyl alcohol, and the like; absorbants, such as silicon
dioxide; preservatives, such as methylparaben, propylparaben, and
sodium benzoate; surfactants, such as sodium lauryl sulfate,
polysorbate 80, and the like; colorants such as F.D. & C. dyes
and lakes; flavorants; and sweeteners.
[0180] The invention will now be further described by way of the
following non-limiting examples.
Example 1
Delayed-Release Carbohydrate Formulations for Athletic Performance
Enhancement
[0181] The goal of the Example is to develop a nutrition fluid that
delivers carbohydrates and other nutrients to an athlete in a
manner that promotes peak athletic performance. The proposed
approach is to engineer controlled release of digestible
carbohydrates from aqueous dispersion of suitable micro or
nanospheres. Important digestible carbohydrates include: the
monosaccharides--glucose, fructose and galactose; the
dissacharides--sucrose, maltose and lactose; and the
polysaccharide, starch. Starch is broken down in to dextrins by
salivary amylase (in the mouth) and pancreatic amylase (in the
small intestine). Dextrin is acted upon by the brush border enzymes
in the small intestine, which also convert the double sugars into
simple sugars. The monosaccharides are finally transported across
the intestinal epithelium into the bloodstream. The proposed
research seeks controlled release of digestible carbohydrates,
especially the simple sugars, glucose and fructose, for sustained
uptake into the blood.
[0182] A basic understanding of the physiology of the
gastrointestinal (GI) tract is useful in the design of the delivery
system. The retention time of food in the stomach is up to 2 hours
and depends, among other factors, on the calorific value of the
meal (see, e.g., Hadi, N. A.; Giouvanoudi, A.; Morton, R.; Horton,
P. W.; Spyrou, N. M. Variations in gastric emptying times of three
stomach regions for simple and complex meals using scintigraphy.
IEEE Transactions on Nuclear Science 2002, 49, 2328-2331). The
controlled release system should be able to withstand the acidic pH
(1-3) of the stomach during gastric retention, without releasing
the sugar payload. Residence time in the small intestine, where
most of the nutrient absorption occurs, is about 3 h. For nutrient
delivery over a longer time period, it is necessary to prolong
intestinal retention which may be achieved by encapsulating the
nutrient in a carrier with mucoadhesive properties. Hydrophilic
polymers containing carboxylic acid groups exhibit good
mucoadhesive properties. A key step in the design of a controlled
release system for sugar is the selection of a carrier material for
encapsulating carbohydrates. Polysaccharides and their derivatives
are polymers of choice as carriers for sustained-release drug
delivery and scaffolds in tissue engineering because of their
non-toxic nature and excellent biocompatibility (see, e.g.,
Dumitriu, S.; Dumitriu, M. Hydrogels as support for drug delivery
systems. In Polysaccharides in Medicinal Applications; Dumitriu, S.
Ed.; Dekker: New York, 1996; pp 705-764; Coviello, T.; Matricardi,
P.; Marianecci, C.; Alhaique, F. Polysaccharide hydrogels for
modified release formulations. J. Control. Rel. 2007, 119, 5-24 and
Kong, H.; Mooney, D. J. Polysaccharide-based hydrogels in tissue
engineering. In Polysaccharides, 2.sup.nd ed.; Dumitriu, S., Ed.;
Dekker: New York, 2005; pp 817-837). They have also been used for
flavor encapsulation in food formulations (see, e.g., Madene, A.;
Jacquot, M.; Scher, J.; Desobry, S. Flavour encapsulation and
controlled release--a review. International Journal of Food Science
and Technology 2006, 41, 1-21).
[0183] Blends of polysaccharides are used to synthesize aqueous
dispersions of micro- or nanoparticles. Hydrophobically modified
polysaccharides such as hydroxypropyl cellulose or hydroxyethyl
cellulose are known to spontaneously form nanoparticles in water.
Interpenetrating polymer networks of these polymers, with
polysaccharides containing carboxylic acid groups, are synthesized.
FIG. 1 shows the chemical structures of three important
carboxy-containing polysaccharides. The monomeric unit of the
carboxymethylcellulose backbone consists of D glucose residues
linked through .beta.-(1.fwdarw.4) bonds. Alginates are composed of
(1.fwdarw.4)-linked .beta.-D-mannuronic acid and
.alpha.-L-guluronic acid monomers which vary in amount and
sequential distribution along the polymer chain depending on the
source of alginate. Hyaluronic acid is a straight polymer
consisting of alternating (1.fwdarw.4)-linked
2-acetamide-2-deoxy-.beta.-D-glucose and (1.fwdarw.3) linked
.beta.-D-glucuronic acid.
[0184] To increase stability of the particles in the GI tract, the
particles are crosslinked to form hydrogels. Different crosslinking
mechanisms are investigated to achieve the desired release
kinetics. Crosslinking is performed using free radical initiators
such as persulfate salts, or redox systems involving ascorbic acid,
or a naturally occurring crosslinker such as genipin. Ionic
crosslinking is also explored. Anionic polysaccharides such as
gellan can be used for ionic crosslinking, instead of chemicals
such as borax which may not be desirable in a food formulation.
[0185] It is expected that the carboxy containing hydrogel
particles are in a collapsed state in the acidic environment of the
stomach. Hence, the encapsulated sugar molecules are retained
within the particles in the stomach. The hydrogel particles will
achieve an expanded state when they reach the small intestine (pH
5-7), and will release the encapsulated sugar at a rate faster than
that in the stomach.
[0186] Several researchers have investigated the synthesis of
polysaccharide particles and hydrogels for controlled release. Most
of these studies were, however, focused on incorporating relatively
hydrophobic drugs or protein macromolecules in the carriers. An
objective of the proposed research is to encapsulate small
hydrophilic molecules such as sugars. The equilibrium partitioning
of sugar molecules between the hydrogel particles and the aqueous
phase is determined. Due to similarities in the chemical structures
of the polysaccharide carrier and the encapsulated monosaccharides,
it is expected that the encapsulation efficiency of polysaccharide
hydrogels are higher than those of other hydrogels.
[0187] There are only a few studies that have reported delayed
release systems for carbohydrates. Fox and Allen (Fox, G. J.;
Darlene, A. Method and composition for controlling the release of
carbohydrates by encapsulation. U.S. Pat. No. 5,536,156, Jul. 16,
1996) have coated carbohydrate microparticles with an edible
delayed-release coating. The coated carbohydrate, when orally
ingested, causes a time delayed release of the carbohydrate into
the digestive system. The coated particles were 30 to 100 .mu.m in
size and were stored in solid particulate form. In contrast,
Applicants seek to develop controlled release particles that are
dispersed in an aqueous medium. Lake and Smith (Lake, M.; Smith, U.
Composition and method for long-term glycemic control. Int. Pat.
Appl. WO/2006/022585, Feb. 3, 2006) have reported the preparation
of starch granules that can be used for improved long-term control
of blood glucose in a diabetic patient. The delayed-release starch
formulation was designed to reduce the incidence of nocturnal
hypoglycemia, wherein the patient would ingest a therapeutic amount
of starch granules at bedtime. Zecher (Zecher, D. C. Controlled
release carbohydrate embedded in a crosslinked polysaccharide. Int.
Pat. Appl. WO/2000/032064, Aug. 6, 2000) has reported a similar
controlled release carbohydrate composition consisting of
covalently crosslinked polysaccharides. However, the crosslinked
carbohydrates were not in a particulate form, and were not in the
form of aqueous suspensions.
[0188] The following sections will describe methods for the
synthesis of polysaccharide hydrogels.
[0189] Hydrophobized polysaccharides are highly promising in the
synthesis of nanoparticles because of their self-assembling
properties in aqueous environment. Akiyoshi and Sunamoto (Akiyoshi,
K.; Sunamoto, J. Supramolecular assembly of hydrophobized
polysaccharides. Supramolecular Science 1996, 3, 157-163) found
that polysaccharides that were functionalized with hydrophobes such
as cholesterol spontaneously formed nanoparticles when dispersed in
water. The size, density and colloidal stability of the
nanoparticle could be controlled by tailoring the grafting density
and degree of hydrophobicity of the hydrophobe. Polysaccharides
such as pullulan, dextran and mannan were partly substituted by
various hydrophobic groups such as long alkyl chains and
cholesterol. For example, pullulan with a molecular weight of 55
kDa, when functionalized with cholesterol (.about.1.7 cholesterol
moieties per 100 units of glucose) spontaneously formed
nanoparticles that were 20-30 nm in size (Akiyoshi, K.; Deguchi,
S.; Tajima, H.; Nishikawa, T.; Sunamota, J. Self-assembly of
hydrophobized polysaccharide: Structure of hydrogel nanoparticle
and complexation with organic compounds. Proc. Japan Acad. 1995,
71, 15-19). The cholesterol bearing pullulan self-aggregated to
form monodisperse stable nanoparticles after ultrasonification of
the suspension in water. No coagulation occurred even after heating
at 90.degree. C. for 1 h. These nanoparticles were used for hosting
hydrophobic substances such as antitumor adriamycin (Akiyoshi, K.;
Taniguchi, I.; Fukui, H.; Sunamoto, J. Hydrogel nanoparticle formed
by self-assembly of hydrophobized polysaccharide. Stabilization of
adriamycin by complexation. European Journal of Pharmaceutics and
Biopharmaceutics 1996, 42, 286-290) and various water-soluble
proteins, but encapsulation of small water-soluble molecules was
not reported.
[0190] Simi and Abraham (Simi, C. K.; Abraham, T. E. Hydrophobic
grafted and crosslinked starch nanoparticles for drug delivery.
Bioprocess and Biosystems Engineering 2007, 30, 173-180) have
grafted fatty acid on to starch using potassium persulfate as
catalyst. Nanoparticles resulting from the modified starch
molecules were further stabilized by crosslinking with sodium
tripolyphosphate. The nanoparticles were used for encapsulation of
a hydrophobic drug.
[0191] Thielemans et al. (Thielemans, W.; Belgacem, M. N.;
Dufresne, A. Starch nanocrystals with large chain surface
modifications. Langmuir 2006, 22, 4804-4810) were also successful
in surface-modification of nanoscale starch particles using stearic
acid chloride (a hydrophobe) and poly(ethylene glycol) methyl ether
(a hydrophilic molecule). Woo et al. (Woo, B. H.; Jiang, G.; Jo, Y.
W.; DeLuca, P. P. Preparation and characterization of a composite
PLGA and poly(acryloyl hydroxyethyl starch) microsphere system for
protein delivery. Pharmaceutical Research 2001, 18, 1600-1606) have
reported the synthesis of polysaccharide microspheres using
acryloyl-modified hydroxyethyl starch. The microspheres were
investigated for their ability to load protein for controlled
protein delivery.
[0192] Basheer et al. (Besheer, A.; Hause, G.; Kressler, J.; Mader,
K. Hydrophobically modified hydroxyethyl starch: Synthesis,
characterization, and aqueous self-assembly into nano-sized
polymeric micelles and vesicles. Biomacromolecules 2007, 8,
359-367) reacted hydroxyethyl starch with fatty acids (lauric,
pahnitic, and stearic acids) under mild reaction conditions using
dicyclohexyl carbodiimide (DCC) and dimethylaminopyridine (DMAP).
The resulting polymers self-assembled to form 20-30 nm micelles and
250-350 nm polymeric vesicles. However, chemicals such as DCC and
DMAP are potentially toxic and cannot be used in edible
formulations.
[0193] Chakraborty et al. (Chakraborty, S.; Sahoo, B.; Teraoka, I.;
Gross, R. A. Solution properties of starch nanoparticles in water
and DMSO as studied by dynamic light scattering. Carbohydrate
Polymers 2005, 60, 475-481) have studied the solution properties of
starch nanoparticles in water using dynamic light scattering. The
nanoparticles were obtained from Ecosynthetix (Lansing, Mich.), and
were synthesized from corn starch using glyoxal as crosslinker. A
mixture of starch, glycerol (18 wt % of dry starch), and glyoxal
(0.1-10 wt %) was extruded to obtain crosslinked starch granules.
The granules were cryogenically ground and sieved to obtain
particles smaller than 150 nm in diameter. Dynamic light scattering
or the particles in water indicated two main populations, with mean
diameters of 40 and 300 nm, consisting of isolated starch
nanoparticles and their aggregates, respectively. At higher
concentration (.about.3% w/w), a third peak appeared at around 1
.mu.m, because of particle aggregation. Control of particle
aggregation is an important step in the design of carbohydrate
nanoparticles.
[0194] A key feature of the proposed polysaccharide hydrogels is
their pH responsiveness. Ideally, the hydrogels should not swell in
the acidic environment of the stomach, but should swell upon entry
into the small intestine and release the encapsulated sugars at a
controlled rate. This section reviews an extreme case where the
polysaccharide matrix was insoluble in acidic environments, while
it completely dissolved at higher pH values.
[0195] Scleroglucan is a branched homopolysaccharide that gives
only D-glucose upon complete hydrolysis. The polymer consists of a
main chain of (1.fwdarw.3)-linked .beta.-D-glucopyranosyl units. At
every third unit along the main chain, the polymer bears a single
(1.fwdarw.6)-linked .beta.-D-glucopyranosyl unit as a branch. The
glucopyranose side chain of scleroglucan was oxidized by means of a
two-step reaction: first with periodate, to form an aldehyde
derivative, and then with chlorite, which resulted in the
carboxylated derivative called sclerox (see, e.g., FIG. 2 and
Coviello, T.; Palleschi, A.; Grassi, M.; Matricardi, P.;
Bocchinfuso, G.; Alhaique, F. Scleroglucan: A versatile
polysaccharide for modified drug delivery. Molecules 2005, 10,
6-33). By varying the ratio between oxidizing agent and
polysaccharide, the polymer could be oxidized to a different
extent. It was found that above a 60% oxidation, sclerox became
sensitive to environmental conditions giving a reversible sol-gel
transition mediated by pH. Permeation of model molecules occurred
at different rates through the sol and the gel, and consequently,
release from sclerox tablets showed different profiles in the two
environments simulating the gastric and the intestinal fluids,
respectively.
[0196] In acidic medium the formation of a swelled layer around the
dosage form acquired a fundamental role in determining the rate of
delivery, while at higher pH values erosion and dissolution became
predominant. Addition of an acid substance, such as citric acid, in
the formulation reduced the release rate in simulated intestinal
fluid. The delivery rate was still too rapid in relation to the
transit time through the GI tract. Thus another strategy was used.
The polysaccharide was first derivatized to introduce aldehydic or
carboxylic groups on the side chain. These groups were then
crosslinked to produce more stable three-dimensional networks.
[0197] Pitarresi et al. have reported crosslinking of methacrylic
anhydride functionalized carbohydrates by UV irradiation.
Hyaluronic acid was first derivatized with methacrylic anhydride.
Relatively low molecular-weight hyaluronic acid (174 kDa) was
dissolved in water to form a 2% (w/v) solution. A 20-fold excess of
methacrylic acid (with respect to the moles of repeating unit of
hyaluronic acid) was added with simultaneous addition of 5 N NaOH
(to maintain the pH between 8 and 9). The reaction was kept at
4.degree. C. and stirred for 24 h. The reaction mixture was then
precipitated in ethanol and the product was recovered and purified
by centrifugation and gel permeation chromatography.
[0198] Giezen et al. have disclosed a process for producing
biopolymer nanoparticles in which a starch or a starch derivative
was crosslinked using dialdehyde or polyaldehyde (see, e.g.,
Giezen, F. E.; Jongboom, R. O. J.; Fell, J.; Gotlieb, K. F.;
Boersma, A. Biopolymer nanoparticles. U.S. Pat. No. 6,677,386, Jan.
13, 2004). A plasticizer, glycerol, was used during the process,
along with an acid such as maleic acid, oxalic acid, or citric
acid. It should be noted that chemicals such as dialdehydes and
glycerol are not suitable as food ingredients. The crosslinked
nanoparticles had an average particle size below 400 nm. The
viscosity of an aqueous dispersion containing 10 wt % of these
particles had a viscosity below 150 mPas (measured at a shear rate
of 186 s.sup.-1).
[0199] The formulation viscosity is expected to increase with an
increase in particle concentration. As a first approximation,
viscosity of a suspension is related to the particle concentration
through the Einstein's equation, .eta.=.eta..sub.w(1+2.5.phi.),
where .eta. is the viscosity of the dispersion, .eta..sub.w is the
viscosity of the aqueous phase, and .phi. is the volume fraction of
particles in the dispersion. The particle volume fraction is given
by
.phi. = [ 1 + ( .rho. p .rho. w ) ( 1 m - 1 ) ] - 1 ,
##EQU00001##
where .rho..sub.p is the density of the particles, .rho..sub.w is
the aqueous phase density, and m is the mass fraction of particles
in the dispersion. Dispersion viscosity also depends on the
interparticle distance, H, which is the average distance between
the surfaces of two neighboring particles in the dispersion. For a
population of monodisperse particles with hexagonal close packed
structure, the interparticle distance is given by
H = D { ( 0.74 .phi. ) 1 / 3 - 1 } ##EQU00002##
where D is the particle diameter. Therefore, for a given mass
fraction of polymer in the dispersion (that is, a fixed .phi.) the
dispersion viscosity is expected to be higher when the particles
are smaller in size. In this Example, the viscosity of the
dispersion is tailored to be close to that of water (.about.1
mPas).
[0200] Magnani et al. have synthesized polysaccharide hydrogels
using alginates, hyaluronane, and carboxymethylcellulose (see,
e.g., Magnani, A.; Rappuoli, R.; Lamponi, S.; Barbucci, R. Novel
polysaccharide hydrogels: characterization and properties. Polym.
Adv. Technol. 2000, 11, 488-495 and Barbucci, R.; Consumi, M.;
Lamponi, S.; Leone, G. Polysaccharides based hydrogels for
biological applications. Macromol. Symp. 2003, 204, 37-58). The
crosslinking procedure consisted of activating the carboxylate
moieties by 2-chloro-1-methylpyridinium iodide and using
1,3-diaminopropane as a crosslinker. Aqueous solution of the sodium
hyaluronate, alginate and carboxymethyl cellulose were transformed
in carboxylic acid by treatment with a strong acid exchange resin
Dowex 50 WX8 (Fluka) at 4.degree. C. The solution was added to a 5%
tetrabutylammonium hydroxide solution to achieve a pH between 8 and
9. After lyophilization the tetrabutylammonium salt of the
polysaccharide was dissolved in dimethylformamide (DMF). A
stoichiometric quantity of 2-chloro-1-methylpyridinium iodide was
added to the solution kept at 0.degree. C. The crosslinking diamine
was then added in excess to the solution and the reaction mixture
was maintained under stirring at room temperature for 4 h. The
reaction was facilitated by the addition of a small amount of
triethylamine, which acted as hydrogen iodide captor. Macroscopic
gels were obtained. The synthesis of micro- or nanoparticles was
not reported. Moreover, the chemicals involved are not suitable for
food formulation.
[0201] Kabra et al. prepared macrogels of hydroxylpropyl cellulose
by crosslinking the polymer with divinyl sulfone at a temperature
above the lower critical solution temperature (LCST, 41.degree. C.)
(see, e.g., Kabra, B. G.; Gehrke, S. H.; Spontak, R. J.
Microporous, responsive hydroxypropyl cellulose gels. 1. Synthesis
and microstructure. Macromolecules 1998, 31, 2166-2173). Cai et al.
used the same reaction to prepare aqueous suspensions of
hydroxylpropyl cellulose nanoparticles (see, e.g., Cai, T.; Hu, Z.;
Marquez, M. Synthesis and self-assembly of nearly monodisperse
nanoparticles of a naturally occurring polymer. Langmuir 2004, 20,
7355-7359). High molecular weight (.about.10.sup.6 Da) was
dissolved in an aqueous solution of sodium hydroxide (pH 12).
Dodecyltrimethylammonium bormide and dinvyl sulfone were added as
the surfactant and crosslinker, respectively. The solution was
heated to a temperature above the LCST for about 3 h resulting in
nanoparticles with diameters ranging from 170 nm to 430 nm. The
particle concentration was below 0.1 wt %. Gao et al. have also
reported in situ crosslinking of self-associated hydroxypropyl
cellulose nanoparticles using divinyl sulfone (see, e.g., Gao, J.;
Haidar, G.; Lu, X.; Hu, Z. Self-association of
hydroxypropylcellulose in water. Macromolecules 2001, 34,
2242-2247).
[0202] De Nooy et al. have used the reaction between a carboxylic
acid, an aldehyde, and an isocyanide (the Passerini three-component
condensation) to prepare polysaccharide hydrogels (see, e.g., De
Nooy, A. E. J.; Masci, G.; Crescenzi, V. Versatile synthesis of
polysaccharide hydrogels using the Passerini and Ugi multicomponent
condensations. Macromolecules 1999, 32, 1318-1320). Carboxylic acid
containing carbohydrates such as carboxymethyl cellulose or
hyaluronic acid were used to prepare the hydrogels. Polysaccharides
such as scleroglucan or pullulan that do not contain carboxylic
acid groups were partially oxidized using TEMPO to introduce
aldehyde and carboxylic acid groups. The Ugi condensation reaction
involves an additional component, an amine. Formaldehyde or
glutaraldehyde were used along with amines such as
1,5-diaminopentane or ammonium chloride for the condensation
reaction. Chitosan was also used in hydrogel synthesis because of
the amine groups present in its structure. All the gels synthesized
were macroscopic gels. Micro- or nanoparticle synthesis was not
reported. Moreover, aldehydes and isocyanides are generally not
considered safe as food ingredients.
[0203] Covalently crosslinked hydrogels prepared using other
crosslinking reactions have also been reported. Dou et al. have
synthesized carboxy-functionalized nanoparticles of dextran,
hydropropyl cellulose, and hydroethyl cellulose (see, e.g., Dou,
H.; Tang, M.; Yang, W.; Sun, K. One-pot synthesis,
characterization, and drug loading of polysaccharide-based
nanoparticles with carboxy functional groups. Colloid Polym. Sci.
2007, 285, 1043-1047). Their procedure, however, involves the use
of chemicals such as nitric acid, acrylic acid, cerium (IV)
ammonium nitrate, and N,N'-methylene bisacrylamide, which are
unsuitable for food formulation. Yu and Hoffman have reported the
synthesis of chemically crosslinked sodium alginate/chondroitin
6-sulfate hydrogels for controlled release of a model cationic
protein, lysozyme (see, e.g., Yu, X. J.; Hoffman, A. S.
Polysaccharide hydrogels as drug delivery matrixes. Proceedings of
the 22.sup.nd International Symposium on Controlled Release of
Bioactive Materials, 1995; Controlled Release Society; pp 352-353).
The hydrogel was found to be in a condensed state in divalent
cation solutions and in a decondensed state in PBS. Chen et al.
have reviewed some polysaccharide hydrogels that were used for
nasal delivery of peptides and proteins (see, e.g., Chen, J.; Jo,
S.; Park, K. Polysaccharide hydrogels for protein drug delivery.
Carbohydrate Polymers 1995, 28, 69-76). These hydrogels, which were
designed for nasal delivery of insulin, included 45 .mu.m starch
microspheres (see, e.g., Illum, L.; Jorgensen, H.; Bisgaard, H.;
Krogsgaard, O.; Rossing, N. Bioadhesive microspheres as a potential
nasal drug delivery system. Int. J. Pharm. 1987, 39, 189-199),
epichlorohydrin crosslinked dextran (see, e.g., Edman, P.; Bjork,
E. Routes of delivery: Case studies: (1) Nasal delivery of peptide
drugs. Adv. Drug Delivery Rev. 1992, 8, 165-177) and hyaluronic
acid ester microspheres (see, e.g., Illum, L.; Farraj, N. F.;
Fisher, A. N.; Gill, I.; Miglietta, M.; Benedetti, L. M. Hyaluronic
acid ester microspheres as a nasal delivery system for insulin. J.
Control. Rel. 1994, 29, 133-141). The microspheres of hyaluronic
esters were 10 to 100 .mu.m in diameter (see, e.g., Benedetti, L.
M.; Topp, E. M.; Stella, V. J. Microspheres of hyaluronic acid
esters-Fabrication methods and in vitro hydrocortisone release. J.
Control. Rel. 1990, 13, 33-41).
[0204] Selection of a suitable crosslinker is a key step in the
preparation of polysaccharide hydrogels for food formulations.
Clearly, toxicity of the crosslinking chemical precludes its use.
Genipin is a naturally occurring crosslinker for proteins and
polysaccharides, and is obtained from gardenia fruit extracts. It
has attracted significant interest in the synthesis of
polysaccharide hydrogels. It has low acute toxicity (LD.sub.50 i.v.
382 mg/kg in mice) and is much less toxic than most other chemical
crosslinking agents such as glutaraldehyde. Its structure is shown
in FIG. 2.
[0205] Meena et al. used genipin to crosslink agarose in aqueous
medium at pH .about.7 at ambient conditions (see, e.g., Meena, R.;
Prasad, K.; Siddhanta, A. K. Preparation of genipin-fixed agarose
hydrogel. J. Appl. Polym. Sci. 2007, 104, 290-296). Genipin was
purchased from Challenge Bioproducts Co., Taiwan. The amount of
genipin used was about 0.8 wt % of the mass of agarose.
Genipin-fixed agarose showed decreased swelling in acidic medium
(pH 1.2), representative of the gastric environment. The extent of
swelling was 4.8 g/g, whereas the uncrosslinked agarose swelled up
to 6 g/g. The authors have reported that, under ambient conditions,
maximum crosslinking was achieved after about 85 h.
[0206] Alternatively, crosslinking can be achieved using free
radicals. Free radical initiators such as ammonium persulfate are
listed in GRAS list of chemicals, and can be used in food
formulations.
[0207] Based on the well known borax mediated crosslinking of
polymers containing hydroxyl groups, Palleschi et al. have
synthesized hydrogels of scleroglucan using borax (see, e.g.,
Palleschi, A.; Coviello, T.; Bocchinfuso, G.; Alhaique, F.
Investigation of a new scleroglucan/borax hydrogel: structure and
drug release. Int. J. Pharm. 2006, 322, 13-21). They have studied
the release kinetics of model drugs theophylline, vitamin B12 and
myoglobin from the crosslinked hydrogels. These hydrogels were
macroscopic gels, and not micro- or nanospheres.
[0208] Gellan can also be used as an ionic crosslinking agent.
Gellan is an anionic microbial polysaccharide that is well known
for its gelling properties in the presence of counterions,
especially divalent ions, like calcium. Gellan has been used as a
crosslinker for scleroglucan.
[0209] Carrageenans are linear sulfated biopolymers, composed of
D-galactose and 3,6-anhydro-D-galactose units. .kappa.-Carrageenan
beads are prepared by gelling with monovalent ions (often K.sup.+)
and sometimes divalent ions. Alginates are linear polysaccharides
produced by algae, which contain varying amounts of
(1.fwdarw.4)-linked .beta.-D-mannuronic acid and
.alpha.-L-guluronic acid residues. Mohamadnia et al. have
synthesized ionically crosslinked beads of carbohydrate biopolymers
.kappa.-carrageenan (FIG. 5) and sodium alginate (see, e.g.,
Mohamadnia, Z.; Zohuriaan-Mehr, M. J.; Kabiri, K.; Jamshidi, A.;
Mobedi, H. pH-Sensitive IPN hydrogel beads of carrageenan-alginate
for controlled drug delivery. J. Bioactive Compat. Polym. 2007, 22,
342-356 and Mohamadnia, Z.; Zohuriaan-Mehr, M. J.; Kabiri, K.;
Jamshidi, A.; Mobedi, H. Ionically crosslinked carrageenan-alginate
hydrogel beads. Journal of Biomaterials Science: Polymer Edition
2008, 19, 47-59). Alginate gelation takes place when divalent or
trivalent cations (usually Ca.sup.2+) interact ionically with
guluronic acid residues, resulting in the formation of a
three-dimensional network. Alginate-Ca.sup.2+ hydrogels have been
studied for controlled release oral drug formulations (see, e.g.,
Bajpai, S. K.; Sharma, S. Investigation of swelling/degradation
behavior of alginate beads crosslinked with Ca.sup.2+ and Ba.sup.2+
ions. React. Func. Polym. 2004, 59, 129-140).
[0210] Langer et al. have described the synthesis of
interpenetrating polymer networks consisting of ionically or
covalently crosslinked hydrogels (see, e.g., Langer, R. S.; Anseth,
K.; Elisseeff, J. H.; Sims, D. Semi-interpenetrating or
interpenetrating polymer networks for drug delivery and tissue
engineering. U.S. Pat. No. 6,224,893, May 1, 2001). Carbohydrates
such as hyaluronic acid, dextran, heparin sulfate, chondroitin
sulfate, heparin, alginate, gellan and carrageenan were used to
synthesize the ionically crosslinked hydrogels. The covalently
crosslinked hydrogels consisted of chitosan polymer and
isothiocyanate crosslinker. The hydrogels were in the form of
viscous solutions capable of retaining biologically active
molecules or drugs when injected in vivo. The formation of micro-
or nanoparticles was not reported. The use of isothiocyante
crosslinker may not be appropriate for food grade hydrogel
synthesis.
[0211] A blend of hydrophobically modified polysaccharide such as
hydropropyl cellulose and a carboxy containing polysaccharide such
as alginate or carboxymethyl cellulose is used to prepare the
hydrogel particles. The hydrophobically modified polysaccharide
results in spontaneous particle formation due to phase separation
in water, while the polysaccharide containing carboxylic acid
groups imparts a pH-responsive behavior and will also increase
intestinal transit time. A review of the formation of hydrogels
(both macroscopic gels and aqueous dispersions) using a blend of
polysaccharides follows.
[0212] Ichikawa et al. have synthesized nanoparticle suspensions of
0.5 wt % concentration by self-assembly of chitosan (with a degree
of deacetylation .about.77%) and carboxymethyl cellulose
hydrolysates (see, e.g., Ichikawa, S.; Iwamoto, S.; Watanabe, J.
Formation of biocompatible nanoparticles by self-assembly of
enzymatic hydrolysates of chitosan and carboxymethyl cellulose.
Biosci. Biotechnol. Biochem. 2005, 69, 1637-1642). The polymers
were hydrolyzed with the enzymes chitosanase and cellulase,
respectively. Electrostatic interactions between the carboxylate
groups of carboxymethyl cellulose with the amino groups of chitosan
resulted in spontaneous formation of nanoparticles just by mixing
solutions of the two polymers. Particle size depended on the mixing
ratio of the solutions, and also by the molecular weight of the
polymers. It was necessary to hydrolyze the polymers and lower the
molecular weight before mixing in order to prevent the formation of
macroscopic gel.
[0213] Sergio et al. have reported the preparation of hydrogels
from mixtures of acidic polysaccharides such as alginates, and
basic polysaccharides such as oligosaccharide derivatives of
chitosan (see, e.g., Sergio, P.; Ivan, D.; Eleonora, M. Hydrogels
of polysaccharide mixtures for tissue engineering and as carriers
of active compounds. Int. Pat. Appl. WO/2007/135114, Nov. 29,
2007). They have described the synthesis of microcapsules with a
mean diameter of 870 .mu.m using an electrostatic `bead generator`.
An alternative chemical method of synthesizing the particles
consisted of, for example, mixing solutions of alginate and lactose
derivative of chitosan prepared in 0.15 M NaCl and 10 mM HEPES (pH
7.4). The total polymer concentration was 2% and the weight ratio
of polyanion to polycation was 3:1. The particles were typically
large in size such that they could be imaged by optical
microscopy.
[0214] White et al. have prepared hydrogel films (for application
in the fields of dermatology, plastic surgery, urology and
orthopaedics) using basic polysaccharide such as chitosan and
anionic polysaccharide such as hyaluronic acid (see, e.g., White,
B. J.; Rodden, G. I. Compositions of semi-interpenetrating polymer
network. Int. Pat. Appl. WO/2005/061611, Jul. 7, 2005).
[0215] Vieira et al. have prepared hydrogels of oxidized alginate
and oxidized alginate blended with chitosan, and studied the
interaction of these drugs with the antifolate drug pyrimethamine
(see, e.g., Vieira, E. F. S.; Cestari, A. R.; Airoldi, C.; Loh, W.
Polysaccharide-based hydrogels: Preparation, characterization and
drug interaction behavior. Biomacromolecules 2008, 9, 1195-1199).
Sodium alginate was partially oxidized using sodium periodate to
obtain 2,3-dialdehyde alginate. The periodate was completely
removed by extensive dialysis. Gelation of 2,3-dialdehyde alginate
was achieved with CaCl.sub.2 or chitosan/CaCl.sub.2 in the presence
of borax. The synthesis of micro- or nanoparticles is not
reported.
[0216] Meena et al. have discussed the synthesis of a hydrogel
system based on grafting of agar and sodium alginate blend with
acrylamide (see, e.g., Meena, R.; Chhatbar, M.; Prasad, K.;
Siddhanta, A. K. Development of a robust hydrogel system based on
agar and sodium alginate blend. Polym. Int. 2008, 57, 329-336).
Agar and sodium alginate were dissolved separately in distilled
water. Agar was dissolved using microwave irradiation at 90.degree.
C. for 2 min, while the sodium alginate was dissolved at ambient
temperature. Blends of agar and sodium alginate were prepared by
mixing agar them in different ratios. The resulting blends were
cooled, forming gels which were cut into small pieces and
dehydrated with isopropanol. Dewatered, hardened gel particles were
filtered through a nylon cloth under reduced pressure and dried in
air, followed by drying in an oven at 50.degree. C. for 2 h. The
dried blend samples were ground using a mortar and pestle to obtain
20-40 mesh particles.
[0217] Hydrogels consisting of a gellan co-crosslinked with
scleroglucan have also been reported (see, e.g., Alhaique, F.;
Coviello, T.; Rambone, G.; Carafa, M.; Murtas, E.; Riccieri, F. M.;
Dentini, M.; Desideri, P. A gellan-scleroglucan co-crosslinked
hydrogel for controlled drug delivery. Proceedings of the
International Symposium on Controlled Release of Bioactive
Materials 1998, 25.sup.th 866-867). The use of both gellan and
scleroglucan in the hydrogel resulting in better stiffness and
stability of the macroscopic hydrogel, and resulted in a slower
release of the guest molecule. The release rate was undesirably
high from the Ca.sup.2+ crosslinked gellan alone.
[0218] Kim et al. have synthesized polysaccharide-based hydrogel
using photocrosslinking of modified dextran (see, e.g., Kim, S. H.;
Won, C. Y.; Chu, C. C. Synthesis and characterization of
dextran-based hydrogel prepared by photocrosslinking. Carbohydrate
Polymers 1999, 40, 183-190). Dextran contains (1.fwdarw.6)-linked
.alpha.-D-glucopyranosyl residues, and three hydroxyl groups per
glucose residue in the structure. Dextran was first reacted with
bromoacetyl bromide. The bormoacetylated dextran was then reacted
with sodium acrylate for incorporating vinyl groups.
Photocrosslinking was achieved by irradiating the acrylated dextran
by UV light. These were macroscopic gels, and not micro- or
nanoparticles.
[0219] Similarly, Reis et al. introduced pendent vinyl groups by
reacting starch with glycidyl methacrylate (see, e.g., Reis, A. V.;
Guilherme, M. R.; Moia, T. A.; Mattoso, L. H. C.; Muniz, E. C.;
Tambourgi, E. B. Synthesis and characterization of starch-modified
hydrogel as potential carrier for drug delivery system. J. Polym.
Sci.: Part A: Polym. Chem. 2008, 46, 2567-2574). Starch is a
polysaccharide composed of two structural components: amylase and
amylopectin. Amylase is a linear chain consisting of 250-300
(1.fwdarw.4)-linked .alpha.-D-glucose residues. Amylopectin is a
branched molecule consisting of about 1400 D-glucose residues with
.alpha.(1.fwdarw.4) and .alpha.(1.fwdarw.6) linkages. It
constitutes about 80% of the total starch and can be easily
hydrolyzed. Crosslinkable vinyl groups were introduced by the
reaction of starch with glycidyl methacrylate resulting in
structures schematically shown in FIG. 7.
[0220] Chen et al. synthesized polymerizable saccharide monomers,
such as sucrose, by reaction of the sugar with epoxy acrylate, or
methacryloyl chloride and acetyl chloride, and used these monomers
to form hydrogels (see, e.g., Chen, J.; Bongjo, S.; Park, K.
Hydrophilic, hydrophobic, and thermoreversible saccharide gels and
foams, and methods for producing same. U.S. Pat. No. 6,018,033,
Jan. 25, 2000). Cai et al. have similarly modified
hydropropylcellulose by covalently attaching vinyl groups that
allowed chemical linking of the polysaccharide chains into
nanoparticles through a free radical polymerization process (see,
e.g., Cai, T.; Hu, Z.; Ponder, B.; St. John, J.; Moro, D. Synthesis
and study of and controlled release from nanoparticles and their
networks based on functionalized hydroxypropylcellulose.
Macromolecules 2003, 36, 6559-6564).
[0221] As mentioned previously, reports on encapsulation of
hydrophilic molecules in particulate carriers of aqueous
dispersions are not numerous. Edlund and Albertsson varied the
crosslink density of hemicellulose-based hydrogel microspheres and
found that the crosslinked network was not able to retard the rapid
release of the small and hydrophilic molecules of compounds such
caffeine (see, e.g., Edlund, U.; Albertsson, A.-C. A microspheric
system: hemicellulose-based hydrogels. Journal of Bioactive and
Biocompatible Polymers 2008, 23, 171-186). On the contrary, release
of a macromolecule such as protein (bovine serum albumin) could be
controlled by varying the network mesh size, with the release
proceeded by Fickian diffusion. Other studies have shown the same
dependence of size and hydrophilicity of entrapped molecules.
[0222] Applicants synthesized hydroxypropyl cellulose microgels
using relatively non-toxic crosslinking agents such as trisodium
trimetaphosphate (TSTMP) and sodium tripolyphosphate (STPP).
Hydroxypropyl cellulose (HPC) is prepared by base-catalyzed
reaction of propylene oxide with cellulose. HPC is permitted in
foods for human consumption, and is described under section
121.1160 of the U.S. Food and Drug Administration regulations
[Klug, E. D. Hydroxypropyl Cellulose. In Encyclopedia of Polymer
Science and Technology; Bikales, N. M., Ed.; Wiley Interscience:
New York, 1971; Vol. 15, pp 307-314]. Up to 0.4 wt % of unreacted
TSTMP and STPP are permissible in food products according to FDA
regulations. Other reagents permitted by FDA for making food grade
starch, such as phosphoryl chloride, adipate, and adipic-acetic
mixed anhydride, may also be used for the crosslinking reaction.
Carcinogens such as epichlorohydrin, although used in the past for
crosslinking starch, can obviously not be used. See, e.g., FIG. 10
which depicts crosslinking of hydroxyl containing polysaccharides
using TSTMP in the presence of sodium hydroxide.
[0223] Crosslinking of starch using trisodium trimetaphosphate has
been typically carried out in aqueous media at pH of 11.5 [Xie, S.
X.; Liu, Q.; Cui, S. W. Starch modification and application. In
Food Carbohydrates: Chemistry, Physical Properties, and
Applications; Cui, S. W. Ed.; Taylor & Francis: New York 2005;
p. 358]. The reaction is allowed to proceed at 40.degree. C. for 2
to 6 h. The applicants found that hydroxypropyl cellulose
microparticles could be obtained, at relatively high concentrations
(up to 10 wt %, without macrophase separation), using significantly
higher sodium hydroxide concentration and reaction temperature.
Sodium hydroxide not only participates in the crosslinking
reaction, but also, evidently, lowers the LCST of hydroxypropyl
cellulose resulting in particle formation even at room temperature
(at sufficiently high concentrations of NaOH).
[0224] Hydroxypropyl cellulose powder, obtained from Sigma-Aldrich,
was used for microparticle synthesis. The HPC polymer had a
number-average molecular weight, M.sub.n, of 10,000 g/mol, a
weight-average molecular weight, M.sub.w, of 80,000 g/mol, a degree
of substitution, DS, of 2.5, and a molar substitution, MS, of 3.7.
The degree of substitution, DS, is defined as the average number of
hydroxyl groups substituted per anhydroglucose unit [Klug, E. D.
Hydroxypropyl Cellulose. In Encyclopedia of Polymer Science and
Technology; Bikales, N. M., Ed.; Wiley Interscience: New York,
1971; Vol. 15, pp 307-314]. The molar substitution, MS, is defined
as the average number of propylene oxide molecules combined per
anhydroglucose unit.
[0225] About 15 mg of refined soy lecithin (MP Biomedicals) was
dissolved in 5 mL of a sodium hydroxide solution (pH=12) to obtain
a pale yellow translucent solution. Four hundred milligram of HPC
was added to this solution and stirred to result in a viscous
solution. In another vial, a 12% (w/v) solution of TSTMP was
prepared in distilled water. Five milliliters of this TSTMP
solution was then added to the HPC/soy lecithin solution. The
mixture was stirred to obtain a homogeneous solution, which was
heated at 50.degree. C. for 1 h and subsequently cooled to room
temperature. The pH of the resulting dispersion, measured using a
stainless steel ISFET pH probe (IQ Scientific Instrument), was 7.8.
The pH was adjusted to 7 using a few microliters of 4 M
hydrochloric acid. The HPC dispersion consisted of: 400 mg of HPC
(3.2 mmol of hydroxyl groups), 15 mg (0.05 mmol) soy lecithin, 600
mg (2.0 mmol) of TSTMP, and about 12 mg (0.3 mmol) sodium hydroxide
in about 10 mL of distilled water. The number-average particle
diameter was 3.5 .mu.m and the weight-average particle diameter was
3.7 .mu.m. The viscosity of the dispersion was about 11 cP. Ten
milliliters of a 20% (w/v) dextrose solution in distilled water was
then added to this dispersion, and the mixture was heated at
60.degree. C. for 10 min. The number-average particle diameter
remained nearly the same (.about.5 .mu.m) after addition of
dextrose. The viscosity of the final dispersion was about 5 cP. The
average diameter of the particles in the dispersion was determined
using a ALVS-NIBS High Performance Particle Sizer (ALV-GmbH,
Langen/Germany). Dispersion viscosity was determined using a
Ubbelohde Viscometer (Cannon Instrument Co., Pennsylvania).
[0226] There were no significant differences in the particle sizes
or the dispersion viscosities when the formulations were heated at
50.degree. C. for 3 h instead of 1 h.
[0227] In another formulation, 10 mL of a 4% (w/v) solution of HPC
in distilled water was taken in a glass vial. Sodium hydroxide
pellets (310 mg, 7.75 mmol) were added and dissolved in to this
solution. The addition of sodium hydroxide resulted in a cloudy
homogeneous dispersion. TSTMP (600 mg, 1.96 mmol) and soy lecithin
(14 mg, 0.043 mmol) were subsequently added and dissolved. The
dispersion was heated at 50.degree. C. for 1 h, after which it was
cooled to room temperature. The procedure resulted in the formation
of macroparticles that settled to the bottom of the vial.
Immediately after cooling, the dispersion was stirred (using a
magnetic stirrer) and neutralized to pH 7 using 4 M hydrochloric
acid. The number- and weight-average particle diameters in the
supernatant phase were about 610 nm and 690 nm, respectively. The
viscosity of the HPC dispersion was about 1.6 cP. Ten milliliters
of a 20% (w/v) dextrose solution in distilled water was then added
to this dispersion, and the mixture was heated at 60.degree. C. for
10 min. The number-average particle diameter in the dextrose loaded
dispersion was about 1.6 .mu.m and the weight-average particle
diameter was about 2.2 .mu.m after addition of dextrose. The
viscosity of the final dispersion was about 2 cP.
[0228] In another embodiment, heating a solution of 4 g of HPC
(31.9 mmol of hydroxyl groups) in 100 g of water with 2.1 g (52.5
mmol) of sodium hydroxide and 1 g (3.27 mmol) of TSTMP at
110.degree. C. for 2 h, resulted in the formation of hydrogel
microspheres. The dispersion was cooled to room temperature and
neutralized using about 4 mL of 4 M hydrochloric acid to result in
a solution with a viscosity of about 22 cP and a weight-average
particle diameter of about 3.4 .mu.m. Addition of 104 mL of 20%
(w/v) dextrose solution gave a final dispersion with a sugar
concentration of 10% (w/v), a viscosity of 6.8 cP and a
weight-average particle diameter of about 4.1 .mu.m. The
formulation was heated at 60.degree. C. for 10 min after the
addition of sugar solution.
[0229] In another formulation, 8 g of HPC (63.7 mmol of hydroxyl
groups) dissolved in 100 g of water was heated with 2.23 g (55.8
mmol) of sodium hydroxide and 1 g (3.27 mmol) of TSTMP. Heating was
carried out in a sealed glass reactor at 110.degree. C. for 2 h.
After cooling, the unreacted sodium hydroxide was neutralized using
about 20 mL of 4 M hydrochloric acid, to yield a dispersion of
crosslinked HPC microspheres with a weight-average particle
diameter of about 4.3 .mu.m. The viscosity of the dispersion was
about 31.2 cP. A 20% (w/v) dextrose solution (120 mL) was then
added to obtain a formulation with 10% (w/v) dextrose, 3.3% (w/v)
HPC, about 2.5% (w/v) sodium chloride. The dispersion was heated at
60.degree. C. for 10 min after sugar addition. The weight-average
particle diameter in the final dispersion was about 4.5 .mu.m, and
the dispersion viscosity was about 31 cP. The dispersion viscosity
was sensitive to the order in which the solutions were mixed. If
the dextrose solution was added after the second heating step
(60.degree. C. for 10 min), the viscosity of the resulting
dispersion was higher (about 55 cP).
[0230] Microparticle hydrogels of hydroxypropyl cellulose and
sodium alginate (CAS no. 9005-38-3; American International
Chemical, Inc., F-200) are synthesized as follows. Ten milligrams
of HPC (0.080 mmol of hydroxyl groups) was dissolved in 1 mL of
distilled water. To this solution was added 1 mL of 2.5 M NaOH
solution (2.5 mmol NaOH), 20 mg (0.065 mmol) of trisodium
trimetaphosphate, 10 mg of sodium alginate and 2 mg (6.1 .mu.mol)
of soy lecithin. The solution was stirred thoroughly. A cloudy
dispersion was obtained that remained stable even after adding a
few drops of concentrated hydrochloric acid (leading to a final pH
of about 2, simulating the acidic environment of the stomach).
[0231] Hydroxypropyl cellulose self-assembles in water at a
temperature greater than 41.degree. C. This temperature, above
which spontaneous self-assembly of the polymer chain occurs, is
called the lower critical solution temperature (LCST). Thermal
self-assembly of HPC is a reversible process. Individual polymer
chains constituting the microparticles get solvated by water
molecules when the solution is cooled below the LCST. Crosslinking
the HPC chains using trisodium metaphosphate (TSTMP) prevents
dissolution of the microparticles when the solutions are cooled
below the critical solution temperatures.
[0232] In another strategy, crosslinking may be achieved by
functionalizing the polysaccharide using acryloyl (or methacryloyl)
groups using acryloyl chloride (or methacryloyl chloride).
Formation of acryloyl esters results from the reaction of acryloyl
chloride with the hydroxyl groups of the polysaccharide (FIG. 11).
It is important, however, to completely remove unreacted acryloyl
chloride from the functionalized polymer, because of toxicity of
acryloyl chloride. The vinyl functionalized HPC may then be
crosslinked in water, above the LCST, using a relatively benign
free-radical redox-initiator such as ascorbic acid and hydrogen
peroxide, or thermal initiator such as potassium persulfate.
[0233] Thus, 1 g of hydroxypropyl cellulose (8 mmol) was taken in a
round bottom flask equipped with a magnetic stir bar and fitted
with a rubber septum. The polymer was dissolved in 20 mL of
anhydrous dichloromethane to obtain a cloudy, viscous solution. The
air in the flask was purged with dry nitrogen. About 1 mL (7 mmol)
of triethyl amine was injected in to the reactor, followed by
drop-wise addition of about 520 .mu.L (6.4 mmol) of acryloyl
chloride. The mixture was stirred at room temperature, whereupon
the cloudy solution became clear few minutes after the addition of
acryloyl chloride. The solution was stirred overnight, after which
the acrylated hydroxypropyl cellulose product was recovered and
purified by repeated precipitations in cold (.about.0.degree. C.)
diethyl ether and acetone. The product was dried in vacuo at
40.degree. C. About 40 mg of the acrylated HPC polymer was
dissolved in 2 mL distilled water to obtain a cloudy solution at
room temperature. About 65 mg (200 mmol) of soy lecithin was added
to this solution and dissolved. The solution of HPC and soy
lecithin was de-oxygenated by bubbling nitrogen gas, after which a
2 mL of a degassed solution of ammonium persulfate (9.1 mg, 40
mmol) was injected. The solution was heated at 70.degree. C. for 2
h to obtain a dispersion of crosslinked acrylated hydroxypropyl
cellulose particles. The number-average and weight-average particle
diameters were 1.28 .mu.m and 1.34 .mu.m, respectively.
[0234] In an emulsion-based synthesis of hydroxypropyl cellulose
microgels, 80 mg of acrylated hydroxypropyl cellulose was dissolved
in 2 mL of dichloromethane. Distilled water (4 mL) was added to
this solution and stirred to obtain an emulsion. Crosslinking of
the acrylated hydroxypropyl cellulose was carried out at 35.degree.
C. using a redox system of ammonium persulfate and dextrose.
Dextrose (21.6 mg, 12 mmol) was dissolved in the emulsion. Two
milliliters of a solution of ammonium persulfate (27.4 mg, 0.12
mmol) in distilled water (2 mL) was injected in to the emulsion to
initiate the crosslinking reaction. Dichloromethane was removed
from the resulting dispersion using a rotary evaporator. A cloudy
dispersion of crosslinked acrylated hydroxypropyl cellulose
microgels was obtained. The crosslinked particles settled to the
bottom of the vial on standing, and could therefore be isolated in
a powder form by decanting the supernatant. The crosslinking may
also be carried out using redox systems such as persulfate/glucose,
hydrogen peroxide/ascorbic acid, etc.
[0235] Scanning electron microscopy of a 400 mg HPC, 100 mg TSTMP,
200 mg NaOH, 10 mL water solution heated at 110.degree. C. for 2 h,
wherein the dispersion was neutralized with concentrated HCl acid
revealed large (.about.1 .mu.m) cubic particles seen under SEM. HPC
has a low glass transition temperature and readily forms a film on
the SEM substrate at room temperature. However, it was difficult to
image the nanoparticles using SEM.
Example 2
[0236] Further to the various aspects of the invention detailed
above, a basic understanding of the relative importance of
carbohydrates (CHOs) as energy sources is useful in the design of
the delivery systems of the invention. Food formulations that can
improve endurance capacity and exercise performance of athletes
have been the focus of several studies in sports and exercise
sciences..sup.i Athletes require a continuous supply of fuel during
exercise to avoid fatigue. Proteins, fats and carbohydrates have
all been considered as important components of an athlete's
diet,.sup.ii,iii but the current emphasis on carbohydrate-rich diet
for athletes has its basis in studies conducted about 90 years ago.
These early studies suggested a relationship between blood glucose
concentration and fatigue. Levine et al., in 1924, found that a
carbohydrate-rich diet for participants of a 25-mile marathon race,
resulted in improved running performance (measured as the time to
complete the race) and prevented hypoglycemia in the
runners..sup.iv On the other hand, Christensen and Hansen showed
that a high fat diet during the exercise caused hypoglycemia and
neuroglucopenia, which resulted in severe fatigue and exhaustion
after exercise..sup.v,vi,vii They found that these symptoms could
be prevented by using a carbohydrate diet prior to exercise.
[0237] The relative importance of carbohydrates, fats, and proteins
as energy sources for exercise, has been elaborated in the 1995
review by Coyle..sup.viii A more recent review by Jeukendrup
focuses on carbohydrates as energy sources..sup.i While protein
catabolism generally contributes to less than 5% of the energy
required for muscle contraction,.sup.ix carbohydrates in the form
of muscle glycogen, liver glycogen and blood glucose, and fats in
the form of plasma fatty acids and intramuscular triglyceride, are
the main sources of energy for steady-state aerobic
exercise..sup.viii The human body stores a large amount of energy
as fat (>300,000 kJ),.sup.x,ix but body fat is not readily
available for oxidation even during moderate exercise. Muscle's
limited ability to oxidize fat at sufficiently high rates to
sustain moderate to high intensity exercise (i.e., greater than 60%
of the maximal oxygen uptake rate, {dot over
(V)}.sub.O.sub.2.sub.max), makes muscle glycogen and blood glucose
the primary sources of energy during these exercises. Fatigue
occurs when muscle glycogen and blood glucose stores become
depleted.
[0238] The energy store in liver glycogen 80 g) is about 1280 kJ,
that in muscle glycogen (.about.400 g) is about 6400 kJ, and blood
glucose including the glucose content of extracellular fluid
(.about.10 g) is about 160 kJ..sup.ix,xi Thus, an 80-kg athlete,
with a maximum oxygen uptake rate of 4.5 L/min, who is performing
an intense aerobic exercise at about 80% of {dot over
(V)}O.sub.2.sub.max, will expend energy at a rate of about 75
kJ/min, causing depletion of the endogenous carbohydrate store
within about 105 min. The athlete will be unable to oxidize fat at
rates sufficient to meet the energy requirements of even
moderate-intensity exercise (60-75% {dot over
(V)}.sub.O.sub.2.sub.max)..sup.viii These factors are expected to
adversely affect exercise performance after 105 min. Carbohydrate
consumption can spare the endogenous carbohydrate store and avoid
fatigue.
[0239] Many studies have reported that carbohydrate consumption
before and during exercise can delay fatigue in athletes during
high intensity
exercises..sup.xii,xiii,xiv,xv,xvi,xvii,xviii,xix,xx,xxi,xxii,xxiii
Jeukendrup and co-workers found that improved physical performance
was observed in studies where carbohydrates were consumed at a rate
of 40-75 g/h and no further improvement was observed when the
carbohydrate ingestion rate exceeded 75 g/h..sup.i They also
determined that moderate rates of glucose ingestion (35 g/h) during
exercise suppressed endogenous glucose production, and high rates
of glucose ingestion (175 g/h) completely blocked endogenous
glucose production..sup.xxiv Further, they also found that a high
exogenous carbohydrate oxidation rate (>1 g/min) and hence
reduced muscle glycogen consumption was obtained for a combined
glucose and fructose diet when compared to a glucose
diet..sup.xxv
[0240] Pre-exercise carbohydrate consumption increases both liver
and muscle glycogen stores, and is therefore commonly practiced to
delay fatigue and improve exercise performance..sup.xxvi However,
it can also cause an increase in plasma insulin concentration,
which will increase muscle glucose uptake at the onset of exercise,
but will subsequently lead to hypoglycemia..sup.xxvi Febbraio et
al. have demonstrated that pre-exercise ingestion of carbohydrate
is beneficial only when the carbohydrate ingestion is maintained
throughout exercise..sup.xxvi Continuous ingestion of CHO may not
be convenient for athletes. Most studies in the literature employ
an intravenous method for continuous ingestion of CHO, or require
participants to consume CHO bolus every 10-15 min. Both of these
are not practical. Formulations available in the market are not
capable of sustained delivery of CHO. As described herein, the
inventors have developed one or more formulations that can provide
sustained delivery of CHOs.
[0241] A higher plasma insulin concentration also adversely affects
fat catabolism. Mobilization, uptake and oxidation of fatty acid
determine the utility of the largest energy store in the human
body--the adipose tissue triglycerides--as an energy source to
muscles during exercise. The adipose tissue triglycerides must
first be hydrolyzed, by lipase enzyme, to release free fatty acids
into the bloodstream for uptake by working muscle..sup.ix,x
Intramuscular triglycerides can also undergo lipolysis and act as a
fatty acid source for oxidation in mitochondria. At low exercise
intensities (<25% {dot over (V)}.sub.O.sub.2.sub.max) and in the
fasted state, almost all of the energy required for exercise is
derived from plasma fatty acid. Fat oxidation can provide up to 50%
of the energy needed for exercise at 70% {dot over
(V)}.sub.O.sub.2.sub.max (with about equal contribution from plasma
fatty acids and intramuscular triglycerides), and less than
one-third of the energy needed for more strenuous exercise lasting
10-30 min. After depletion of muscle glycogen, muscle's ability to
oxidize fat is limited to an energy utilization rate of about 50%
{dot over (V)}.sub.O.sub.2.sub.max.
[0242] The availability of plasma fatty acids for oxidation by
muscles decreases as the intensity of exercise is increased,
possibly because of catecholamine-stimulated vasoconstriction in
adipose tissue blood vessels, insufficient adipose tissue blood
flow,.sup.x and therefore insufficient albumin delivery to carry
fatty acids from adipose tissue to the working muscle..sup.ix The
muscle's limited ability to oxidize fat has also been attributed to
the rate limiting step of carnitine palmitoyltransferase stimulated
transport of fatty acids across mitochondrial membrane..sup.viii
The presence of carbohydrate in muscle is known to reduce fat
oxidation and fat transport across mitochondrial membrane.
Increases in plasma insulin concentration because of pre-exercise
CHO loading reduce lipolysis.sup.xxvii and fat
availability,.sup.xxviii and adversely affect exercise performance.
Even a very small increase in plasma insulin concentration can
suppress the lipolytic rate by more than 50% below basal
levels..sup.x
[0243] Coyle has demonstrated that when carbohydrate was ingested
throughout exercise, at 74% {dot over (V)}.sub.O.sub.2.sub.max, so
that the glucose concentration in the bloodstream remained high,
muscle glycogen use was minimal during the later stages of exercise
(3 to 4 h period), indicating that blood glucose was the
predominant carbohydrate source during this period..sup.viii Other
studies have shown that muscle glycogen is not necessary for
exercise..sup.xxix
[0244] The absorption of CHO from the small intestine into the
systemic circulation is the rate limiting factor in using exogenous
glucose as energy source during exercise. The maximum rate at which
exogenous glucose can be oxidized during exercise is about 1
g/min..sup.xxi
[0245] Carbohydrates such as, glucose, sucrose, and maltodextrins,
with high glycemic indices are equally effective in maintaining
blood glucose concentrations and carbohydrate oxidation and
improving exercise performance..sup.viii Fructose ingestion is
typically not effective for improving performance compared with
glucose or sucrose because of the relatively slow rate at which the
liver converts fructose to glucose. Fructose ingestion results in a
4-fold increase in liver glycogen storage than glucose..sup.xxx
While fructose is predominantly metabolized in the liver, glucose
bypasses the liver and is either stored or oxidized by the muscle.
Jandrain et al. have found that when ingested repeatedly during
moderate-intensity prolonged exercise, fructose is metabolically
less available than glucose, despite a high rate of conversion to
circulating glucose..sup.xxxi Recently, Jeukendrup and coworkers
have found that ingesting moderate amounts of glucose plus fructose
does not increase exogenous CHO oxidation above that of an
isocaloric amount of glucose alone..sup.xxxii
[0246] However, other studies have shown that the rate of exogenous
CHO oxidation can be increased by using a mixture of different
monosaccharides (e.g., glucose, fructose, and sucrose)..sup.xxxiii
Jentjens et al. found that when glucose was ingested at a rate of
1.8 g glucose per minute, the rate of exogenous CHO oxidation was
limited to 0.83 g/min..sup.xxxiv On the other hand, when a mixture
of glucose and fructose was ingested, a total exogenous CHO
oxidation rate of 1.26 g/min could be achieved--a 52% increase. An
earlier study by Adopo et al. had shown that ingestion of a mixture
of glucose and fructose resulted in higher exogenous CHO oxidation
rates than an isocaloric amount of glucose..sup.xxxv The oxidation
rate of the exogenous glucose and fructose was 21% higher than the
rate when only glucose was consumed. Because different
monosaccharides are transported across the intestinal lumen by
specific transport proteins, a mixture of monosaccharides may
result in a higher overall uptake by cells than a single
carbohydrate. For example, while glucose and galactose are
transported through intestinal cell membranes by a transport
protein called sodium-dependent glucose transporter 1 (SGLT1),
fructose is transported by a different transport protein called
glucose transporter 5 (GLUT5). In principle, supplying a 1:1
mixture of glucose and fructose molecules will reduce traffic in
the SGLT1 transport pathway by a factor of 2, compared to the case
where only glucose molecules are provided. Although the net rate of
absorption of CHOs may increase using a mixture of glucose and
fructose, fructose may not be immediately available as energy
source, because of the relatively slow rate of hepatic conversion
of fructose to glucose.
[0247] The blood flow rate to the small intestine could also be a
limiting factor in CHO absorption. There is a significant decrease
in the blood flow rate to small intestine during high intensity
exercise (cf. Table 4)..sup.xxxvi The reason for a limiting
exogenous glucose oxidation rate during exercise could also be due
to reduced blood flow rate to small intestine. It is also likely
that hepatic glycogen synthesis and glycogenolysis do not allow a
glucose output greater than about 1.0 g/min, regardless of the
supply rate from the small intestine.
TABLE-US-00004 TABLE 4 Blood flow distribution during rest and
dynamic exercise in an athlete Rest Heavy exercise % of total % of
total cardiac cardiac mL/min output mL/min output Splanchnic
(gastric, small 1.4 24 0.3 1 intestinal, colonic, pancreatic,
hepatic, and splenic).sup.xxxvii Renal 1.1 19 0.9 4 Brain 0.75 13
0.75 3 Coronary 0.25 4 1 4 Skeletal muscle 1.2 21 22 86 Skin 0.5 9
0.6 2 Other 0.6 10 0.1 0.5 Total cardiac output 5.8 100 25.65
100
[0248] Pfeiffer, B. et al., Int. J. Sport Nutr. Exerc. Metab.
(2009) 19(5):485-503 discusses the effect of carbohydrate gels on
gastrointestinal tolerance. Hultson, C. et al., Int. J. Sport Nutr.
Exerc. Metab. (2009) 19(3):275-284 shows that there is no placebo
effect from carbohydrate intake during prolonged exercise. Currel,
K. et al., Med. Sci. Sports Exerc. (2008) 40(2):275-281 pertains to
endurance performance with ingestion of more than one
carbohydrate.
[0249] Experimental Methods
[0250] Microparticles of a temperature responsive polymer, such as
hydroxypropyl cellulose (HPC), were prepared by heating an aqueous
solution of the polymer above its lower critical solution
temperature. The polymer chains within the particles were
covalently crosslinked using FDA approved trisodium
trimetaphosphate (TSTMP), to obtain microparticle hydrogels. The
particles were loaded with dextrose (D-glucose) and the rates of
release of entrapped dextrose were studied for formulations with
different chemical compositions and particle concentrations. The
sugar that was present within the water-swollen hydrogel particles
were available for delayed release. The remaining sugar was present
in the aqueous phase, and was available for immediate absorption
across the intestinal lumen. The hydrogel microparticles were
coated with pH responsive, mucoadhesive polymer, such as sodium
alginate, to provide a diffusional barrier against gastric release.
Both in vitro release kinetics and in vivo release kinetics (at two
different rates of energy expenditure) were experimentally
determined. Glucose concentration versus time profiles for the
delayed-release formulations of the present invention showed clear
differences and advantages over conventional immediate release
formulations available in the market, and other controls.
[0251] Materials.
[0252] Hydroxypropoyl cellulose (HPC-SL, USP grade) was received
from Nippon Soda Co. Ltd. Refined soy lecithin was purchased from
MP Biomedicals Inc., LLC (catalog no. 102147). Sodium alginate
polymers (sodium alginate NF, F-200, SAHMUP and sodium alginate NF,
SALMUP) were received from American International Chemical, Inc.
Trisodium trimetaphosphate (TSTMP, reagent grade) and sodium
hydroxide (reagent grade, >98%) were purchased from
Sigma-Aldrich. The glucose oxidase/peroxidase enzymes (PGO enzymes
capsules, product no. P7119), o-dianisidine dihydrochloride
(catalog no. D3252), dextrose (catalog no. D9434) and hydrochloric
acid (37%, catalog no. 320331) were obtained from Sigma-Aldrich.
Thin-N-Thik.RTM. 99 starch and Resista.RTM. 682 starch, anhydrous
citric acid, Staleydex.RTM. 333 dextrose, and Krystar.RTM. 300
crystalline fructose, were received from Tate & Lyle. Food
grade soy lecithin, UltraLec.RTM. P Deoiled Lecithin was received
from Archer Daniels Midland Company. The food grade surfactant,
diacetyl tartaric acid ester of monoglyceride (DATEM, Panodan.RTM.
150 LP K-A) was received from Danisco. Sodium hydroxide (FCC grade)
was purchased from VWR. Sodium benzoate (FCC grade) was purchased
from Fischer Scientific. Food grade potassium sorbate and trisodium
trimetaphosphate were purchased from Spectrum Chemical Mfg. Corp.
All the chemicals were used without further purification. A widely
used commercial sports drink, GATORADE.RTM., was used as a positive
control for the in vivo experiments. GATORADE.RTM. consists of
water, high fructose corn syrup (glucose-fructose syrup), sucrose
syrup, citric acid, natural flavor, salt, sodium citrate,
monopotassium phosphate, modified food starch, red dye # 40, and
glycerol ester of rosin. The total sugar concentration is 5.83%
(w/v). The sodium and potassium concentrations are 0.45 mg/mL and
0.125 mg/mL, respectively.
[0253] Hydroxypropyl Cellulose (HPC).
[0254] Hydroxypropyl cellulose is a temperature-responsive polymer.
When heated above the lower critical solution temperature (LCST) of
the polymer solution, the hydrated polymer chains lose water
because of thermal disruption of polymer-water hydrogen bonds. The
polymer chains precipitate out of solution, as they become
hydrophobic, to form microparticles. Particle formation by
hydrophobic interaction is reversible--the polymer molecules become
soluble again when the dispersion is cooled below the LCST. The
effect of different additives on the lower critical solution
temperature of an aqueous HPC solution was determined using
Differential Scanning calorimetry. The LCST of an aqueous solution
of HPC (8% w/v) was 48.degree. C. When 4 mL of 3.2% (w/v) soy
lecithin solution was added to an 8% (w/v) HPC solution (10 mL), no
change in the LCST was observed. When 3 g of TSTMP solution in
water (1.77% w/v) was added to the solution containing HPC and soy
lecithin, the LCST decreased to 37.degree. C. Finally, 0.5 g of a
1.36% w/v sodium hydroxide solution was added and the dispersion
was heated for 1 h at 50.degree. C., with stirring at 300 rpm. A
solid precipitate of polymer particles was observed after 1 h of
heating, which could be easily re-dispersed after cooling to room
temperature. The pH of the dispersion was adjusted to about 7 by
adding 40 .mu.L of 4 N hydrochloric acid. Dextrose (1.75 g) was
added to the dispersion and was dissolved by stirring. The LCST of
the crosslinked HPC in dispersion, after addition of dextrose, was
about 32.degree. C. From these measurements of the effect of
additives on the LCST of HPC, it is evident that particle formation
occurs even without the use of a crosslinker. Chemical crosslinking
is, however, desirable to maintain particle integrity over a wider
range of ionic strength, temperature and pH conditions.
[0255] The degree of substitution (DS) and molar substitution (MS)
are important parameters that affect particle formation and
crosslinking in HPC dispersions. Each glucose unit in the cellulose
molecule has three hydroxyl groups. The degree of substitution is
defined as the average number of hydroxyl groups per anhdryoglucose
unit that have reacted with the propylene oxide..sup.xxxviii
Therefore, the degree of substitution is always less than or equal
to three. Molar substitution is defined as the average number of
propylene oxide molecules that have reacted per glucose unit. The
molar substitution is generally greater than the degree of
substitution, and can be greater than 3. The ratio of molar
substitution to degree of substitution gives the average length of
the hydroxypropyl side chains (x+1, cf. FIG. 12), in the
polymer.
[0256] Based on the structure of the HPC polymer, shown in FIG. 12,
it is evident that the average molecular weight of each repeat unit
in the polymer is equal to (162.15+58.08MS). Each repeat unit has
three hydroxyl groups. Hence, the number of moles of hydroxyl group
per gram of the HPC polymer is given by 3/(162.15+58.08MS). For
HPC-SL, the degree of substitution is 1.9, and the molar
substitution is about 2.1. Hence, the concentration of hydroxyl
groups is about 10.6 mmol per gram of the polymer.
[0257] Dispersion Synthesis.
[0258] At the reaction temperature of 50.degree. C., the HPC chains
aggregated to form microparticles. The individual polymer chains in
the particles were covalently crosslinked using the reaction shown
in FIG. 13. At the end of the reaction, the particles settled at
the bottom of the vial. They could, however, be easily re-dispersed
by gentle stirring, after cooling to the room temperature.
[0259] An IQ150-77 pH/mV/Temperature system (IQ Scientific
Instruments) with a general purpose stainless steel ISFET sensor
probe was used for pH measurements. Particle sizes in the
dispersions were measured using ALV-NIBS High Performance Particle
sizer. Scanning electron microscopy was done using a JEOL JSM 6300
scanning electron microscope. A drop of the sample was air dried on
an aluminum stub for about 12 hours at room temperature. The dry
particles were sputter coated with a conducting layer of gold
before the SEM analysis. The viscosities of the dispersions were
determined using an Ubbelohde viscometer (Cannon instruments Co.,
size 1C). The time taken for the liquid to elute between two
fiducial points on the viscometer was measured using a stopwatch,
and the viscosity of the formulation was calculated as the product
of the `viscometer constant`, the experimentally determined liquid
density, and the elution time. Differential scanning calorimetry
(DSC) was performed using a TA Instruments Differential Scanning
calorimeter. The DSC measurements were made in an inert atmosphere
of ultra high purity nitrogen. PerkinElmer aluminum pans (#
02190062) were used for both the sample and the reference. The
samples were heated to 75.degree. C., held at this temperature for
1 minute, and then cooled to 20.degree. C. at a rate of 10.degree.
C./min. The difference in heat flow between the sample and
reference was measured to obtain the DSC thermogram.
[0260] In vitro release kinetics, of glucose encapsulated in the
hydrogel microparticles, was determined using PermeGear
Side-Bi-Side horizontal diffusion cell. The diffusion cell
consisted of a donor and receiver chamber separated by a membrane.
The membrane was placed between the two chambers and the chambers
were held together with a stainless steel clamp. The donor and
receiver chamber had a volume of 7 mL each, and the diameter of the
orifice was 15 mm. Both the donor and receiver chamber were
surrounded by jackets through which water from a temperature
controlled water bath was circulated. For the release kinetics
experiments that are detailed herein, a polyethersulfone membrane
was used because of its hydrophilicity and acid resistance.
Polyethersulfone membranes with 450 nm pore size, and 25 mm
diameter were purchased from Sterlitech Corporation. The diffusion
cell assembly was mounted on a magnetic stir plate. The contents of
the receiver chamber were stirred using a magnetic stir bar. The
contents of the donor chamber were left unstirred. For the
determination of glucose concentration as a function of time,
100-.mu.L samples were withdrawn from the receiver chamber using a
microsyringe, and replaced with an equal volume of distilled
water.
[0261] Glucose concentrations in the in vitro experiments were
determined using a colorimetric glucose oxidase method, following a
Sigma-Aldrich protocol..sup.xxxix The glucose oxidase/peroxidase
enzyme solution was prepared by dissolving 1 capsule of Sigma's PGO
Enzymes in 100 mL of water in an amber bottle. Each capsule
contained 500 units of glucose oxidase (Aspergilus niger), 100
purpurogallin units of peroxidase (horseradish), and buffer salts.
The bottle was inverted several times with gentle shaking to
dissolve the capsule. The o-dianisidine solution was prepared by
dissolving 50 mg of o-dianisidine dihydrochloride in 20 mL of
water. The PGO-enzymes reaction solution was prepared by mixing 100
mL of the PGO enzyme solution and 1.6 mL of the o-dianisidine
dihydrochloride solution. The solution was mixed by inverting
several times or with mild shaking. A glucose standard of 0.05
mg/ml in water was prepared. The glucose-containing sample was
added to the PGO enzymes reaction solution. The reaction was
allowed to proceed to completion in approximately 45 minutes at
room temperature. The final absorbance was measured using a
PerkinElmer Lambda 650 UV-vis spectrophotometer at 450 nm
wavelength. The glucose concentration of the sample was determined
as follows:
Sample Glucose Concentration ( mg / mL ) = Absorbance ( Test )
.times. Dilution of Sample .times. 0.05 mg / mL Absorbance (
Standard ) ##EQU00003##
FIG. 14 shows the reaction scheme for the enzymatic determination
of glucose.
[0262] Glucose is oxidized to gluconic acid and hydrogen peroxide
by glucose oxidase (FIG. 14). Hydrogen peroxide reacts with
o-dianisidine in the presence of peroxidase to form a colored
product. The intensity of the brown color measured at 450 nm is
proportional to the original glucose concentration.
[0263] In Vivo Release Kinetics.
[0264] OneTouch Ultra blood glucose biosensor marketed by LifeScan
was used to determine blood glucose concentration. The OneTouch
Ultra Blood Glucose Monitoring System uses advanced electrochemical
biosensor test strips that require only 1 .mu.L of blood and 5 s
for concentration analysis..sup.xl The test strips features a
design that automatically pulls blood into the test strip.
[0265] For the non-exercise arm of the study, the subject was
instructed to:
[0266] 1. Avoid strenuous exercise for 24 h before the
experiment.
[0267] 2. Fast overnight, for at least 10 h, before the
experiment.
[0268] 3. While seated, have blood drawn and analyzed using a
conventional spring loaded single use lancet and handheld glucose
meter for the purpose of determining blood glucose concentration.
Initial measurements were obtained and recorded up to three times
in 45 min to establish a baseline glucose level.
[0269] 4. Consume 400 mL of test formulation, water (control),
aqueous dextrose solution or GATORADE.RTM. (positive controls)
within approximately 2 min.
[0270] 5. While seated, have blood drawn and analyzed for blood
glucose concentration using the handheld glucose meter.
Measurements were obtained and recorded usually every 5 min for the
first 90 min, and every 15 min thereafter for up to 240 min to
obtain the blood glucose concentration vs. time profile.
[0271] After subtracting the fasted state (baseline) value from the
blood glucose concentrations that were measured at different times,
the area under the concentration versus time profiles were
determined by trapezoidal rule using a simple Matlab code.
[0272] A Precor Model 966i tread mill, or similar, was used for the
dynamic exercise studies. In the exercise arm of the study, the
subject was instructed to:
[0273] 1. Avoid strenuous exercise for 24 h before experiment.
[0274] 2. Fast overnight, for at least 10 h, before the
experiment.
[0275] 3. Enter the testing facility and sit comfortably for up to
45 min.
[0276] 4. While seated, have blood drawn and analyzed using a
conventional spring loaded single use lancet and handheld glucose
meter for the purpose of determining blood glucose level. Initial
measurements will be obtained and recorded up to 3 times in 45 min
to establish a baseline glucose level.
[0277] 5. Following establishment of baseline glucose level, run on
a treadmill at a speed of approximately 5 mph (.about.60% {dot over
(V)}.sub.O.sub.2.sub.max) for up to 15 min. At prescribed time
points the subject was requested to: briefly stop running; have
blood drawn and analyzed using a conventional spring loaded single
use lancet and handheld glucose meter for the purpose of
determining blood glucose level; and immediately resume running
until completion of the warm-up period. Initial measurements were
obtained and recorded up to three times in 15 minutes to establish
an exercise baseline glucose concentration. Immediately following
the warm-up period consume 400 mL of test formulation within
approximately 2 minutes.
[0278] 6. Resume running at the previous pace and at prescribed
time points the subject was requested to: briefly stop running,
have blood drawn and analyzed using a conventional spring loaded
single use lancet and handheld glucose meter for the purpose of
determining blood glucose level and immediately resume running
after each sampling until completion of the exercise arm. Subject
was instructed to run at the pre-set pace for as long as possible
(up to 195 min). Measurements were obtained and recorded on a
regular basis to obtain the blood glucose concentration versus time
profile.
[0279] After subtracting the baseline value from the blood glucose
concentrations that were measured at different times, the area
under the concentration vs. time profiles were determined.
Example 2.1
[0280] Hydroxypropyl cellulose (HPC-SL, 4 g) was dissolved in 50 g
of distilled water in a 250-mL Erlenmyer flask, at room
temperature, using a magnetic stirrer. To the HPC solution in the
Erlenmyer flask, an aqueous solution of soy lecithin was added, and
the mixture was stirred for 5 min until a homogenous pale yellow
solution was obtained. To this solution, an aqueous solution of
TSTMP was added in three aliquots, with stirring for 2 min between
each addition. The cloudiness of the formulation increased after
the addition of TSTMP, indicating particle formation. Finally, an
aqueous solution of sodium hydroxide was added, and the dispersion
was stirred for 5 min. The pH of this dispersion was measured using
a pH meter and was about 11.6. The resulting formulation was heated
for 2 h on a water bath maintained at 50.degree. C. using a hot
plate (Corning Instruments PC 620D) and stirred at 300 rpm using a
magnetic stirrer. A solid precipitate formed during the reaction.
The Erlenmyer flask was taken out form the water bath, allowed to
cool to room temperature, and mixed using a magnetic stirrer until
the solid precipitate formed during the reaction re-dispersed to
form a uniform, homogeneous dispersion. The pH of this dispersion
was measured to be 10.9. The pH of the dispersion was adjusted to
7.8 using 4 N HCl solution (.about.30 to 50 .mu.L). Dextrose powder
(8.95 g) was then added, and the mixture was stirred at room
temperature for 5 min until the solid dissolved. The dispersion was
heated again, at 50.degree. C., for 20 min, while mixing at 300
rpm. The precipitate formed was re-dispersed using a magnetic
stirrer, after cooling to room temperature.
Example 2.2
[0281] Ten milliliters of 8% (w/v) HPC solution was taken in a
glass vial. While stirring using a magnetic stirrer (300 rpm) and
stir bar (5 mm L.times.2 mm D), an aqueous solution of soy lecithin
was added to the vial. To this solution, an aqueous solution of
TSTMP was added drop-wise, with continued stirring. Finally, 0.5 mL
of NaOH solution was added, and the resulting solution was heated
at 50.degree. C. for 1 h. After 1 hr, the mixture was cooled. A
white solid settled at the bottom of the solution. The
microparticles in the settled solid phase re-dispered upon mixing
at 200 rpm (at room temperature, for about 1 h), to result in a
clear homogeneous solution. The dispersion thus obtained was
neutralized with a few microliters of 4 N HCl solution. The final
HPC concentration in the dispersion was about 4.4% (w/v). Dextrose
powder (1.85 g) was added to 18.5 mL of a 4.4% (w/v) HPC
dispersion. The dispersion was stirred until the dissolution of
dextrose was complete. At least 48 h elapsed before performing the
release studies.
[0282] Hydroxypropyl cellulose microparticles, with an average
diameter of about 5.4 .mu.m, were obtained (cf. FIGS. 15a and 15b).
The viscosity of the 4.4% (w/v) HPC dispersion was found to be
15.46 cP at room temperature (before the addition of dextrose). A
few particles with a diameter greater than 5.4 .mu.m (c.f. FIG.
15c) were observed in the SEM images.
Example 2.3
[0283] Polysaccharides such as hydrophobically modified food starch
(Thin-N-Thik.RTM. 99 Starch and Resista.RTM. 682 starch) were used
to form the hydrogel microparticles. An 8% (w/v) starch solution
was prepared by dissolving 800 mg of starch in 10 mL of distilled
water at room temperature to obtain a cloudy, but homogeneous,
dispersion. Solutions of refined soy lecithin, TSTMP, and sodium
hydroxide were then prepared in distilled water. To 10 mL of the
starch dispersion taken in a glass vial, 4 mL of the soy lecithin
solution was added and the mixture was stirred for 5 min. To this
mixture was added 3 mL of TSTMP solution. After stirring for 5 min,
0.5 mL of sodium hydroxide solution was added. The glass vial was
placed in an oil bath at 50.degree. C. and heated for 60 min.
Agitation was provided using a magnetic stirrer and speed of 300
rpm was maintained during the reaction. After 60 min, the vial was
removed form the oil bath, allowed to cool to room temperature and
the pH was adjusted to 7 using a few microliters of 4 M
hydrochloric acid solution. The particle sizes in the resulting
dispersions are given in Table 5.
TABLE-US-00005 TABLE 5 Particle sizes in hydrophobically modified
starch dispersions. Mass average particle Type of Starch diameter
(nm) Thin-N-Thik .RTM. 99 898 Starch Resista .RTM. 682 681.4
starch
Example 2.4
[0284] The following formulation was synthesized in a 2-liter glass
reactor using distilled water and food grade chemicals. Stock
solutions of sodium alginate (SALMUP), soy lecithin, trisodium
trimetaphosphate, sodium hydroxide, citric acid, and dextrose were
prepared in distilled water. A stock solution of sodium benzoate
and potassium sorbate was prepared by dissolving sodium benzoate
and potassium sorbate in 250 mL of distilled water. The reaction
was carried out in a European-style, tapered, 3-neck, jacketed
flask (Chemglass, catalog no. CG-1576-11) of 2-liter capacity. The
contents of the reactor were mixed using an overhead stirrer
(IKA.RTM. RW-20) and an agitator consisting of a polished glass
shaft (Chemglass, catalog no. CG-2078-02) attached to a teflon
stirrer blade (Chemglass, catalog no. CG-2080). A recirculating
water bath was used to control the temperature of water passing
through the reactor jacket.
[0285] One liter of distilled water was poured into the reactor
through a side neck of the flask using a silicone funnel. The speed
of the stirrer was set at 260 rpm. The water flowing through the
reactor jacket was at room temperature. About 100 g HPC-SL powder
was slowly added to a reactor through a silicone funnel over a
period of 20 min, and mixed for about 60 min to obtain a
homogeneous (clear) solution of HPC. A soy lecithin solution was
then added to the reactor. After mixing for about 5 min, TSTMP
solution was added, and the contents of the reactor were further
mixed for 5 min. Finally, sodium hydroxide was added to reactor and
mixed. The pH of the resulting solution was measured to be about
11.1.
[0286] The temperature of the water bath was set to 50.degree. C.
After the temperature of the water bath reached 50.degree. C., the
reactor contents were heated at this temperature for 90 min. After
90 min, a white solid settled to the bottom of the reactor. The hot
water from the jacket of the reactor was drained off, and the
reactor contents were cooled by circulating cold water 10.degree.
C.). The reactor contents were mixed for about 1 h, upon which, the
settled solids re-dispersed in the aqueous phase to result in a
homogeneous dispersion. The pH of the dispersion was measured and
found to be 10.3.
[0287] The pH was adjusted to 7 by addition of the citric acid
solution. Then, 400.1 g of dextrose solution (100% w/v) was added
to the reactor and the contents were mixed for about 10 min.
Finally, sodium alginate solution was added through the funnel. Any
sodium alginate that remained adhered to the funnel was washed into
the reactor using 33 g of dextrose solution. After 10 min of
mixing, solution containing sodium benzoate and potassium sorbate
solution was added. The pH of the dispersion was brought down to
3.8 using the citric acid solution.
[0288] The number average diameter of the particles in the
dispersion was about 4.1 .mu.m, and the mass average diameter was
about 4.3 .mu.m. The viscosity of the dispersion was about 32.2 cP.
The pH was about 3.8. The room temperature density was about 1.2
g/mL. A part of the original dispersion of Example 2.4 was diluted
by adding an equal mass of water to obtain the diluted dispersion,
the viscosity of which was 11.7 cP.
Example 2.5
[0289] Using the procedure described above, another batch of
formulation was synthesized and divided into four parts. Each of
these contained different amounts of the sugars dextrose and
fructose. The overall composition, particle size, and viscosity of
these samples are given in Table 6.
TABLE-US-00006 TABLE 6 Delayed-release formulations containing
dextrose and fructose. A " " indicates the composition contains the
component. Component 2.5A 2.5B 2.5C 2.5D Distilled water Dextrose +
Fructose 5.1% 5.1% 5.1% 5.1% Dextrose/Fructose 1/3 3/1 1/1 1/0
Hydroxypropyl 2.6% 2.6% 2.6% 2.6% cellulose (HPC-SL) Sodium
alginate (SALMUP) Soy lecithin TSTMP NaOH Citric acid Sodium
benzoate Potassium sorbate Final pH 3.8 3.8 3.8 3.8 Number average
4.66 .mu.m 4.56 .mu.m 4.16 .mu.m 4.90 .mu.m particle diameter Mass
average particle 4.94 .mu.m 4.62 .mu.m 4.44 .mu.m 4.96 .mu.m
diameter Room temperature 13.7 cP 12.5 cP 12.4 cP 12.4 cP
viscosity
Example 2.6
[0290] Aqueous solutions of soy lecithin, trisodium
trimetaphosphate (TSTMP), sodium hydroxide, and soldium alginate
were each prepared by dissolving the respective compounds in
distilled water. Anhydrous citric acid was also dissolved in
distilled water. A solution of sodium benzoate and potassium
sorbate in water was also prepared by dissolving sodium benzoate
and potassium sorbate in distilled water. In addition, a solution
of dextrose in water was prepared by dissolving 151 g of dextrose
in 172 g distilled water.
[0291] Synthesis of the hydrogel microparticles was carried out in
a European-style, tapered, 3-neck, jacketed flask (Chemglass,
catalog no. CG-1576-11) of 2-liter capacity. The contents of the
reactor were mixed using an overhead stirrer (IKA.RTM. RW-20) and
an agitator consisting of a polished glass shaft (Chemglass,
catalog no. CG-2078-02) attached to a teflon stirrer blade
(Chemglass, catalog no. CG-2080). A recirculating water bath was
used to control the temperature of water passing through the
reactor jacket.
[0292] Distilled water (750 g) was poured into the reactor, through
a side neck of the flask, using a silicone funnel. The speed of the
stirrer was set at 265 rpm. Water at room temperature was
re-circulated through the reactor jacket. About 75.2 g HPC-SL
powder was slowly added to the water in the reactor using a
silicone funnel, over a period of 20 min, and mixed for about 60
min to obtain a homogeneous solution of HPC. Soy lecithin solution
was added to the reactor. After mixing for 5 min at room
temperature, TSTMP solution was added, and the contents of the
reactor were further mixed for 5 min. Finally, sodium hydroxide was
added to the reactor and mixed. The pH of the resulting solution
was measured to be about 11.1.
[0293] The temperature of the water bath was raised to 50.degree.
C. After the temperature of the water bath reached 50.degree. C.,
the reactor contents were heated at this temperature for 90 min.
After 90 min, a white solid settled to the bottom of the reactor.
The hot water from the jacket of the reactor was drained off, and
the reactor contents were cooled by circulating cold water. The
reactor contents are mixed for about 1 h, upon which, the settled
solids re-dispersed in the aqueous phase to yield a homogeneous
dispersion. The pH of the dispersion was measured and found to be
10.3.
[0294] The pH was adjusted to 6.5 by addition of citric acid
solution. Then, 320 g of dextrose solution was added to the
dispersion and mixed for 10 min. Finally, sodium alginate solution
was added through the funnel. Any sodium alginate that remained
adhered to the funnel was washed into the reactor using 33 g
dextrose solution. After 10 min of mixing, sodium benzoate and
potassium sorbate solution was added. The pH of the dispersion was
brought down to 3.8 using the citric acid solution.
[0295] The number average diameter of the particles in the
dispersion was about 4.5 .mu.M, and the mass average diameter was
about 4.7 .mu.m. The viscosity of the dispersion was about 38.7
cP.
Example 2.7
[0296] The synthesis of the formulation was carried out on a
1.5-liter scale using food grade chemicals. Aqueous solutions of
soy lecithin, TSTMP, sodium hydroxide, sodium alginate, sodium
benzoate and potassium sorbate were first separately prepared.
Anhydrous citric acid was also dissolved in distilled water.
[0297] The reaction was carried out in a European-style, tapered,
3-neck, jacketed glass flask (Chemglass, catalog no. CG-1576-11) of
2-liter capacity. The contents of the reactor were mixed using an
overhead stirrer (IKA.RTM. RW-20) and an agitator consisting of a
polished glass shaft (Chemglass, catalog no. CG-2078-02) attached
to a teflon stirrer blade (Chemglass, catalog no. CG-2080). A
recirculating water bath was used to control the temperature of
water passing through the reactor jacket.
[0298] Distilled water (750 g) was poured into the reactor through
a side neck of the flask using a silicone funnel. The speed of the
stirrer was set at 265 rpm. The water flowing through the reactor
jacket was at room temperature. HPC-SL powder (75.2 g) was added to
a reactor through a silicone funnel over a period of 20 min, and
mixed at room temperature for 60 min, to obtain a homogeneous
(clear) solution of HPC. Soy lecithin solution was then added to
the reactor. After mixing for 5 min, TSTMP solution was added, and
the contents of the reactor were further mixed for 5 min. Finally,
sodium hydroxide was added to the reactor and mixed. The pH of the
resulting solution was measured to be about 11.1.
[0299] The temperature of the water bath was set to 50.degree. C.
After the temperature of the water bath reached 50.degree. C., the
reactor contents were heated at this temperature for 120 min. After
120 min, a white solid settled to the bottom of the reactor. The
hot water from the jacket of the reactor was drained off, and the
reactor contents were cooled by circulating cold water. Further
mixing for about 1 h re-dispersed the settled solids to result in a
homogeneous dispersion. The pH of the dispersion was measured and
found to be 10.3. Addition of the citric acid solution lowered the
pH to about 6.6. Thereafter, dextrose (150 g) was added to the
reactor as a powder and the contents were mixed for about 10 min.
Finally, SALMUP solution was added through the funnel. After 10 min
of mixing, solution containing sodium benzoate and potassium
sorbate was added. Finally, the pH of the dispersion was brought
down to 3.8 using citric acid solution.
[0300] The number average diameter of the particles in the
dispersion was about 4.2 .mu.m, and the mass average diameter was
about 4.8 .mu.m. The viscosity of the dispersion was about 36.7
cP.
Example 2.8
[0301] The formulation of Example 2.1 was used. The kinetics of
release of dextrose from HPC particles was measured at pH 7 and at
a temperature of 28.degree. C. This temperature was chosen because
it was below the LCST of HPC in the dispersion.
[0302] A water bath operating at 28.degree. C. was used to
circulate water through the heating jackets of the donor and
acceptor compartments of the diffusion cell. Seven milliliters of
the HPC formulation was added to the donor compartment of the
diffusion cell, and 7 mL of distilled water (pH .about.7) was added
to the receptor compartment. At regular intervals small aliquots
(0.1 mL) of the liquid in the receptor compartment were withdrawn
using a microsyringe, and replaced with equal volumes of distilled
water (0.1 mL). The aliquots were added to a 250 mL volumetric
flask and were diluted using distilled water (by a factor of 2500).
The concentrations of glucose were determined using the
colorimetric glucose oxidase method.
[0303] The acceptor compartment glucose concentration vs. time
profile for the HPC dispersion was compared with the profile
obtained for a glucose control, which had the same overall glucose
concentration as the HPC dispersion (8.7 wt %).
[0304] The concentration vs. time profiles are quite different for
the test formulation and the control indicating that the
formulation of Example 2.1 showed delayed release of glucose. As
expected, the final glucose concentration was lower for the HPC
dispersion (.about.38 mg/mL) than the glucose control (.about.54
mg/mL). The experimental procedure employed non-sink conditions for
determining the kinetics of glucose transport. A part of the
glucose in the HPC dispersion remains sequestered within the
particles at equilibrium. Therefore, the final concentration in the
donor compartment is lower than that in the case of the glucose
control.
Example 2.9
[0305] Sodium alginate (SALMUP) powder was added to 20 g of the
formulation described in Example 2.1. The pH of the final
dispersion was adjusted to 3.8 using 4 N hydrochloric acid. The
formulation contained 8.7% (w/w) dextrose, 3.9% (w/w) HPC, SALMUP,
TSTMP, and soy lecithin. The same formulation was diluted by an
equal mass of water. The kinetics of glucose transport was measured
at 28.degree. C. using a diffusion cell. The diluted formulation
contained 4.3% (w/w) dextrose, 1.9% (w/w) HPC, SALMUP, TSTMP, and
soy lecithin. The effect of dilution on the kinetics of glucose
transport was studied using a diffusion cell, following the
procedure described in Example 2.8. FIG. 17 compares the acceptor
compartment glucose concentration profiles for the original
formulation and the diluted formulation.
[0306] The plateau value of the concentration profile is lower for
the diluted formulation (about 50%) than the original formulation,
as expected for a 1:1 dilution. It is also evident from the
concentration profiles shown in FIG. 17 that the diluted
formulation released glucose at a faster rate compared to the
original formulation.
Example 2.10
[0307] The kinetics of release of dextrose from HPC particles was
measured at pH 7 and at a temperature of 37.degree. C. This
temperature was chosen because it was above the LCST of HPC in the
dispersion, and close to the temperature of the human body. The
formulation of Example 2.1 was used. Almost all of the encapsulated
glucose is released after about 10 h (FIG. 18). The HPC particles
undergo a transition from a hydrophilic to a hydrophobic state when
the temperature is raised above the LCST (e.g., 37.degree. C.). The
glucose molecules that were present within the particles are
expelled along with the water, as the particles undergo shrinking.
It is evident from FIG. 18 that the glucose concentration in the
receptor compartment increases at a slower rate when the HPC
dispersion was present in the donor compartment instead of a
glucose solution with same total concentration of glucose
(control).
Example 2.11
[0308] The kinetics of release of dextrose from HPC particles was
measured at pH 2 and at a temperature of 37.degree. C. The pH value
of 2 is similar to the pH of the gastric fluid in the fasted state.
The dispersion of Example 2.4 was used. The pH of the dispersion
was adjusted to 2 using 4 N hydrochloric acid. Water in the jacket
of the diffusion cell was maintained at 37.degree. C. using a water
bath. Seven milliliter of the HPC dispersion was added to the donor
compartment and 7 mL of acidic water (pH 2, hydrochloric acid) was
added to the receptor compartment. At selected time intervals, 0.1
mL aliquots of the fluid in the receptor compartment were
withdrawn, which were replaced by an equal volume of distilled
water. The withdrawn aliquots were diluted by a factor of 10 using
a volumetric flask, and the glucose concentrations were determined
using a GM8 analyzer (Analox Instruments). FIG. 19 shows the
concentration profiles for the dispersion and the glucose solution
(control). Clearly, the rate of release is slower for the HPC
dispersions than the glucose solution.
Example 2.12
[0309] The effect of pH on the kinetics of release of dextrose from
HPC particles was further illustrated using the dispersions of
Example 2.1. The pH of the original dispersion was adjusted to
.about.7 using 4 N hydrochloric acid. Sodium alginate (SALMUP)
powder was added to 20 g of this dispersion, and dissolved using a
magnetic stirrer. A few minutes before the determination of the
glucose release kinetics (using a diffusion cell), about 10 g of
the dispersion that contained sodium alginate was taken, and its pH
was adjusted to 3.8 using 4 N hydrochloric acid. The pH of the
remaining 10 g of the dispersion was adjusted to 2 using 4 N
hydrochloric acid. All three dispersions so obtained contained
about 8.7% (w/w) dextrose, 3.9% (w/w) HPC, SALMUP, TSTMP, and soy
lecithin. The kinetics of glucose release, at 28.degree. C., was
determined using a diffusion cell, as discussed in Example 2.8. The
donor compartment contained 7 mL of the dispersion. The receptor
compartment contained 7 mL of distilled water, acidified to a pH
that was the same as that of the dispersion in the donar
compartment. The concentrations of glucose in the receptor
compartment were determined using the colorimetric glucose oxidase
method. FIG. 20 compares the receptor compartment glucose
concentration profiles for the three dispersions: at pHs 7, 3.8,
and 2. There is almost no difference in the concentration profiles
for pH values of 7 and 3.8. However, when the pH of the dispersion
was substantially lower than the pKa of the carboxylic acid group
(of alginate), a significantly different concentration time profile
was observed. The data clearly demonstrates the role of sodium
alginate in creating a diffusional barrier for glucose molecules
within the HPC particles. The barrier property is enhanced at
acidic pH that the dispersion will encounter in the gastric
environment after consumption.
Example 2.13
[0310] About 12.85 oz (380 mL, 450 g) of the dispersion of Example
2.4 was consumed after an overnight fast (.about.10 h). The
formulations were comprised of materials that are considered to be
food additives that are `generally recognized as safe` (GRAS) by
the U.S. Food and Drug Administration. Amounts of all ingredients
fall within allowable levels as determined by U.S. FDA and WHO.
During the hour before the consumption of the HPC dispersion, the
fasted-state blood glucose concentrations were measured to
establish a baseline. The blood glucose concentration was
determined at definite intervals after consumption of the
dispersion (FIG. 21a). FIG. 21a also shows the blood glucose
concentration profile for the same subject, after consumption of
380 mL of GATORADE.RTM.. In FIG. 21b, the blood glucose
concentrations are normalized by subtracting the fasted-state,
baseline glucose concentration. Both the HPC dispersion and
GATORADE.RTM. exhibited a high glycemic index (a measure of the
rate at which the ingested carbohydrate is available for intestinal
absorption, reflected in the rate of increase in the blood glucose
concentration).
[0311] The GATORADE.RTM. control, however, showed a sharp decrease
in the blood glucose concentration at around 70 min, evidently
because of insulin response (hyperinsulinemia) to high glycemic
index carbohydrate..sup.viii In the re-fed state (after consuming
the HPC dispersion), the insulin concentration in the blood
increases in response to the elevated blood sugar levels. Insulin
enhances the uptake of glucose from the blood into the body cells,
causing a decrease in the blood glucose concentration. The
hypoglycemic response of insulin is countered by glucagons, which
triggers liver glycogenolysis, eventually returning the blood
glucose concentration to the homeostatic level. Evidently, this
process occurred over a period of 45 min (between 50 and 95 min
after consuming GATORADE.RTM.; cf. FIG. 21b). In contrast, the HPC
dispersion was able to supply glucose to the blood stream at a
controlled rate, and was able to sustain blood glucose levels above
the fasted-state concentration up to about 170 min. Although the
HPC dispersion contained an overall sugar amount that was about 75%
higher than the GATORADE.RTM. control, the insulin response was
relatively milder. The minimum in the blood glucose concentration
was not observed until around 185 min.
Example 2.14
[0312] FIG. 22 compares the blood glucose concentration profile for
an HPC dispersion of Example 2.5 with that of the GATORADE.RTM.
control. The formulation was comprised of materials that are
considered to be food additives that are `generally recognized as
safe` (GRAS) by the U.S. Food and Drug Administration. Amounts of
all ingredients fall within allowable levels as determined by U.S.
FDA and WHO. Equal volumes (380 mL) of the two formulations were
consumed. Both the formulations contained similar sugar
concentrations. The HPC dispersion of the present example has
approximately half the total number of HPC particles in the HPC
dispersion of Example 2.13. Hence, blood glucose concentrations
were maintained above the fasted-state concentration only up to
about 75 min, which was nevertheless about 25 min longer than the
control.
Example 2.15
[0313] This example illustrates the influence of delayed release of
two different types of carbohydrates, namely dextrose and fructose,
on the blood glucose concentration profile. Although fructose
cannot promote muscle glycogen synthesis rapidly,.sup.viii it can
be used to control the insulin response, and sustain blood glucose
concentrations significantly above the fasted-state value, even 2 h
after carbohydrate consumption (cf. FIG. 23). Two different HPC
dispersions of Example 2.5 (2.5A and 2.5D) are compared in FIG. 23.
One dispersion contained dextrose alone as the sugar, while the
other dispersion contained both dextrose and fructose. The total
sugar concentration in the second dispersion was the same as that
in the first dispersion. The same volume (380 mL) of both
dispersions was consumed, and the glycemic response was measured.
The formulations were comprised of materials that are considered to
be food additives that are `generally recognized as safe` (GRAS) by
the U.S. Food and Drug Administration. Amounts of all ingredients
fall within allowable levels as determined by U.S. FDA and WHO. It
is seen in FIG. 23 that the formulation that contained mainly
fructose had a lower glycemic index, as expected. The blood glucose
concentrations were above the fasted-state levels up to about 230
min. The delayed release mechanism provided by the HPC dispersion,
along with the metabolic lag in the hepatic conversion of fructose
to glucose result in the blood glucose level greater than the
baseline value.
Example 2.16
[0314] The blood glucose concentration profile for the HPC
dispersion of Example 2.7 was compared with an aqueous solution of
glucose used as a control (cf. FIG. 24). The formulations were
comprised of materials that are considered to be food additives
that are `generally recognized as safe` (GRAS) by the U.S. Food and
Drug Administration. Amounts of all ingredients fall within
allowable levels as determined by U.S. FDA and WHO. Both the
formulations contained similar overall sugar concentrations
(.about.10 wt %). The blood glucose concentration remained above
the fasted-state level for a longer time (.about.115 min) for the
HPC dispersion than the glucose control (.about.60 min). The
insulin response is also significantly less pronounced for the
delayed release formulation in comparison to the immediate release
control.
Example 2.17
[0315] The effect of timing of ingestion of the HPC dispersion is
illustrated using the dispersion of Example 2.7. The formulation
was comprised of materials that are considered to be food additives
that are `generally recognized as safe` (GRAS) by the U.S. Food and
Drug Administration. Amounts of all ingredients fall within
allowable levels as determined by U.S. FDA and WHO. A 380 mL (450
g) bolus of the formulation was consumed. The blood glucose
concentration versus time profile for the bolus dose was compared
with the concentration profile for an experiment in which the same
amount of formulation was ingested in three parts--each part
consisting of 127 mL (150 g) of the dispersion. It is seen from
FIG. 25 that both the strategies resulted in supra-baseline levels
of blood glucose concentration for at least up to 110 min. The
glycemic response was lower, as expected, when smaller amounts of
the carbohydrate dispersion were consumed frequently.
Example 2.18
[0316] The effect of timing of ingestion of the HPC dispersion is
illustrated using the dispersion of Example 2.7, using
GATORADE.RTM. as control. In two separate experiments, 380 mL (450
g) of each formulation was ingested in three parts--each part
consisting of 127 mL (150 g) of the formulation. The blood glucose
concentrations are compared in FIG. 26.
Example 2.19
[0317] The effect of delayed release carbohydrate feeding before
medium-intensity exercise (.about.60% {dot over
(V)}.sub.O.sub.2.sub.max) on the blood glucose concentration
profiles is illustrated in this example. Three hundred and eighty
milliliters of the HPC dispersion of Example 2.4 was consumed prior
to the exercise. The control for this experiment was 380 mL of a
GATORADE.RTM. formulation to which was added 17.5 g of dextrose to
approximate the dextrose concentration of the HPC dispersion of
Example 2.4. As can be seen from FIG. 27, the blood glucose
concentration is significantly higher at 50 min for the HPC
dispersion compared to the control. This study also measured energy
output as a function of elapsed time to fatigue where the subject
ran at a rate equivalent to .about.60% {dot over
(V)}.sub.O.sub.2.sub.max for as long as possible. The HPC
dispersion produced an elapsed time 55% greater than the water
control, and 31% greater than the positive control, GATORADE.RTM.
formulation to which was added 17.5 g of dextrose.
Example 2.20
[0318] The glycemic impact of the HPC dispersions were
characterized using area under the blood glucose concentration
profiles. FIG. 28 depicts the parameters of the concentration time
profiles that were used to compare the blood glucose concentration
profiles for different formulations illustrated in Examples 2.13 to
2.19. C-C.sub.b is the blood glucose concentration relative to the
baseline value, C.sub.b, which is the blood glucose concentration
in the fasted-state (before the start of the experiment). The time
t=0 corresponds to the start of the experiment (e.g., the ingestion
of CHO formulation). The time, t.sub.exp, is the total time
duration of the experiment over which blood glucose concentrations
were measured. The time, t.sub.baseline, is the time at which the
blood glucose concentration crosses the baseline, i.e., falls below
the fasted-state value. A higher value of t.sub.baseline is
desirable because the glucose in blood is available as an energy
source, for a longer time, after CHO ingestion.
[0319] Two different area-under-the-curve values were calculated
for all the reported blood glucose concentration profiles:
AUC.sub.+ and AUC.sub.total.
[0320] AUC.sub.+ is the area of the region denoted by `+` in FIG.
28, and was calculated using the following equation:
AUC + = .intg. 0 t baseline ( C - C b ) t ##EQU00004##
where the integral is evaluated between the limits t=0 and
t=t.sub.baseline.
[0321] AUC.sub.total is the total area under the concentration
profile, and is the algebraic sum of the areas denoted by `+` and
`-` in FIG. 28. AUC.sub.total was computed using:
AUC total = .intg. 0 t exp ( C - C b ) t ##EQU00005##
where the integral was evaluated between the limits t=0 and
t=t.sub.exp.
[0322] AUC.sub.+ represents the effect of exogenous CHO on blood
glucose concentration. The blood glucose concentration initially
increases because of CHO consumption. The exogenous blood glucose
is converted to tissue glycogen because of insulin response, and is
also used to provide energy to working muscles during exercise,
leading to a decrease in the blood glucose concentration. The fall
of blood glucose concentration below the fasted-state value is
because of insulin response (in experiments where the subject
remained seated), or when the rate of blood glucose oxidation by
working muscles exceeds the rate of supply of glucose to blood
stream (by exogenous and endogenous sources, such as glucose
absorption across intestinal lumen and glycogenolysis,
respectively), or both.
[0323] Table 7 summarizes the effect of different formulations on
the blood glucose concentration, in terms of t.sub.baseline,
AUC.sub.+, and AUC.sub.total, and the average deviation in the
blood glucose concentration during the duration of the experiment
C-C.sub.b, calculated using:
C - C b = .intg. 0 t exp ( C - C b ) t t exp . ##EQU00006##
Both AUC.sub.total and C-C.sub.b depend on the duration of the
experiment, t.sub.exp. Hence, comparison of these values, between
different experiments, is generally made only if the experiments
were of similar duration (e.g., with similar t.sub.exp values).
Unless stated otherwise, 380 mL of the formulation was ingested as
a single bolus dose.
[0324] It is evident from Table 7 that the HPC dispersions, in
general, showed a t.sub.baseline that was higher than immediate
release CHO controls, or water. The AUC.sub.+ and AUC.sub.total
values were also significantly higher for the HPC dispersions
compared to the controls, during both rested and exercising
states.
TABLE-US-00007 TABLE 7 Analysis of blood glucose concentration
profiles for various HPC dispersions and controls. AUC.sub.+
AUC.sub.total t.sub.baseline (mg- t.sub.exp (mg- C - C.sub.b
Formulation (min) min/dL) (min) min/dL) (mg/dL) Experiments in
which subject remained seated HPC 4.2 wt %, 167 2355 230 2000 8.7
glucose 8.7 wt % (Example 2.13) GATORADE .RTM. 50 1274 230 255 1.1
(sugars 5.8 wt %) (Example 2.13) HPC 2.6 wt %, 75 1447 230 458 2.0
glucose 5.1 wt % (Example 2.14) HPC 2.6 wt %, 99 431 230 1218 5.3
glucose 1.3 wt %, fructose 3.8 wt % (Example 2.15) HPC 4.2 wt %,
115 1466 213 4489 21.1 glucose 9.8 wt % (Example 2.16) Glucose
solution 61 1409 160 1248 7.8 (10 wt % concentration) (Example
2.16) HPC 4.2 wt %, 132 1856 160 1635 10.2 glucose 9.8 wt %;
dispersion ingested in three equal parts (Example 2.17) GATORADE
.RTM. 102 961 160 760 4.8 (sugars 5.8 wt %); ingested in three
equal parts (Example 2.18) Experiments in which subject ran on a
treadmill at 60% VO.sub.2max HPC 4.2 wt %, 78 800 159 926 5.8
dextrose 8.7 wt % (Example 2.19) GATORADE .RTM. + 71 400 116 328.0
2.8 dextrose (sugars 10 wt %) (Example 2.19) Water 54 ~-93 98 ~229
~-2.3 (Example 2.19)
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[0405] Having thus described in detail preferred embodiments of the
present invention, it is to be understood that the invention
defined by the above paragraphs is not to be limited to particular
details set forth in the above description as many apparent
variations thereof are possible without departing from the spirit
or scope of the present invention.
[0406] Each patent, patent application, and publication cited or
described in the present application is hereby incorporated by
reference in its entirety as if each individual patent, patent
application, or publication was specifically and individually
indicated to be incorporated by reference.
* * * * *
References