U.S. patent application number 10/025364 was filed with the patent office on 2002-06-13 for multicomponent biological vehicle.
This patent application is currently assigned to UNIVERA PHARMACEUTICALS, INC.. Invention is credited to Jia, Qi.
Application Number | 20020071868 10/025364 |
Document ID | / |
Family ID | 26769282 |
Filed Date | 2002-06-13 |
United States Patent
Application |
20020071868 |
Kind Code |
A1 |
Jia, Qi |
June 13, 2002 |
Multicomponent biological vehicle
Abstract
The present invention provides a novel vehicle for the delivery
of biologically active agents. The vehicle, Maxcell.TM., is
formulated from a novel combination of natural plant extracts and
is comprised of Aloe vera polysaccharide fraction Immuno-10, cAMP,
piperine, calcium phosphate and glycyrrhizinic acid.
Inventors: |
Jia, Qi; (Arvada,
CO) |
Correspondence
Address: |
SWANSON & BRATSCHUN L.L.C.
1745 SHEA CENTER DRIVE
SUITE 330
HIGHLANDS RANCH
CO
80129
US
|
Assignee: |
UNIVERA PHARMACEUTICALS,
INC.
100 Technology Drive, Suite 160
Broomfield
CO
80021
|
Family ID: |
26769282 |
Appl. No.: |
10/025364 |
Filed: |
December 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10025364 |
Dec 19, 2001 |
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09301892 |
Apr 29, 1999 |
|
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60083420 |
Apr 29, 1998 |
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Current U.S.
Class: |
424/486 |
Current CPC
Class: |
A61K 9/205 20130101;
A61K 9/4866 20130101 |
Class at
Publication: |
424/486 |
International
Class: |
A61K 009/14 |
Claims
What is claimed is:
1. A biological vehicle for the delivery of one or more
biologically active agents, said vehicle comprising at least one
compound that enhances the bioavailability of said biological
agents, at least one compound that enhances intracellular signaling
processes, and at least one compound that enhances cell
proliferation and cell survival.
2. The biological vehicle of claim 1 wherein said compound that
enhances the bioavailability of said active agent acts by
increasing the permeability of gastrointestinal epithelial cells to
said active biological ingredient.
3. The biological vehicle of claim 1 wherein said compound that
enhances the bioavailability of said active agent acts as a
bioadhesive polymer, wherein said bioadhesive polymer binds to and
coordinates said active biological agents; whereby said
coordination increases the delivery of said active biological
agents to the cell surface.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/301,892, filed Apr. 29, 1999, entitled
"Multicomponent Biological Vehicle." This application also claims
the benefit of U.S. Provisional Patent Application Ser. No.
60/083,420, filed Apr. 29, 1998, entitled "Multicomponent
Biological Vehicle."
FIELD OF INVENTION
[0002] The present invention relates generally to a vehicle for the
delivery of biologically active agents. In particular this
invention includes a biological vehicle formulated from a novel
combination of plant extracts that can be used for the delivery of
a variety of biologically active agents. Also included in this
invention are methods for using the vehicle and formulations
containing the vehicle.
BACKGROUND OF THE INVENTION
[0003] A vehicle is a substance, usually without biological
activity, which is used as a medium for the administration of
pharmacologic agents. Ideally a vehicle should be nonirritating and
compatible with common medications. Criteria for vehicle selection
include solubility of the active agent in the vehicle and the
ability of the vehicle to penetrate physical barriers such as the
stratum corneum and the lipid portion of the cell membrane.
Consideration must also be given to any interactions between the
vehicle and the active agent, the efficiency with which the vehicle
releases the incorporated active ingredient, the molecular size and
the composition of the vehicle. Traditional vehicles for
subcutaneous administration include oils which tend to be
irritating.
[0004] Occasionally, a vehicle will have a synergistic effect, such
that the final sum total of the activity in each system is greater
than the sum of the components. This is highly desirable in that
less of the pharmacologic agent can be used to achieve the same
effect, thereby reducing or eliminating any side effects associated
with higher dosages.
[0005] Aloe is an intricate plant which contains many biologically
active substances. (Cohen et al. (1992) in Wound
Healing/Biochemical and Clinical Aspects, 1st ed. W B Saunders,
Philadelphia). Over 300 species of Aloe are known, most of which
are indigenous to Africa. Studies have shown that the biologically
active substances are located in three separate sections of the
aloe leaf--a clear gel fillet located in the center of the leaf, in
the leaf rind or cortex of the leaf and in a yellow fluid contained
in the pericyclic cells of the vascular bundles, located between
the leaf rind and the internal gel fillet, referred to as the
latex. Historically, Aloe products have been used in dermatological
applications for the treatment of burns, sores and other wounds.
These uses have stimulated a great deal of research in identifying
compounds from Aloe plants that have clinical activity, especially
anti-inflammatory activity. (See, e.g., Grindlay and Reynolds
(1986) J. of Ethnopharmacology 16:117-151; Hart et al. (1988) J. of
Ethnopharmacology 23:61-71). As a result of these studies there
have been numerous reports of Aloe compounds having diverse
biological activities, including anti-tumor activity, anti-gastric
ulcer, anti-diabetic, anti-tyrosinase activity, (see, e.g., Yagi et
al. (1977) Z. Naturforsch 32c:731-734), and antioxidant activity
(International Application Serial No. PCT/US95/07404).
[0006] Recent research has also shown that Aloe vera, a term used
to describe the extract obtained from processing the entire leaf,
isolated from the Aloe vera species of Aloe can be used as a
vehicle for delivering the corticosteroid, hydrocortisone,
administered both topically and subcutaneously to the site of
inflammation. (Davis et al. (1991) JAPMA 81: 1). Davis et al.
studied the topical and systemic anti-inflammatory activity of Aloe
vera alone and in combination with hydrocortisone acetate. This
study revealed that Aloe vera contributed in an additive way to the
activity of the steroid, suggesting that Aloe vera may be useful as
a biological vehicle for hydrocortisone. These studies also
revealed that Aloe vera assists in the penetration of
hydrocortisone through the stratum corneum. Davis has also shown
that Aloe vera can be used as a biological vehicle for the delivery
of the estrogenic hormones, estrogen and .beta.-estradiol and
androgenic hormone, testosterone propionate. (U.S. application Ser.
No. 08/662,654, filed Jun. 13, 1996, entitled, "Method of Using
Aloe Vera as a Biological Vehicle," now issued as U.S. Pat. No.
5,708,038, which is incorporated herein by reference in its
entirety). The significance of these findings is that if used in
combination with Aloe vera, the dosage of the steroid can be
reduced, while maintaining its biological activity, thereby
reducing or eliminating any toxic side effects associated with
higher dosages. Since Aloe vera contains many hydrophilic
compounds, such as enzymes, amino acids and carbohydrates, as well
as, hydrophobic compounds, such as vitamins and sterols, Davis et
al. postulate that pharmacologic agents of both solubilities can be
placed in Aloe vera and carried through the epidermal barrier.
(Davis et al (1991) JAPMA 81:1).
SUMMARY OF THE INVENTION
[0007] The present invention includes a novel biological vehicle
that can be used for the delivery of a variety of biologically
active agents. The vehicle, called Maxcell.TM., is comprised of
Aloe polysaccharide fraction Immuno-10 (see U.S. Pat. No.
6,133,440, entitled "Process for the Preparation of
Immunomodulatory Polysaccharides from Aloe," which is incorporated
herein by reference in its entirety), aqueous extract of Ziziphus
jujuba fruits, hydrophilic solvent extract of Glycyrrhiza uralensis
rhizome, organic solvent extract of Piper nigrum or Piper longum,
and calcium inorganic and/or organic salts. The components of
Maxcell.TM. combine to facilitate the harmony between biologically
active agents and targeted cells and organs by guiding the
biological active agents to their sites of action; by preventing
the clearance of the agents from blood; and by protecting cells
against harsh properties of biological active agents. The
biological vehicle functions as a bioadhesive polymer that
selectively binds to the active ingredients thereby forming a
complex; the complex thus formed then selectively binds to
mannose-6-phosphate receptors on the cell membrane. This serves to
promote efficient delivery of the active ingredients to the target
cells.
[0008] The biological vehicle of this invention can be used to
deliver a variety of biologically active agents including, but not
limited to nutrients and dietary supplements, such as essential
minerals like iron, chromium, selenium, zinc, copper, magnesium,
manganese, calcium; vitamins such as vitamins A, E, K and D, and
vitamins B1, B2, B6 and B12; essential amino acids and derivatives
thereof such as arginine, lysine, leucine, phenylalanine, taurine,
and N, N-dimethyl glycine; anti-oxidants such as like
bioflavonoids, polyphenols, beta-carotene, curcumine, catechins,
anthocyanidins; hormones such as melatonin; single plant extracts
such as like Echinacea, garlic, Gingko biloba, Goldenseal, Saw
palmetto, Ginseng (Panex, Siberian, & American), Cat's claw
Astragalus, St John's Wort; and combinations of the above nutrients
and dietary supplements. The components of Maxcell.TM. not only
function as a classical delivery system to target bioavailability,
but also emphasize the response from the targeted living cells
through renewal, enhancement and protection functions.
Additionally, the components of Maxcell.TM. also combine to produce
synergistic effects. Also included in the invention is a method for
preparing the vehicle, methods for using the vehicle and
nutritional supplement formulations containing the vehicle.
[0009] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention as
claimed.
FIGURES
[0010] FIG. 1 illustrates the ability of Maxcell.TM. and Immuno-10
to stimulate kidney cell renewal (%) as a function of dosage in
.mu.g/mL.
[0011] FIG. 2 illustrates the ability of Maxcell.TM. to protect the
liver cells of mice against damage caused by high dosage of
CCl.sub.4.
[0012] FIG. 3 illustrates the ability of Immuno-10 and Maxcell.TM.
to protect cells against UV-B damage. Both Immuno-10 and
Maxcell.TM. provide 18% to 31% protection of cells against UV-B
damage.
[0013] FIG. 4 illustrates Coenzyme Q-10 bioavailability enhancement
by Maxcell.TM.. This figure depicts the increase of rat serum Q-10
level (%) as a function of time.
[0014] FIG. 5 illustrates Coenzyme Q-10 bioavailability enhancement
by Maxcell.TM.. This figure depicts the rat serum Q-10
concentration (.mu.g/mL) increase as a function of time.
[0015] FIG. 6 illustrates graphically Coenzyme Q-10 bioavailability
enhancement by Maxcell.TM.. This figure depicts the rat serum Q-10
concentration (.mu.g/mL) increase as a function of time for Q-10
only (.diamond-solid.), Q-10 in combination with Immuno-10
(.box-solid.) and Q-10 in combination with Maxcell.TM. (A).
[0016] FIG. 7 illustrates graphically the combined effect of
Maxcell.TM. and the Super Immune formulation on the stimulation of
TNF-.alpha. release from mouse peritoneal macrophages. This figure
depicts the release of TNF-.alpha. as a function of dosage.
[0017] FIG. 8 illustrates the results of the Bio Energy (BioE)
formulation Anti-Fatigue test.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present invention describes a novel biological vehicle
that can be used for the delivery of a variety of biologically
active agents, including but not limited to nutrients, dietary
supplements and other compounds with biological activity. The
vehicle, called Maxcell.TM., is comprised of Aloe polysaccharide
fraction Immuno-10, aqueous extract of Ziziphus jujuba fruits,
hydrophilic solvent extract of Glycyrrhiza uralensis rhizome,
organic solvent extract of Piper nigrum or Piper longum, and
calcium inorganic and/or organic salts. The components of
Maxcell.TM. combine to: enhance the therapeutic effects of the
biologically active agents, enhance intracellular communication and
response to biologically active agents, facilitate the transport of
biologically active agents into the blood stream following
ingestion, bind to and protect biologically active agents, guide
the agents to their sites of action and prevent the clearance of
the biologically active agents. The biological vehicle can be used
to deliver a variety of biologically active agents, including but
not limited to nutrients and dietary supplements, such as essential
minerals like iron, chromium, selenium, zinc, copper, magnesium,
manganese, calcium; vitamins such as vitamins A, E, K and D and
vitamins B1, B2, B6 and B12; essential amino acids and derivatives
thereof such as arginine, lysine, leucine, phenylalanine, taurine,
and N, N-dimethyl glycine; anti-oxidants such as like
bioflavonoids, polyphenols, beta-carotene, curcumine, catechins,
anthocyanidins; hormones such as melatonin; single plant extracts
such as like Echinacea, garlic, Gingko biloba, Goldenseal, Saw
palmetto, Ginseng (Panex, Siberian, & American), Cat's claw
Astragalus, St John's Wort; and combinations of the above nutrients
and dietary supplements.
[0019] The components of Maxcel.TM. not only function to enhance
bioavailability, but also combine to promote cell renewal,
stimulate the immune system and confer protection against toxins
and the side effects of drugs. Additionally, the components of
Maxcell.TM. also combine to produce synergistic effects.
[0020] Certain terms used to describe the invention herein are
defined as follows.
[0021] The term "Aloe" refers to the genus of South African plants
of the Liliaceae family of which the Aloe barbadensis plant is a
species.
[0022] The term "Aloe vera" is defined as the dried inner filet of
the leaf of various species of the Aloe plant.
[0023] The term "Aloe vera polysaccharide," refers to
polysaccharides isolated from the Aloe plant.
[0024] The term "Aloe vera polysaccharide fraction Immuno-10" also
referred to as "Immuno-10," refers to a complex carbohydrate
preparation produced from the inner filet of the leaf of Aloe
species. It functions as a bioadhesive polymer by binding to and
coordinating the active ingredients; the complex thus formed then
selectively binds to receptors on the cell membrane. This serves to
promote efficient delivery of the active ingredients to the target
cells. In addition to drug delivery functions, Aloe vera
polysaccharide fraction Immuno-10 has been shown to prevent the
suppression of contact hypersensitivity in mice exposed to
ultraviolet B radiation. Immuno-10 also inhibits ultraviolet B
irradiation-induced tumor necrosis factor (TNF-.alpha.) release in
a human epidermoid carcinoma cell line and promotes wound-healing
and cellular proliferation, particularly in the immune system.
[0025] The typical process for the isolation of Aloe vera
polysaccharide involves alcohol precipitation from the juice of
Aloe barbadensis. The instant application makes use of a novel and
more efficient procedure, described in U.S. Pat. No. 6,133,440,
entitled "Process for the Preparation of Immunomodulatory
Polysaccharides from Aloe," which is incorporated herein by
reference in its entirety. The composition and chemical structure
of the activated polysaccharides in Immuno-10 isolated using this
method is as follows:
[0026] Size exclusion chromatography analysis shows that the
average molecular weight of the polysaccharides in Immuno-10 is
70.about.80 kDa with a range between 50.about.200 kDa. Analysis of
the monosaccharide composition indicates that the polysaccharides
in Immuno-10 contain D-galactose (approx 5% or less), D-glucose
(approx. 5% or less) and D-mannose (approximately 90%). The
polysaccharides in Immuno-10 may also contain trace amounts of
xylose and arabinose. Pharmaceutical grade Immuno-10, which is more
highly purified, contains mainly D-galactose and D-mannose in a
ratio of 1 to 9.6.+-.2.2. Proton and .sup.13C NMR-spectroscopy
analysis indicates that the monosaccharide linkages are primarily
.beta.-1,4 linkages. The main structure of Immuno-10 polysaccharide
is .beta.-1,4 glucomannan. Furthermore, the polysaccharide is
highly acetylated (approximately 1 acetyl group per sugar residue
on average). The 2, 3 and 6 positions of the monosaccharide units
can be independently substituted with an --OH or an --OAc.
[0027] A "vehicle" refers to a substance which acts as a physical
or physiological carrier for biologically active agents, also
referred to as pharmacologic agents. A vehicle aids in the
penetration of biologically active agents through physical
barriers, such as the stratum corneum, gastrointestinal,
respiratory, urinary and blood-brain barriers. A "biological
vehicle" also enhances the biological activity of the active agent
regardless of the agent under consideration. Thus, a biological
vehicle not only acts as a carrier, but also has an additive or a
synergistic effect as it enhances the effects of other
pharmacologic agents. The vehicle of the instant invention is
comprised of Aloe vera polysaccharide extract (preferably
Immuno-10), cAMP, glycyrrhizinate, piperine alkaloids and calcium
inorganic or organic salts.
[0028] The term "biologically active agent," also referred to as
"pharmacologic agent," refers to any substance that can cause a
physiological effect in a living organism. Biologically active
agents may be derived from natural sources, such as extracts from
plants and herbs, or they may be synthesized from precursor
components in the laboratory. They include nutrients and
nutritional supplements, including but not limited to amino acids,
amino acid derivatives, vitamins (both water and fat soluble) and
minerals, antioxidants, metabolic intermediates, co-enzymes,
cofactors, hormones or any other nutritional supplement known in
the art. Biologically active agents also include herb and plant
extracts, including but not limited to Echinacea, garlic, Gingko
biloba, Goldenseal, Saw palmetto, Ginseng, Gotu kola (Centella
asiatica), Withania, Kava Kava, Astragalus, St John's wort; and
compositions which contain combinations of the above nutrients and
dietary supplements.
[0029] "Glycyrrhizinate" also referred to as "glycyrrhizinic acid"
or its salts, including but not limited to its potassium and
ammonium salts, is obtained from the rhizome of Glycyrrhiza
uralensis and other species from the same genus. The glycyrrhizinic
acid/salt content is typically from 5% to 98% in the crude or
refined extract. The Glycyrrhiza species known as licorice, is one
of the oldest traditional medicines and is used as a tonic,
antiphlogistic, mucolytic, expectorant and analgesic for treatment
of gastrointestinal and respiratory disorders. Glycyrrhizinic acid
and its salts provide protection against rapid DNA damage and
inhibit mutagenicity. They also provide protection against gastric
ulcers induced by drugs and have been documented as having
protective effects against toxins, viruses, tumors and allergic
agents.
[0030] "Piperine alkaloids" are a mixture of nitrogen containing
compounds existing in the fruit of Piper longum and Piper nigrum,
both indigenous to India. These Ayurvedic herbs are well known as
stimulant, carminative and tonic agents. Piperidine is one of the
major piperine alkaloids in the hydrophobic extract. The piperidine
content in the crude or refined extract is typically between 5% to
97%. Piperidine has been documented for its enhancement of drug
bioavailability. It is believed that this effect is due to an
increase in the permeability of intestinal epithelial cells, and
also to the inhibition of drug metabolism pathways. Piperine
alkaloids are also known to function as appetite stimulants,
anti-colic and anti-tussive agents, and can induce resistance
against infection.
[0031] "cAMP" (cyclic adenosine monophosphate) can be found in the
largest amounts ever reported in the fruit of Ziziphus jujuba,
where it is present at 100 to 1000 .mu.mol/g. The fruits of
Ziziphus jujuba have been utilized in traditional medicine to
tonify the spleen, augment energy, nourish the blood and calm the
spirit. cAMP is known as being one of the key molecules involved in
intracellular signal transduction. cAMP is synthesized in cells in
response to stimulation from certain hormones or biologically
active agents. It binds to and activates a number of protein
kinases, which are responsible for a myriad of cellular changes.
cAMP has been implicated in switching the direction of nerve growth
cone turning, immunostimulation, modulating leukocyte adhesion,
modulating superoxide production in macrophages, modulating
lipolysis and modulating tumor necrosis factor. Increased levels of
cAMP in cells have been shown to potentiate hormonal response in
vitro and in vivo and have been used as a treatment for selective
weight control, neurodegenerative conditions, viral infections,
allergic diseases, thrombosis, platelet aggregation, cardiovascular
disease and high intraocular pressure. (See Song et al. (1997)
Nature 388:275; Laudanna et al. (1997) J. Biol. Chem. 272:24141; Si
et al. (1997) 36:1; Wang et al. J. Biol. Chem (1997) 272:5959;
Hoffman et al. (1984 Am. J. Physiol. 274:E772).
[0032] "Calcium (inorganic and organic salts)" supply calcium as a
nutritional supplement and are also believed to augment
intracellular stores of calcium. Intracellular calcium is
synergistic with cAMP as a second messenger during signal
transduction. In this way, calcium is believed to potentiate and
enhance cellular response to extracellular signals sent by
biologically active agents.
[0033] In one embodiment of the instant invention, a biological
vehicle is prepared by combining Aloe vera polysaccharide extract
(preferably Immuno-10), isolated from Aloe vera gel, Ziziphus
jujuba fruit extract containing cAMP, Glycyrrhiza uralensis rhizome
extract containing glycyrrhizinate, Piper nigrum or Piper longum
fruit extract containing piperine alkaloids and calcium inorganic
or organic salts. In a preferred embodiment the individual
components are combined respectively in substantially a
10:35:4:4:22 weight:weight ratio. The corresponding biologically
active components are formulated respectively as
Immuno-10:cAMP:glycyrrhizinate:piperine alkaloids:calcium in
substantially a 2:0.005:6:1:10 weight:weight ratio.
[0034] The resulting biological vehicle may be administrated orally
at approximately 100 to 2500 mg/day. In a preferred embodiment the
vehicle is administrated at approximately 750 mg/day. The vehicle
may be combined with any number of biological agents in order to
supply said biological activities. In preferred embodiments, the
vehicle and the active biological agents are combined, with the
optional inclusion of pharmaceutically acceptable excipients, in
the form of, but not limit to, capsules, tablets, soft gel
capsules, time-release capsules and liposomes. In a preferred
embodiment the amount of Maxcell.TM. in the composition is from 1%
to 99.7% on a weight basis.
[0035] The biologically active agents can be nutrients and
nutritional supplements, such as amino acids, vitamins and
derivatives thereof, minerals, anti-oxidants, metabolic
intermediates, co-enzymes and co-factors, hormones and derivatives
thereof, or any other nutritional supplements known in the art. It
is also possible to combine the biological vehicle with herb and
plant extracts, including, but not limited to Echinacea, garlic,
Gingko biloba, Goldenseal, Saw palmetto, Ginseng (Panex, Siberian
and American), Gotu kola (Centella asiatica), Withania, Kava Kava,
Astragalus, St John's wort, and compositions which contain
combinations of the above nutrients and dietary supplements.
[0036] Example 1 describes one example of the preparation of the
Maxcell.TM. vehicle.
[0037] Example 2 describes the ability of Maxcell.TM. to promote
cell renewal. As can be seen in FIG. 1, at a concentration of 2
mg/mL, Maxcell.TM. stimulates kidney cell proliferation by 55%.
When the Immuno-10 polysaccharide fraction (which is a component of
Maxcell.TM.) is tested alone in the same assay, only moderate
stimulation (14% -17%) is observed. Therefore, it can be seen that
the other ingredients in Maxcell.TM. act in concert with the
Immuno-10 to promote kidney cell proliferation.
[0038] Example 3 illustrates the ability of Maxcell.TM. to protect
liver cells from damage caused by CCl.sub.4 The results are set
forth in FIG. 2.
[0039] The Maxcell.TM. vehicle also confers protection against
ultraviolet B irradiation upon treated cells (Example 4). By
monitoring the amount of TNF-.alpha. released following irradiation
(a response to UV-B damage), it has been determined that
Maxcell.TM. provides up to 31% more protection than control
solutions (data not shown). FIG. 3 illustrates that Immuno-10 and
Maxcell.TM. provide 18% to 31% protection of cells against UV-B
damage, respectively.
[0040] Example 5 (FIGS. 4-6) illustrate the Coenzyme Q-10 (Q-10)
bioavailability enhancement by Maxcell.TM.. FIG. 4 illustrates the
increase of rat serum Q-10 (%) levels as a function of time FIG. 5
illustrates the rat serum Q-10 concentration (.mu.g/mL) increase as
a function of time for Q-10 only and Q-10 in combination with
Maxcell.TM.. FIG. 6 depicts the rat serum Q-10 concentration
(.mu.g/mL) increase as a function of time for Q-10 only
(.diamond-solid.), Q-10 in combination with Immuno-10 (.box-solid.)
and Q-10 in combination with Maxcell.TM. (.DELTA.).
[0041] Example 6 describes the preparation of three different
Maxcell formulations: the Bio Energy formulation, the Super Immune
formulation, and the Anti-Stress formulation.
[0042] Example 7 describes the method used to determine the
combined effect of the Maxcell.TM. vehicle and the Super Immune
formulation (Formulation 2, Example 6) on macrophage activation.
The results are set forth in FIG. 7, which depicts the release of
macrophage TNF-.alpha. measured as a function of dosage. As shown
in FIG. 7, at all concentrations tested macrophage TNF-.alpha.
release was increased by the Maxcell.TM. vehicle. This result
demonstrates the effectiveness of Maxcell.TM. as a biological
vehicle for the delivery of the Super Immune formulation.
[0043] Example 8 describes the method used to determine the effect
of the Bio Energy formulation (BioE) on stamina and the combined
effect of the Maxcell.TM. vehicle and the Bio Energy formulation
(BioE) on stamina. The results are set forth in FIG. 8. As shown in
FIG. 8, the Bio Energy formulation increases the stamina of the
mice when compared to treatment with saline or glucose.
Additionally, when used in combination with Maxcell.TM. the stamina
of the mice is enhanced by virtue of the increase in
bioavailability of the Bio Energy formulation.
[0044] The following examples are presented for illustrative
purposes only and are not intended to limit the scope of the
invention.
EXAMPLES
Example 1
Preparation of the Maxcell.TM. Vehicle
[0045] Extraction of Ziziphus jujuba fruit.
[0046] Ziziphus jujuba fruit (purchased from the Pacific Ocean
Grocery Store, Denver, Colo.) was dried and crushed in a coffee
grinder. The product was then freeze dried at room temperature,
then finely ground to a small particle powder with a total weight
of 857 g. The powder was soaked in 4.5 L of distilled water for 2
hours at 60.degree. C., then centrifuged at 3,000 rpm for 15
minutes at room temperature. The supernatant was filtered into an
Erlenmeyer flask. The remaining plant residue was extracted again
in the same manner and the two extract solutions were combined and
lyophilized to yield 376.7 g of a yellow powder with a sweet
taste.
[0047] Preparation of Maxcell.TM..
[0048] 100 g of Aloe polysaccharide fraction Immuno-10, prepared as
described in U.S. Pat. No. 6,133,440, entitled "Process for the
Preparation of Immunomodulatory Polysaccharides from Aloe," which
is incorporated herein by reference in its entirety, was combined
with 350 g Ziziphus extract, 40 g of solvent extract from Piper
nigrum, 220 g calcium phosphate and 40 g of monoammonium
glycyrrhizinate (>65% pure).
[0049] The daily dosage of this formulation (750 mg) provides 100
mg Immuno-10, 350 mg Ziziphus extract, 5 mg piperine extract, 245
mg calcium phosphate and 50 mg of monoammonium glycyrrhizinate
(>65% pure).
Example 2
Stimulation of Cell Proliferation by Maxcell.TM.
[0050] This experiment was performed using BHK-21: Baby Hamster
Kidney cell line in DMEM (Dulbecco's modified eagle
medium)/2.5.about.10% FBS with 3000.about.5000 cells/well. The
cells were grown in a 96 well flat-bottom tissue culture treated
plate. The MaxCell.TM. solution was prepared in DMEM/FBS/PGS at
concentrations of 8.2 .mu.g/mL, 24.7 .mu.g/mL, 74.1 .mu.g/mL, 222.2
.mu.g/mL, 666.7 .mu.g/mL and 2000 .mu.g/mL. Fibroblast growth
factor (FGF) was used as a positive control in DMEM/FBS/PGS at a
concentration of 150 ng/mL. 200 .mu.L of DMEM/FBS/PGS was added to
all of the perimeter wells of the 96 well cell culture plate, as an
experimental control. All experiments were performed in triplicate
for FGF and MaxCell.TM. (6 doses each)/plate. The BHK cells were
diluted to 3000.about.5000 cells/50 .mu.L (harvested and counted
prior to need) in DMEM/FBS/PGS. The samples were placed in a
37.degree. C. incubator for three days, after which 25 .mu.L/well
of MTT was added to all sample and control wells. The plates were
incubated plates for 4.5 additional hours followed by addition of
100 .mu.L extraction solution in the dark, then placed on a wet
paper towel and covered with foil at 37.degree. C. The following
day the plates were read on dual wavelength 570-630 nm. The result
are set forth in FIG. 1.
Example 3
Protection of Liver cells by MaxCell.TM.
[0051] ICR mice, 8 weeks of age, were used in this experiment. The
animals received food and water ab libitum. They were administrated
MaxCell.TM. in saline at 200 mg/kg, silymarin in saline at 200
mg/kg, and saline only, intraperitoneally for 4 days. After the
fourth dose the animals received an intraperitoneal injection of
carbon tetrachloride (CCl.sub.4) at 20 mg/kg in 0.2 mL to induce
liver damage. After 24 hours of treatment with CCl.sub.4, the
animals were euthanized by CO.sub.2 asphyxiation and blood was
drawn by cardiac puncture into a heparinized syringe. The serum
glutamic oxalacetic and glutamic pyruvic transminases levels were
determined as described by Reitman and Frankel (1957) 28:56-63. The
result are set forth in FIG. 2.
Example 4
Effect of MaxCell.TM. on Immuno-10 Protection against UV
Radiation-induced Local and Systemic Suppression of Inflammatory
Responses
[0052] This experiment was performed as described by Wolf et al.
(1993) The Journal of Investigative Dermatology 100:254-259, which
is incorporated herein by reference in its entirety. Wolf, et al.'s
experiment is as follows. UV radiation was provided by a bank of
sic FS40 sunlamps (National Biology Corp., Twinsburg, Ohio), which
have a peak emission at 313 nm and deliver 65% of their total
energy within the UVB (280-320 nm) wavelength range; their UVB
irradiance was 5 W/m.sup.2 at a 20 cm distance, as determined by an
IL 200 radiometer equipped with an SEE 240 detector fitted with an
SES280 filter and a quartz diffuser (International Light, Inc.,
Newburyport, Mass.). One day after the dorsal hair of the mice had
been removed with electric clippers, they were exposed to the
radiation source while housed five per cage in individual
compartments. Because of shielding by the cage lids, the final
irradiance received by the animals was approximately 3 W/m.sup.2.
The sunscreen preparations were liberally applied (approximately
200 .mu.L/mouse) and rubbed on the shaved dorsal surface and tail
of the mice 20 minutes before exposure to UVR.
[0053] Measurement of Inflammatory Response.
[0054] The inflammatory response was determined by measuring the
double skin-fold thickness of the dorsal skin with a spring-loaded
micrometer (Mitutoyo, Tokyo, Japan) prior to and 24 and 48 hours
after UVR exposure. Skin swelling was determined by subtracting the
average skin thickness before UV irradiation from that after UV
irradiation. The protection from inflammation by a sunscreen was
defined as complete protection where there was no significant skin
swelling in a sunscreen-treated UV-irradiated group 24 and/or 48
hours after UVR exposure when compared with sunscreen-treated,
unirradiated mice; the protection was defined as partial protection
when there was significant skin swelling, but the swelling was
significantly different from that of the UC-irradiated group
without sunscreen.
[0055] The Immuno-10 was prepared as described in U.S. patent
application Ser. No. 09/169,449, filed Oct. 9, 1998, entitled
"Process for the Preparation of Immunomodulatory Polysaccharides
from Aloe," now U.S. Pat. No. 6,133,440. The dosage of Maxcell.TM.
and Immuno-10 was 0.5 mg/mL. The results are set forth in FIG.
3.
Example 5
Enhancement of the Bioavilability of Coenzvme Q (Q-10) by
Maxcell.TM.
[0056] CD male rats, 8 weeks of age, were used for this experiment.
The animals were fasted 18 hours and cannulated. Water was provided
ab libitum. Positive control animals (4 rats) received one oral
dose of 50 mg/kg Q-10 in 400 .mu.L of soy bean oil. Negative
control animals (4 rats) received one oral dose of 50 mg/kg saline.
Study animals (2.times.4 for each group) received one oral dose of
200 mg/mL of MaxCell.TM. (prepared as described in Example 1) or
Immuno-10 (prepared as described in U.S. patent application Ser.
No. 09/169,449, filed Oct. 9, 1998, entitled "Process for the
Preparation of Immunomodulatory Polysaccharides from Aloe") with 50
mg/kg Q-10 in 400 .mu.L of Soybean oil. Approximately 600 .mu.L of
blood was taken at 1, 2, 3, 4, 6, and 8 hours. The blood was
analyzed to determine the amount of coenzyme Q in the plasma as
described by Kaplan et al. (1995) Physiol. Res. 44:39-45. The
results are set forth in FIGS. 4-6.
Example 6
Maxcell.TM. Formulations
[0057] Three different formulations using the instant biological
vehicle (Maxcell.TM.) were prepared. These formulations should be
regarded solely as examples, and are not meant to limit the ways in
which Maxcell.TM. may be used.
1 Daily Dosage Component Amount Effective Range Formulation 1. Bio
Energy Dose: 3 tablets per day Ginseng extract (Siberian and/or
Panex) 250 mg 10-1,000 mg Co-enzyme Q10 18 mg 5-250 mg
.alpha.-ketoglutaric acid 300 mg 50-1,000 mg Potassium magnesium
aspartate 300 mg 50-1,000 mg Chromium polynicotinate 300 .mu.g
50-1,000 .mu.g Maxcell.TM. 750 mg 100-2,500 mg Formulation 2.
"Super Immune" Formulation Dose: 3 capsules or tablets daily
Astragalus extract 500 mg 50-1,500 mg Schisandra extract 300 mg
50-1,500 mg Ganoderma extract 150 mg 50-1,000 mg Maxcell.TM. 750 mg
100-2,500 mg L-arginine 200 mg 50-1,000 mg
[0058] The Schisandra extract contains schisandrins and the
Ganoderma extract contains ganoderic acid and glucans.
2 Formulation 3. "Anti-Stress" Formulation Tablet or capsule Daily
Dosage Dose: 2-3 tablets per day Amount Effective Range Ziziphus
jujuba Mill var. spinosa 200 mg 50-600 mg seed extract Gotu kola
(Centella asiatica) whole 200 mg 50-600 mg plant extract Withania
somnfera root extract 300 mg 50-600 mg Piper methyisticum rootstock
extract 200 mg 50-500 mg Taurine 200 mg 100-1,000 mg Maxcell .TM.
250 mg 100-2,500 mg
Example 7
Synergistic Effect of the Maxcell.TM. Vehicle on the Immune
Stimulation Activity of the Super Immune Formulation
[0059] This experiment was performed as described in U.S. Pat. No.
6,133,440, entitled "Process for the Preparation of
Immunomodulatory Polysaccharides from Aloe," which is incorporated
herein by reference in its entirety. Briefly, resident mouse
peritoneal macrophages were isolated from ICR mice and plated at
200,000 cells per well in a 96-well plate. The cells were washed
three times to remove non-adherent cells after a 2 hour incubation.
The macrophages were then incubated with the Super Immune
formulation, both with and without Maxcell.TM. overnight. The
Maxcell.TM. vehicle was prepared as described in Example 1 and the
Super Immune formulation was prepared as described in Example 6.
Maxcell.TM. and the Super immune formulation were tested at 1.95
.mu.g/mL, 3.91 .mu.g/mL, 7.81 .mu.g/mL, 15.63 .mu.g/mL and 31.25
.mu.g/mL. The TNF-.alpha. released into the media was determined by
ELISA. Lipopolysaccharides (LPS) were used as a positive control.
The results are set forth in FIG. 7.
Example 8
Improvement in Fatigue by the Bio Energy Formulation
[0060] ICR male mice, 8 weeks of age, were used for this
experiment. The animals were fasted 18 hours prior to treatment
with the Bio Energy formulation. Water was provided ab libitum.
Positive control animals (8 mice) received one oral dose of 600
mg/kg glucose via gavage feeding. Negative control animals (8 mice)
received one oral dose of 600 mg/kg saline via gavage feeding.
Study animals (10 for each group) received 600 mg/mL of the
BioEnergy formulation (prepared as described in Example 6) with
MaxCell.TM., 600 mg/mL of the BioEnergy formulation without
MaxCell.TM., and 134 mg/mL of MaxCell.TM. alone.
[0061] The animals received food and water ab libitum after being
dosed. After 60 minutes, a 2 gram weight was glued to the end of
the tails of the mice. Individual mice were placed into a tub (50
cm.times.30 cm.times.25 cm) of 20 +/-0.5.degree. C. water and
allowed to swim until their heads went under water. The time of
swim was recorded. The animals were then towel-dried and put into a
warmed cage on top of a heating pad, until completely dry. The test
results are depicted in FIG. 8.
* * * * *