U.S. patent application number 13/494237 was filed with the patent office on 2013-02-28 for products for anti-inflammation support.
This patent application is currently assigned to Rock Creek Pharmaceuticals, Inc.. The applicant listed for this patent is Jonnie R. Williams. Invention is credited to Jonnie R. Williams.
Application Number | 20130053355 13/494237 |
Document ID | / |
Family ID | 47744572 |
Filed Date | 2013-02-28 |
United States Patent
Application |
20130053355 |
Kind Code |
A1 |
Williams; Jonnie R. |
February 28, 2013 |
PRODUCTS FOR ANTI-INFLAMMATION SUPPORT
Abstract
This disclosure provides products, including pharmaceutical
compositions and dietary supplements, which are useful for
anti-inflammatory support.
Inventors: |
Williams; Jonnie R.;
(Bradenton, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Williams; Jonnie R. |
Bradenton |
FL |
US |
|
|
Assignee: |
Rock Creek Pharmaceuticals,
Inc.
Gloucester
MA
|
Family ID: |
47744572 |
Appl. No.: |
13/494237 |
Filed: |
June 12, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61528380 |
Aug 29, 2011 |
|
|
|
Current U.S.
Class: |
514/167 ;
514/334 |
Current CPC
Class: |
A61K 31/07 20130101;
A23L 29/03 20160801; A61K 31/4545 20130101; A23L 33/10 20160801;
A61K 31/4545 20130101; A23V 2002/00 20130101; A61P 29/00 20180101;
A23V 2002/00 20130101; A61K 31/444 20130101; A61K 31/593 20130101;
A61K 31/593 20130101; A23P 10/20 20160801; A61K 31/07 20130101;
A61K 36/81 20130101; A23L 33/155 20160801; A23L 33/105 20160801;
A23V 2200/324 20130101; A61K 47/22 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/167 ;
514/334 |
International
Class: |
A61K 31/444 20060101
A61K031/444; A61P 29/00 20060101 A61P029/00; A61K 31/593 20060101
A61K031/593 |
Claims
1. A product comprising: a compound of Formula I ##STR00005##
wherein R represents hydrogen or C.sub.1-C.sub.5 alkyl; R'
represents hydrogen or C.sub.1-C.sub.7 alkyl; and X represents
halogen or C.sub.1-C.sub.7 alkyl, or a pharmaceutically acceptable
salt thereof; and a vitamin.
2. The product of claim 1, wherein R represents hydrogen or
C.sub.1-C.sub.3 alkyl.
3. The product of claim 1, wherein R' represents hydrogen or
C.sub.1-C.sub.4 alkyl.
4. The product of claim 1, wherein X represents halogen or
C.sub.1-C.sub.3 alkyl.
5. The product of claim 1, wherein the compound is anatabine.
6. The product of claim 5, wherein the compound is
S-(-)-anatabine.
7. The product of claim 5, wherein the compound is
R-(+)-anatabine.
8. The product of claim 1, wherein the vitamin is Vitamin A.
9. The product of claim 1, wherein the vitamin is Vitamin D3.
10. The product of 8, further comprising Vitamin D3.
11. The product of claim 5, wherein the anatabine is synthetic
anatabine.
12. The product of claim 5, wherein the anatabine is provided in
the form of an extract from a plant.
13. The product of claim 5, which comprises anatabine in an amount
of about 1 mg.
14. The product of claim 1, which comprises Vitamin A in an amount
of about 417 IU.
15. The product of claim 1, comprising Vitamin D3 in an amount of
about 33 IU.
16. The product of claim 1, further comprising one or more
additional ingredients selected from the group consisting of
mannitol, natural and artificial mint flavors, sucralose, silicon
dioxide, stearic acid, hydroxypropyl methylcellulose, magnesium
stearate, titanium dioxide, natural glaze, methyl parabens,
propylparabens, triethyl citrate, citric acid, BHT, mono and
diglycerides, and polysorbate 80.
17. The product of claim 1, which is a dietary supplement.
18. The product of claim 1, which is a pharmaceutical
composition.
19. A method of providing anti-inflammation support, comprising
administering to an individual in need thereof a product
comprising: a compound of Formula I ##STR00006## wherein R
represents hydrogen or C.sub.1-C.sub.5 alkyl; R' represents
hydrogen or C.sub.1-C.sub.7 alkyl; and X represents halogen or
C.sub.1-C.sub.7 alkyl, or a pharmaceutically acceptable salt
thereof; and a vitamin.
20. The method of claim 19, wherein the individual is human.
Description
[0001] This application claims the benefit of and incorporates by
reference Ser. No. 61/528,380 filed on Aug. 29, 2011.
BRIEF DESCRIPTION OF THE DRAWINGS
[0002] FIG. 1. Graph showing effects of anatabine on
TNF.alpha.-induced NF.kappa.B activity in vitro.
[0003] FIG. 2. Graph showing effects of a crude extract of
smokeless tobacco on TNF.alpha.-induced NF.kappa.B activity in
vitro.
[0004] FIG. 3. Graph showing effects of nicotine and of an alkaloid
extract of smokeless tobacco on TNF.alpha.-induced NF.kappa.B
activity in vitro.
[0005] FIG. 4. Graph showing the results of a cytotoxicity assay
measuring release of lactate dehydrogenase (LDH) using supernatant
from the cells assayed in FIG. 1.
[0006] FIG. 5. Graph showing the results of a cytotoxicity assay
using supernatant from the cells assayed in FIG. 2.
[0007] FIG. 6. Graph showing the results of a cytotoxicity assay
using supernatant from the cells assayed in FIG. 3.
[0008] FIG. 7. Graph comparing effects of anatabine, CELEBREX.RTM.,
and aspirin on lipopolysaccharide (LPS)-induced NF.kappa.B activity
in human white blood cells.
[0009] FIG. 8. Graph demonstrating effects of increasing
concentrations of anatabine ("anatabloc" in the figure) on the
release of interleukin 1-beta (IL-1.beta.) from human blood
cells.
[0010] FIG. 9. Graph demonstrating time course of effect of
anatabine ("anatabloc" in the figure) on the release of interleukin
1-beta (IL-1.beta.) from human blood cells.
[0011] FIG. 10. Graph demonstrating effects of anatabine, aspirin,
ibuprofen, celecoxib, and diclofenac on IL-1.beta. accumulation
after treatment of whole human blood with LPS.
DETAILED DESCRIPTION
[0012] Products comprising a compound of Formula I (e.g.,
anatabine), described below, and vitamins are useful for
anti-inflammatory support. "Anti-inflammation support" as used
herein includes helping the body to avoid excessive creation of
inflammation, helping the body maintain lower levels of
inflammation, helping the body maintain healthy levels of
C-reactive protein, and the like. Products described herein can be,
e.g., pharmaceutical compositions or dietary supplements. "Dietary
supplement" as used herein includes the type of product identified
in the United States as a "dietary supplement" in the Dietary
Supplement Health and Education Act (DSHEA) of 1994, as well as the
type of product identified in other parts of the world by terms
such as "food supplements," "nutraceuticals," "functional foods,"
or simply "foods."
[0013] Products disclosed herein, including pharmaceutical
compositions and dietary supplements, can be provided for
administration to humans as well as to animals, such as a companion
animal, a service animal, a farm animal, or a zoo animal. Such
animals include, but are not limited to, canines (including dogs,
wolves), felines (including domestic cats, tigers, lions), ferrets,
rabbits, rodents (e.g., rats, mice), guinea pigs, hamsters,
gerbils, horses, cows, pigs, sheep, goats, giraffes, and
elephants.
[0014] In some embodiments, products (e.g., pharmaceutical
compositions, dietary supplements) comprise a compound of Formula
I, which can be provided as a pharmaceutically acceptable or
food-grade salt:
##STR00001##
[0015] wherein:
[0016] R represents hydrogen or C.sub.1-C.sub.5 alkyl;
[0017] R' represents hydrogen or C.sub.1-C.sub.7 alkyl; and
[0018] X represents halogen or C.sub.1-C.sub.7 alkyl.
[0019] In some embodiments,
[0020] R represents hydrogen or C.sub.1-C.sub.3 alkyl;
[0021] R' represents hydrogen or C.sub.1-C.sub.4 alkyl; and
[0022] X represents halogen or C.sub.1-C.sub.3 alkyl.
[0023] The dotted line within the piperidine ring represents a
carbon/carbon or carbon/nitrogen double bond within that ring, or
two conjugated double bonds within that ring. One of the two
conjugated double bonds can be a carbon/nitrogen double bond, or
both of the conjugated double bonds can be carbon/carbon double
bonds. When a carbon/nitrogen double bond is present, R is absent;
and either (i) "a" is an integer ranging from 1-4, usually 1-2, and
"b" is an integer ranging from 0-8, usually 0-4; or (ii) "a" is an
integer ranging from 0-4, usually 0-2, and "b" is an integer
ranging from 1-8, usually 1-4. When a carbon/nitrogen double bond
is not present, R is present; "a" is an integer ranging from 0-4,
usually 1-2; and "b" is an integer ranging from 0-8, usually 0-4 or
1-2. The term "alkyl," as used herein, encompasses both straight
chain and branched alkyl. The term "halogen" encompasses fluorine
(F), chlorine (Cl), bromine (Br), and iodine (I).
[0024] Table 1 below illustrates non-limiting examples of compounds
within Formula I:
TABLE-US-00001 TABLE 1 R R' (position) X (position) a b H CH.sub.3
(3) -- 0 1 CH.sub.3 -- CH.sub.3 (5) 1 0 H -- CH.sub.3CH.sub.2 (4) 1
0 CH.sub.3CH.sub.2 CH.sub.3 (4) -- 0 1 H CH.sub.3 (2) -- 0 2
CH.sub.3CH.sub.2 (5) H CH.sub.3 (3) CH.sub.3 (5) 1 1 CH.sub.3 --
CH.sub.3 (2) 2 0 CH.sub.3 (5)
[0025] Compounds of Formula I may be present in the form of a
racemic mixture or, in some cases, as an isolated enantiomer, such
as illustrated below in Formula IA.
##STR00002##
[0026] An example of a compound of Formula I is anatabine. The
chemical structure of anatabine
(1,2,3,6-tetrahydro-[2,3']bipyridinyl) is illustrated below, in
which * designates an asymmetric carbon.
##STR00003##
[0027] Anatabine exists in tobacco and certain foods, including
green tomatoes, green potatoes, ripe red peppers, tomatillos, and
sundried tomatoes, as a racemic mixture of R-(+)-anatabine and
S-(-)-anatabine, whose structures are illustrated below.
##STR00004##
[0028] An example of a compound of Formula IA is S-(-)-anatabine.
In some embodiments anatabine is provided in the form of a
pharmaceutically acceptable (or food grade) salt of anatabine.
Anatabine may be adsorbed on a cation exchange resin such as
polymethacrilic acid (Amberlite IRP64 or Purolite C115HMR), as
described in U.S.
[0029] U.S. Pat. No. 3,901,248, the disclosure of which is hereby
incorporated by reference in its entirety. Such cation exchange
resins have been used commercially, for example, in nicotine
replacement therapy, e.g., nicotine polacrilex.
[0030] Unless otherwise clear from context, the term "anatabine" as
used herein refers collectively to anatabine, either as a racemic
mixture or an enantiomer, and pharmaceutically acceptable or
food-grade salts of either of them. In general, salts may provide
improved chemical purity, stability, solubility, and/or
bioavailability relative to anatabine in its native form.
Non-limiting examples of possible anatabine salts are described in
P. H. Stahl et al., Handbook of Pharmaceutical Salts: Properties,
Selection and Use, Weinheim/Zurich:Wiley-VCHNHCA, 2002, including
salts of 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid,
2-hydroxyethanesulfonic acid, 2-oxoglutaric acid,
4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic
acid, ascorbic acid (L), aspartic acid (L), benzenesulfonic acid,
benzoic acid, camphoric acid (+), camphor-10-sulfonic acid (+),
capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic
acid (octanoic acid), carbonic acid, cinnamic acid, citric acid,
cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid,
ethanesulfonic acid, formic acid, fumaric acid, galactaric acid,
gentisic acid, glucoheptonic acid (D), gluconic acid (D),
glucuronic acid (D), glutamic acid, glutaric acid,
glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic
acid, hydrochloric acid, isobutyric acid, lactic acid (DL),
lactobionic acid, lauric acid, maleic acid, malic acid (-L),
malonic acid, mandelic acid (DL), methanesulfonic acid,
naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid,
nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic
acid, pamoic acid, phosphoric acid, proprionic acid, pyroglutamic
acid (-L), salicylic acid, sebacic acid, stearic acid, succinic
acid, sulfuric acid, tartaric acid (+L), thiocyanic acid,
toluenesulfonic acid (p), and undecylenic acid.
[0031] As an alternative to preparing anatabine synthetically,
anatabine can be obtained by extraction from tobacco or other
plants, such as members of the Solanaceae family, such as datura,
mandrake, belladonna, capsicum, potato, nicotiana, eggplant, and
petunia. For example, a tobacco extract may be prepared from cured
tobacco stems, lamina, or both. In the extraction process, cured
tobacco material is extracted with a solvent, typically water,
ethanol, steam, or carbon dioxide. The resulting solution contains
the soluble components of the tobacco, including anatabine.
Anatabine may be purified from the other components of the tobacco
using suitable techniques such as liquid chromatography.
[0032] As part of the purification process, tobacco material may be
substantially denicotinized to remove a majority of other alkaloids
such as nicotine, nornicotine, and anabasine. Denicotinizing is
usually carried out prior to extraction of anatabine. Methods that
may be used for denicotinizing tobacco materials are described, for
example, in U.S. Pat. No. 5,119,835, the disclosure of which is
hereby incorporated by reference. In general, tobacco alkaloids may
be extracted from tobacco material with carbon dioxide under
supercritical conditions. The tobacco alkaloids may then be
separated from the carbon dioxide by dissolving an organic acid or
a salt thereof, such as potassium monocitrate, in the carbon
dioxide.
[0033] In some aspects, an isolated form of anatabine is used. An
"isolated form of anatabine," as used herein, refers to anatabine
that either has been prepared synthetically or has been
substantially separated from plant materials in which it occurs
naturally. The isolated form of anatabine should have a very high
purity (including enantiomeric purity in the case where an
enantiomer is used). In the case of synthetic anatabine, for
example, purity refers to the ratio of the weight of anatabine to
the weight of the end reaction product. In the case of isolating
anatabine from plant material, for example, purity refers to the
ratio of the weight of anatabine to the total weight of the
anatabine-containing extract. Usually, the level of purity is at
least about 95%, more usually at least about 96%, about 97%, about
98%, or higher. For example, the level of purity may be about
98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,
99.8%, 99.9%, or higher.
[0034] In some embodiments, products (e.g., pharmaceutical
compositions and dietary supplements) comprise synthetic anatabine.
In some embodiments, products (e.g., pharmaceutical compositions
and dietary supplements) comprise naturally occurring anatabine
(i.e., anatabine extracted from a plant, as described in more
detail below).
[0035] Vitamins and Minerals
[0036] Products (e.g., pharmaceutical compositions, dietary
supplements) disclosed herein also contain one or more vitamins,
such as Vitamin A (retinol), Vitamin B1 (thiamine), Vitamin C
(ascorbic acid), Vitamin D (calciferol), Vitamin D2
(ergocalciferol), Vitamin D3 (cholecalciferol), Vitamin B2
(riboflavin), Vitamin E (tocopherol), Vitamin B12 (cobalamins),
Vitamin K1 (phylloquinone), Vitamin B5 (pantothenic acid), Vitamin
B7 (biotin), Vitamin B6 (pyridoxine), Vitamin B3 (niacin), Vitamin
B9 (folic acid). Methods of synthesizing vitamins are well known,
and vitamins can be obtained from any reputable commercial source.
In some embodiments, products (e.g., pharmaceutical compositions,
dietary supplements) contain synthetic or naturally occurring
anatabine (or other compound of Formula I) and Vitamin A. In some
embodiments, products (e.g., pharmaceutical compositions, dietary
supplements) contain Vitamin D3. In some embodiments, products
(e.g., pharmaceutical compositions, dietary supplements) contain
Vitamin A and Vitamin D3.
[0037] Products disclosed herein (e.g., pharmaceutical
compositions, dietary supplements) optionally can contain one or
more other nutrients, such as pantothenic acid, calcium, iron,
phosphorus, iodine, magnesium, zinc, selenium, copper, manganese,
chromium, molybdenum, chloride, potassium, boron, nickel, silicon,
vanadium, or lutein.
[0038] Additional Components
[0039] Additional components ingredients may be added to products
(e.g., pharmaceutical compositions, dietary supplements) to improve
taste or stability. Optionally, other components such as sweetening
and flavoring agents may be added. Additional components include,
but are not limited to, sweeteners, natural flavorants, artificial
flavorants, colorants, antioxidants, preservatives, chelating
agents, viscomodulators, tonicifiers, odorants, opacifiers,
suspending agents, binders, thickeners, and mixtures thereof,
including, but not limited to, xanthum gum, carboxymethylcellulose,
carboxyethylcellulose, hydroxypropylcellulose, methylcellulose,
microcrystalline cellulose, starches, dextrins, fermented whey,
tofu, maltodextrins, polyols (including sugar alcohols, such as
sorbitol or mannitol), carbohydrates (e.g., lactose), propylene
hlycol alginate, gellan gum, guar, pectin, tragacanth gum, gum
acacia, locust bean gum, gum arabic, gelatin, mannitol, natural
and/or artificial mint flavors, sucralose, silicon dioxide, stearic
acid, hydroxypropyl methylcellulose, magnesium stearate, titanium
dioxide, natural glaze, methylparaben, propylparabens, triethyl
citrate, citric acid, butylated hydroxytoluene (BHT), mono and
diglycerides, polysorbate 80, and the like.
[0040] Forms
[0041] Products (e.g., pharmaceutical compositions, dietary
supplements) disclosed herein may be in many forms to be taken
orally, such as pills, tablets, capsules, soft gels, gelcaps,
liquids, syrups, suspensions, powders, chews, lozenges, gum, bars,
etc., or to be administered by other routes, such as parenterally,
by inhalation spray, topically, via an implanted reservoir, etc.
They can be prepared to be administered in foods or beverages. They
can be supplied as a dried or powdered product for reconstitution
with water or other suitable vehicle before use (e.g., milk, fruit
juice, and the like).
[0042] Optionally, products disclosed herein (e.g., pharmaceutical
compositions, dietary supplements) may be provided in a
time-release formulation to provide anti-inflammatory support over
extended periods. Extended release formulations are known in the
art. For example, swellable particles are taught in U.S. Pat. Nos.
5,582,837, 5,972,389, and 6,723,340. Polymer matrices are taught in
U.S. Pat. Nos. 6,210,710, 6,217,903, and 6,090,411. Typical
materials used for extended release formulations are the polymers
poly(ethylene oxide) and hydroxypropyl methylcellulose. Tablet
formulations for slow release are also described in U.S. Pat. No.
5,942,244.
[0043] Packaging
[0044] Products disclosed herein (e.g., pharmaceutical
compositions, dietary supplements) can be prepared, packaged, and
labeled for use for anti-inflammation support.
[0045] Preparation of Products
[0046] Products disclosed herein (e.g., pharmaceutical
compositions, dietary supplements) as disclosed herein may be
prepared by any suitable technique and is not limited by any
particular method for production. For example, anatabine (or
another compound of Formula I) and vitamins can be combined with
excipients and a binder, and then granulated. The granulation can
be dry-blended with any remaining ingredients and compressed into a
solid form such as a tablet.
[0047] The amount of anatabine (or another compound of Formula I)
and vitamins in products (e.g., pharmaceutical compositions,
dietary supplements) may vary. In some embodiments, the amount of
anatabine (or another compound of Formula I) ranges from about 0.1
mg to about 10 mg (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,
3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10 mg). Anatabine (or another
compound of Formula I) can be provided as free base or in the form
of a salt, such as a citrate salt.
[0048] In some embodiments, the amount of Vitamin A ranges from
about 200 to about 500 IU (e.g., about 200, 250, 300, 350, 400,
401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413,
414, 415, 416, 417, 418, 419, 420, 450, 475, or 500 IU). Vitamin A
can be provided, for example, as retinyl acetate.
[0049] In some embodiments, the amount of Vitamin D3 ranges from
about 15 IU to about 50 IU (e.g., about 15, 20, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 40, 45, or 50 IU). Vitamin D3 can be
provided as cholecalciferol.
[0050] In some embodiments anatabine (or another compound of
Formula I) and Vitamin A are provided in equal proportions (e.g., 1
mg each).
[0051] In some embodiments, one or two lozenges containing 1 mg
anatabine (or another compound of Formula I) can be taken once,
twice, or three times daily. In some embodiments, daily doses do
not exceed 1, 2, 3, 4, 5, or 6 lozenges. In some embodiments, daily
doses can exceed 1, 2, 3, 4, 5, or 6 lozenges.
[0052] In some embodiments a product is in the form of a lozenge
that contains 1 mg anatabine (as anatabine citrate), 417 IU Vitamin
A (as retinyl acetate), 33 IU Vitamin D3 (as cholecalciferol), and
mannitol, natural and artificial mint flavors, sucralose, silicon
dioxide, stearic acid, hydroxypropyl methylcellulose, magnesium
stearate, titanium dioxide, natural glaze, methyl parabens,
propylparabens, triethyl citrate, citric acid, BHT, mono and
diglycerides, and polysorbate 80. In one embodiment this product is
a dietary supplement ("ANATABLOC.TM.").
Example 1
NF.kappa.B-Mediated Transcription Assays
Cytotoxicity Assays
[0053] The effect of a range of doses of anatabine, nicotine, crude
extract of smokeless tobacco, and alkaloid extract of smokeless
tobacco was examined in an NF.kappa.B luciferase assay (inhibition
of TNF.alpha.-induced NF.kappa.B activity). The smokeless tobacco
used in these experiments was plain long-leaf Copenhagen tobacco
purchased from a local vendor. Crude extract was extracted with
methanol and water and clarified by centrifugation and filtration.
The alkaloid extract was prepared from sodium hydroxide and
methanol extraction, organic phase separation and purification. All
treatment samples were prepared as a function of weight (.mu.g/ml),
and all samples were diluted in DMSO. Dilutions were made
immediately before cell culture treatments and, in all cases, the
final amount of DMSO did not exceed 1% in cell culture media.
[0054] Human endothelial kidney cells (HEK293) transfected with an
NF.kappa.B luciferase reporter were challenged with TNF.alpha. for
three hours, then samples were applied to the challenged cells. The
results are shown in FIGS. 1-3.
[0055] Cytotoxicity assays using the supernatants from the treated
cells were conducted using an LDH Cytotoxicity Detection Kit
(Roche) according to the manufacturer's instructions. The results
are shown in FIGS. 4-6.
[0056] As shown in FIG. 1, TNF.alpha. induces an increase in
NF.kappa.B-mediated transcription of luciferase; administration of
anatabine can reduce this transcription to control levels without
cellular toxicity (FIG. 4). Crude extracts of smokeless tobacco,
while not toxic to cells (FIG. 5), do not reduce TNF.alpha.-induced
NF.kappa.B-mediated transcription (FIG. 2). Although not suitable
for administration as pharmaceuticals, both nicotine and an
alkaloid extract of smokeless tobacco reduce TNF.alpha.-induced
NF.kappa.B-mediated transcription (FIG. 3); at higher doses, the
alkaloid extract demonstrates pronounced cytotoxicity (FIG. 6).
Example 2
Effects of Anatabine, CELEBREX.RTM., and Aspirin on LPS-Induced
NF.kappa.B Activity in Human White Blood Cells
[0057] Peripheral blood mononuclear cells were isolated using the
Ficoll plaque method according to the manufacturer's instructions.
The cells were activated with LPS 10 .mu.g/ml and treated with 1.25
mM anatabine, celecoxib (CELEBREX.RTM.), or aspirin. Cells were
then incubated at 37.degree. C. and 5% CO.sub.2 overnight (18
hours) in RPMI medium supplemented with 20 mg/ml PHA, 1%
penicillin/streptomycin, and 1% glutamax and assayed to detect
NF.kappa.B activity. The results are shown in FIG. 7.
Example 3
Effects of Increasing Concentrations of Anatabine on IL-1.beta.
Release from Human Blood Cells
[0058] Stimulation of human blood cells with LPS causes release of
interleukin 1.beta. (IL-1.beta.). Anatabine inhibits this release
in a concentration-dependent manner, as shown in FIG. 8.
[0059] FIG. 9 is a graph demonstrating this inhibition over
time.
Example 4
[0060] Effects of Anatabine, Aspirin, Ibuprofen, Celecoxib, and
Diclofenac on IL-1.beta. Accumulation After Treatment of Whole
Human Blood With LPS
[0061] Whole human blood was treated with LPS (to stimulate
IL-1.beta. production) and either anatabine, aspirin, ibuprofen,
celecoxib, or diclofenac. After 16 hours, IL-1.beta. release was
measured. The results are shown in FIG. 10 and demonstrate a
reduced accumulation of IL-1.beta. in anatabine-treated blood
compared with blood treated with other anti-inflammatory
agents.
Example 5
Dietary Supplement Formulation
[0062] A dietary supplement was prepared by combining the
components listed in Table 2 below and forming into 160 mg tablets
containing 1 mg anatabine, 417 IU Vitamin A (as retinyl acetate),
and 33 IU Vitamin D3 (as cholecalciferol). Anatabine citrate was
prepared synthetically as described in Examples 1-3 of co-pending
application Ser. No. 12/729,346, which is incorporated herein by
reference in its entirety. A granulation was made containing 1.003
mg free base anatabine (Davos/Anthem), 1.003 mg citric acid
anhydrous (Spectrum Chemical Mfg. Corp.), 0.090 mg BHT (Spectrum
Chemical Mfg. Corp.), and 17.958 mg mannitol (Roquette) and
combined with the ingredients in Table 2.
TABLE-US-00002 TABLE 2 Amount Amount Material (mg/tablet) (wt. %)
Anatabine granulation 20 12.5 Mannitol 123.8 77.375 Sucralose 3.2
2.0 Flavors 7.2 4.5 Vitamin A Acetate 1.0 0.625 Vitamin D3 0.4
0.250 Fumed Silica 1.6 1.0 Stearic Acid 2.0 1.25 Magnesium stearate
0.8 0.50 Total 160.0 100
* * * * *