U.S. patent application number 15/190812 was filed with the patent office on 2016-12-29 for bioactivity proanthocyanidin compound isolated from lasia spinosa (l.) thwait.
The applicant listed for this patent is Hong Kong Baptist University. Invention is credited to Zhaoxiang Bian, Albert Sun Chi Chan, Shilin Chen, Chengyuan Lin, Aiping Lu, Huaixue Mu, Hongxi Xu, Dajian Yang.
Application Number | 20160374985 15/190812 |
Document ID | / |
Family ID | 57600834 |
Filed Date | 2016-12-29 |
United States Patent
Application |
20160374985 |
Kind Code |
A1 |
Bian; Zhaoxiang ; et
al. |
December 29, 2016 |
Bioactivity proanthocyanidin compound isolated from Lasia spinosa
(L.) Thwait
Abstract
The present invention disclosed a compound, proanthocyanidin A1
(PA1), which is isolated from Lasia spinosa (L.) Thwait, for its
effects in shortening of clolon length, reducing colonic tissue
damage, suppressing colonic myeloperoxidase activity with colitis.
The said compound can be used in treating inflammatory diseases and
anti-cancer leading compound.
Inventors: |
Bian; Zhaoxiang; (Kowloon,
HK) ; Mu; Huaixue; (Kowloon, HK) ; Lin;
Chengyuan; (Kowloon, HK) ; Xu; Hongxi;
(Kowloon, HK) ; Yang; Dajian; (Kowloon, HK)
; Chen; Shilin; (Kowloon, HK) ; Lu; Aiping;
(Kowloon, HK) ; Chan; Albert Sun Chi; (Kowloon,
HK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hong Kong Baptist University |
Kowloon |
|
HK |
|
|
Family ID: |
57600834 |
Appl. No.: |
15/190812 |
Filed: |
June 23, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62183729 |
Jun 23, 2015 |
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62183728 |
Jun 23, 2015 |
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62183726 |
Jun 23, 2015 |
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Current U.S.
Class: |
514/456 |
Current CPC
Class: |
A61K 31/09 20130101;
A61K 36/888 20130101; A61K 9/0053 20130101; A61K 9/0019 20130101;
A61K 31/7048 20130101; A61K 31/34 20130101; A61K 31/343 20130101;
A61K 31/341 20130101; A61K 31/353 20130101 |
International
Class: |
A61K 31/353 20060101
A61K031/353; A61K 9/00 20060101 A61K009/00; A61K 36/888 20060101
A61K036/888 |
Claims
1. A method for treating an inflammatory disease by administering
an effective dosage of a compound with the structure of:
##STR00002## to a subject in need thereof.
2. The method according to claim 1, wherein said compound is
proanthocyanidin A1.
3. The method according to claim 2, wherein the proanthocyanidin A1
is isolated from a natural plant material comprising Lasia spinosa
(L.) Thwait.
4. The method according to claim 1, wherein the compound inhibits
colitis, inhibits shortening of colon length, reduces colonic
tissue damage, suppresses colonic myeloperoxidase activity and/or
inhibits nitric oxide.
5. The method according to claim 1, wherein the effective dosage
ranges from 0.81 mg/kg/day to 2.43 mg/kg/day.
6. The method according to claim 1, wherein the compound is
administered orally.
7. The method according to claim 1, wherein the subject in need
thereof is a human.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 62/183,728 filed Jun. 23, 2015;
62/183,729 filed Jun. 23, 2015 and 62/183,726 filed Jun. 23, 2015;
the disclosures of which are incorporated herein by reference in
their entirety.
FIELD OF INVENTION
[0002] The present invention relates to a therapeutic effect of
proanthocyanidin Al (PA1). More particularly, it relates to a
compound that is naturally occurring in Lasia spinosa (L.) Thwait
and its effects of treating an inflammatory disease or as an
anti-cancer leading compound.
BACKGROUND OF INVENTION
[0003] Lasia spinosa Linn. Thwait (Araceae) is commonly known as
"Spiny taro", is a perennial herb that grows wild in marshy places
throughout many parts of Asia. The Araceae family is used as
traditional remedies or food and previous work on flavonoid content
in the family showed that Araceae has a profile with flavone
C-glycosides, flavonols, flavones, and proanthocyanidins as the
main classes. Up to date, little information is available about the
chemical constituents of Lasia spinosa Linn. Thwait (Araceae).
Previously the inventors found that the hydro-alcohol extract
exerted significant anti-cancer and anti-inflammatory activities.
In this present invention, isolated major bioactivity compounds of
Lasia spinosa (L.) Thwait and one proanthocyanidin were identified
from the active extract.
[0004] Citation or identification of any reference in this section
or any other section of this application shall not be construed as
an admission that such reference is available as prior art for the
present application.
SUMMARY OF INVENTION
[0005] Accordingly, the present invention relates to a therapeutic
effect of proanthocyanidin A1 (PA1). More particularly, it relates
to a compound that is naturally occurring in Lasia spinosa (L.)
Thwait and its effects of treating an inflammatory disease or as an
anti-cancer leading compound.
[0006] In accordance with one aspect of the present invention,
there is provided a method for treating an inflammatory disease by
administering an effective dosage of a compound with the structure
of:
##STR00001##
to a subject in need thereof.
[0007] In a first embodiment of one aspect of the present
invention, the compound is proanthocyanidin A1 (PA1).
[0008] In a second embodiment of one aspect of the present
invention, in the method for treating an inflammatory disease, the
proanthocyanidin A1 (PA1) is isolated from a natural plant material
comprising Lasia spinosa (L.) Thwait (Araceae).
[0009] In a third embodiment of one aspect of the present
invention, in the method for treating an inflammatory disease, the
compound inhibits colitis, inhibits shortening of colon length,
reduces colonic tissue damage, suppresses colonic myeloperoxidase
activity (MPO) and/or inhibits nitric oxide (NO).
[0010] In a fourth embodiment of one aspect of the present
invention, in the method for treating an inflammatory disease, the
effective dosage ranges from 0.81 mg/kg/day to 2.43 mg/kg/day.
[0011] In a fifth embodiment of one aspect of the present
invention, in the method for treating an inflammatory disease, the
compound is administered orally.
[0012] In a sixth embodiment of one aspect of the present
invention, in the method for treating an inflammatory disease, the
subject in need thereof is a human.
[0013] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described.
[0014] The invention includes all such variation and modifications.
The invention also includes all of the steps and features referred
to or indicated in the specification, individually or collectively,
and any and all combinations or any two or more of the steps or
features.
[0015] Throughout this specification, unless the context requires
otherwise, the word "comprise" or variations such as "comprises" or
"comprising", will be understood to imply the inclusion of a stated
integer or group of integers but not the exclusion of any other
integer or group of integers. It is also 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.
[0016] Furthermore, throughout the specification and claims, unless
the context requires otherwise, the word "include" or variations
such as "includes" or "including", will be understood to imply the
inclusion of a stated integer or group of integers but not the
exclusion of any other integer or group of integers.
[0017] Other definitions for selected terms used herein may be
found within the detailed description of the invention and apply
throughout. Unless otherwise defined, all other technical terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which the invention belongs.
[0018] Other aspects and advantages of the invention will be
apparent to those skilled in the art from a review of the ensuing
description.
BRIEF DESCRIPTION OF DRAWINGS
[0019] The above and other objects and features of the present
invention will become apparent from the following description of
the invention, when taken in conjunction with the accompanying
drawings, in which:
[0020] FIG. 1 shows the structure of compound PAL
[0021] FIG. 2 shows the wound healing assay of (a)-(d) control
data; and (e)-(h) PA1 (cells were treated by compounds PA1 at 6.25
.mu.g/mL which did not alter the cells viability); on human
esophageal carcinoma (KYSE-150) cells. Original magnification,
5.times..
[0022] FIG. 3 shows the effects of PA1 on NO production in
LPS-stimulated RAW 267.4 cells. After macrophages were treated with
100 ng/mL of LPS in the absence or presence of various
concentrations of PA1 (1.56, 3.12, 6.25, 12.5, 25, 50 .mu.g/mL),
which did not alter the cells viability for 20 hours, the
production of NO was determined by Griess reagent. Data were
derived from three independent experiments and presented as
mean.+-.SEM (.sup.####p, compared with control group; *p<0.05,
**p<0.01 and ***p<0.001, compared with LPS-alone group).
[0023] FIG. 4A shows the effects of PA1 on the survival of mice.
Colitis was induced in all groups except the control group. PA1 and
SASP were administered to mice from day 6 to day 13.
[0024] FIG. 4B) shows change of body weight of mice. Colitis was
induced in all groups except the control group. PA1 and SASP were
administered to mice from day 6 to day 13.
[0025] FIG. 4C shows the disease activity index of mice. Colitis
was induced in all groups except the control group. PA1 and SASP
were administered to mice from day 6 to day 13.
[0026] FIG. 4D and FIG. 4E show the colon length of mice with
DSS-induced colitis. Colitis was induced in all groups except the
control group. PA1 and SASP were administered to mice from day 6 to
day 13. The change in body weight was taken as the difference
between the body weight before induction of colitis and that
immediately before the mice were sacrificed on day 14. The DAI
score was determined by combining the scores of (i) body weight
loss, (ii) stool consistency, and (iii) stool bleeding. On day 14,
the mice were sacrificed, and the colon lengths were measured. Data
are expressed as mean.+-.SEM, n=8 (.sup.##p, compared with control
group; *p<0.05 and **p<0.01, compared with DSS group).
[0027] FIG. 5 shows the effects of PA1 on histological
manifestation in DSS-induced colitis in mice. (A) control group;
(B) DSS model group; (C) DSS+SASP 200 mg/kg group; (D) DSS+PA1 10
mg/kg group; (E) DSS+PA1 30 mg/kg. Hematoxylin and eosin staining
images of representative colons are shown at magnifications of
10.times..
[0028] FIG. 6 shows the effects of PA1 on suppressing
myeloperoxidase (MPO) activity in the colon of mice with
DSS-induced colitis. Colitis was induced in all groups except the
control group. PA1 and SASP were administered to the mice from day
6 to day 13. On day 14, the mice were sacrificed, and MPO activity
was determined from colon homogenates. Data are expressed as
mean.+-.SEM, n=8 (*p<0.05, compared with DSS group).
DETAILED DESCRIPTION OF INVENTION
[0029] The present invention is not to be limited in scope by any
of the specific embodiments described herein. The following
embodiments are presented for exemplification only.
[0030] The structures are identified by the .sup.1H, DEPT NMR
spectroscopic data and the structure of proanthocyanidin A1 (PA1)
is shown in FIG. 1.
[0031] Proanthocyanidin A1: [M+H].sup.+: 577.1364 (Calcd. for
577.1346). .sup.1H NMR (400 MHz, CD.sub.3OD): .delta.7.16 (1H, d,
J=2 Hz, H-10), 7.04 (1H, dd, J=2, 8.5 Hz, H-14), 6.98 (1H, s,
H-10'), 6.86 (1H, m, H-14'), 6.84 (1H, s, H-13), 6.82 (1H, s,
H-13'), 6.10 (1H, s, H-6'), 6.08 (1H, d, J=2.4 Hz, H-8), 5.95 (1H,
d, J=2.4 Hz, H-6), 4.76 (1H, d, J=8.0 Hz, H-8), 4.26 (1H, d, J=3.2
Hz, H-4), 4.15 (1H, d, J=3.2 Hz, H-3), 4.08 (1H, m, H-3'), 2.98
(1H, dd, J=5.6, 16.4 Hz, H-4'.beta.), 2.59 (1H, dd, J=2, 8.5 Hz,
H-4'.alpha.); .sup.13C NMR (100 MHz, CD.sub.3OD): 156.77 (s, C-7),
155.27 (s, C-5'), 154.77 (s, C-5), 152.70 (s, C-7'), 150.83 (s,
C-8a), 149.45 (s, C-8'a), 145.42 (s, C-11), 145.37 (s, C-11'),
145.04 (s, C-12), 144.47 (s, C-12'), 130.83 (s, C-9), 129.55 (s,
C-9'), 118.94 (d, C-14'), 118.46 (d, C-14), 114.96 (d, C-13),
114.34 (d, C-10), 114.25 (d, C-13'), 114.05 (d, C-10'), 105.16 (s,
C-8'), 102.66 (s, C-4a), 101.44 (s, C-4a'), 99.03 (s, C-2), 96.77
(d, C-6), 95.21 (d, C-8), 95.16 (d, C-6'), 82.50 (d, C-2'), 67.00
(d, C-3'), 66.27 (d, C-3), 27.86 (d, C-4), 27.79 (t, C-4').
[0032] MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide) and DMSO (dimethyl sulfoxide), SASP (sulsafalazine), LPS
(lipopolysaccharide, L3129), Griess reagent and all chemicals used
were of HPLC grade from Sigma Chemical Co. (St. Louis, Mo., USA).
.sup.1H NMR and .sup.13C NMR spectra were recorded on Bruker-Avance
400 MHz spectrometer, and CD.sub.3OD was used as solvent. Chemical
shifts (.delta.) were reported in ppm with tetramethylsilane as an
internal standard, and J values were given in Hz. High resolution
mass spectra (HRMS) were performed on a VG Autospec-3000
spectrometer. Column chromatography was performed with HSCCC (high
speed countercurrent chromatography), and the preparative HPLC were
used. A waters 2535 Series machine equipped with Alltech
Alltima-C.sub.18 (4.6.times.250 mm, 5 .mu.m) was used for HPLC
analysis, and the preparative Alltech Alltima-C.sub.18
(10.times.250 mm, 5 .mu.m) was used in sample preparation. DSS
(molecular weight: 36 to 50 kDa) was purchased from MP Biomedical
(Santa Ana, Calif., USA). RPMI 1640 medium, Dulbecco's modified
Eagle's medium (DMEM), FBS, penicillin and streptomycin were
purchased from Life Technologies (Carlsbad, Calif., USA).
[0033] Extraction and Isolation
[0034] Air-dried pieces of Lasia spinosa (L.) Thwait root (500 g)
were extracted three times with 75% EtOH (2 L) by reflex extraction
at 60.degree. C. The ethanol extract was concentrated to obtain a
residue (23.1 g) which was then suspended in water (100 mL) in a
separatory funnel prior to be partitioned by different solvents
(3.times.100 mL). The n-butyl alcohol layer (3.5 g, 0.7%) was
chromatographed on silica gel column (semi-preparative column
Preparative RP-C.sub.18) to obtain compound 1 (PA1, 6.16 mg).
[0035] Cell culture
[0036] Murine RAW264.7 macrophages and human esophageal cancer cell
lines KYSE-70, KYSE-150, KYSE-410, KYSE-520 were maintained in RPMI
1640 or DMEM medium supplemented with 100 U/mL penicillin, 100
.mu.g/mL streptomycin and 10% FBS in an incubator at 37.degree. C.
with a humidified atmosphere of 5% CO.sub.2. Cells were
sub-cultured every three days at a dilution of 1:6.
[0037] Cytotoxicity Assay
[0038] In the present invention, PA1 was dissolved in dimethyl
sulfoxide (DMSO) to make stock solutions which were further diluted
in culture medium for this experiment. Cells were seeded in a
96-well plate (3.times.10.sup.3 cells/well) and allowed to attach
to the plate overnight. After the recovery, cells were treated with
1.56, 3.125, 6.25, 12.5, 25, 50 .mu.g/mL of PA1 in culture medium
for 48 h. Then, 20 .mu.L of MTT (5 mg/mL stock in PBS) per well was
added into the medium (200 .mu.L) and incubated for 4 h at
37.degree. C. Finally, the culture medium was removed and 200 .mu.L
of DMSO were added to dissolve the purple formazan crystals.
Absorbance of the solution was measured using a microplate reader
spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, Calif.) at
a wavelength of 570 nm.
[0039] Wound Healing Assay
[0040] 6.times.10.sup.4 cell/well were seeded in 12 well-plate at
30% confluence in completed medium. After 24 hours seeding, the
monolayer was wounded by scoring with a sterile plastic tip (1 mL),
then washed twice with PBS to remove cell debris and then incubated
in a conditioned medium in the absence or presence of PA1 at 6.25
.mu.g/mL for various periods of time up to 72 hours. Cell migration
into the wound surface was monitored by Olympus IX71 microscopy and
digitally photographed.
[0041] Nitric Oxide Production Determination
[0042] Nitric oxide (NO) production was indirectly assessed by
measuring the nitrite levels in the cultured medium determined by a
colorimetric method based on the Griess reagent. The cells were
co-treated with various concentrations of PA1 in the absence or
presence of LPS (100 ng/mL) at 37.degree. C. for 24 hours. Then,
100 .mu.L of each supernatant was mixed with the equal volume of
Griess reagent incubated at room temperature for 15 minutes;
meanwhile, sodium nitrite was used as a standard substance. The
optical densities were measured at 540 nm with a micro-plate
reader.
[0043] Animals
[0044] 7-week-old male C57BL/6 mice weighing about 20-22 g were
purchased from the Laboratory Animal Services Center, the Chinese
University of Hong Kong. The study protocols were approved by the
committee for Care of Laboratory Animals in the School of Chinese
Medicine at the Hong Kong Baptist University.
[0045] Induction of Chronic DSS Colitis and Treatment
[0046] The animals were randomly assigned into five groups (n=8).
Mice in the control group were supplied with distilled water
whereas all other experimental groups were given 2.0% (w/v) dextran
sulphate sodium (DSS) for 5 days. Thereafter, the mice of the DSS,
SASP-treated (200 mg/kg/day) and PA1 (10 or 30 mg/kg/day)-treated
groups were administered by gavage with saline, SASP or PA1 from
day 6 to day 13, respectively.
[0047] Human equivalent dosage is translated from mouse dosage
using the following equation: D.sub.human=D.sub.mouse.times.k
(k=0.081) (Regan-Shaw et al. (2007)). Disclosure thereof is
incorporated herein by its entirety.) Therefore, the human
equivalent dosage is in a range of 0.81 mg/kg/day to 2.43
mg/kg/day.
[0048] Evaluation of Disease Activity Index (DA1)
[0049] The DAI was determined by scoring changes in the body
weight, diarrhea, colon length, and bleeding. Each score was given
in Table 1.
TABLE-US-00001 TABLE 1 Disease activity index scores based on
disease marker intensities Body weight Rectal Score loss (%)
Diarrhea bleeding 0 <1 normal none 1 1-5 2 6-10 Loose stools
slight 3 10-15 4 >15 diarrhea fecal blood
[0050] Histological Analysis
[0051] The colons were dissected longitudinally, gently washed with
ice-cold phosphate buffer saline (PBS), fixed in 4%
paraformaldehyde overnight, and embedded in paraffin. Each of
five-micro-meter sections was stained with hematoxylin/eosin
according to a standard procedure to evaluate colonic damage. The
histological scoring system was shown in Table 2.
TABLE-US-00002 TABLE 2 Histological scoring system for DSS-induced
colitis scoring of severity of histological damage Score
Description 0 none 1 mild 2 moderate 3 severe 0 none 1 mucosa 2
mucosa and submucosa 3 transmural 0 none 1 1/3 damaged 2 2/3
damaged 3 crypt loss by surface epithelium present 4 Both crypt and
surface epithelium lost
[0052] Determination of Neutrophil Infiltration in Colon Tissue
(MPO Assay)
[0053] Myeloperoxidase (MPO) is an enzyme mainly released by
neutrophil, and its activity is directly associated with the
severity of inflammation in a given tissue. In the present
invention, MPO activity was measured as described in the inventor's
earlier publication (Mu, H. X., et al., Anti-inflammatory Actions
of (|)-3'alpha-Angeloxy-4'-keto-3',4'-dihydroseselin (Pd-Ib)
against Dextran Sulfate Sodium-Induced Colitis in C57BL/6 Mice. J
Nat Prod, 2016). The results were normalized to the wet weight of
colon tissue and quantified as units/g tissue.
[0054] Discussion
[0055] In the present invention, the n-butyl alcohol extract of
root of Lasia spinosa (L.) Thwait showed potential anti-cancer and
anti-inflammatory activities. The inventors purified the active
fraction using a column chromatography of the preparative HPLC to
obtain a proanthocyanidin.
[0056] To determine the cytotoxic effect of PA1 against human
esophagus cancer cells (KYSE-70, KYSE-150, KYSE-450 and KYSE-520
cell lines), MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)
assay was performed.
[0057] Furthermore, proanthocyanidin A1 (PA1), was found to be more
cytotoxic against KYSE-450 cells than other cell lines (Table
3).
TABLE-US-00003 TABLE 3 Cytotoxicity of compounds against cancer
cell lines Compound KYSE-150 KYSE-70 KYSE-520 KYSE-450 PA1 -- -- --
48.64 Results are expressed as IC.sub.50 values in .mu.g/mL
[0058] In addition, wound-healing assay was used to test whether
those compounds could affect cell motility. A confluent monolayer
of KYSE-150 cells was scratched to form a wound and incubated in
the absence or presence of PA1 at 6.25 .mu.g/mL. The results
obtained were compared to that of controls. After 72 hours, the
wound edges were indistinguishable in control group, while
compound-treated cells did not migrate into the wound (FIG. 2),
indicating that PA1 had an anti-migration effect on KYSE-150 cells
in the absence of cytotoxicity.
[0059] The effect of PA1 on nitric oxide (NO) production was
investigated using Griess reagent. As shown in FIG. 3, stimulation
with LPS (100 ng/mL) resulted in a significant increase in NO
production compared with the control group while treatments with
PA1 at different concentration led to significant inhibition of NO
production.
[0060] To determine the role of PA1 in inflammatory bowel disease
(IBD), a DSS-induced colitis mouse model was used. In the present
invention, PA1 treated mice showed reduced body weight loss and had
a more rapid recovery that those mice exposed to 2% DSS in drinking
water for 5 days (FIG. 4B). DAI scores indicated that PA1 also led
to a clinical improvement in DSS-induced colitis (FIG. 4C). In
addition, as another important symptomatic parameter in DSS-induced
colitis, shortening of colon length, was rectified after
administration with PA1 (FIG. 4D and 4E). Mortality decreased in
the PA1 treated group, and the results are consistent with the
reduction in mucosal ulceration, crypt damage, edema, and cell
infiltration into mucosal tissue when compared with the DSS group
(FIG. 4A and FIG. 5). Enzyme myeloperoxidase (MPO) reflects the
neutrophil infiltration in colon tissue damage. PA1 treatment was
found to significantly lower the MPO activity (FIG. 6).
[0061] In this invention, PA1 may ameliorate inflammation in
colitis and exert significant anti-tumor invasion activity in
esophageal cancer cell line.
[0062] In summary, PA1 can be developed as a novel therapeutic
agent of colitis and cancer.
INDUSTRIAL APPLICABILITY
[0063] The present invention discloses a chemical entity isolated
from natural sources for its therapeutic and medical uses. More
particularly, it relates to compound that is naturally occurring in
the plant of Lasia spinosa Linn. Thwait (Araceae) and its
biological activity of treating an inflammatory disease or as an
anti-cancer leading compound.
[0064] If desired, the different functions discussed herein may be
performed in a different order and/or concurrently with each other.
Furthermore, if desired, one or more of the above-described
functions may be optional or may be combined.
[0065] While the foregoing invention has been described with
respect to various embodiments and examples, it is understood that
other embodiments are within the scope of the present invention as
expressed in the following claims and their equivalents. Moreover,
the above specific examples are to be construed as merely
illustrative, and not limitative of the reminder of the disclosure
in any way whatsoever. Without further elaboration, it is believed
that one skilled in the art can, based on the description herein,
utilize the present invention to its fullest extend. All
publications recited herein are hereby incorporated by reference in
their entirety.
* * * * *