U.S. patent application number 09/859463 was filed with the patent office on 2005-07-28 for chemical compounds containing a superoxide scavenger and an organic nitrate or nitrite moiety.
This patent application is currently assigned to TEIJIN LIMITED. Invention is credited to Imaizumi, Atsushi, Naughton, Declan P., Sumi, Yoshihiko, Zhang, Zhi.
Application Number | 20050165094 09/859463 |
Document ID | / |
Family ID | 10852643 |
Filed Date | 2005-07-28 |
United States Patent
Application |
20050165094 |
Kind Code |
A1 |
Zhang, Zhi ; et al. |
July 28, 2005 |
Chemical compounds containing a superoxide scavenger and an organic
nitrate or nitrite moiety
Abstract
Compounds for use in the treatment of heart disease include a
superoxide scavenger and an organic nitrate or nitrite moiety. The
compounds can be represented by the formula (A)n(B)m, in which A is
a superoxide scavenger, B is an organic nitrate or organic nitrite
moiety, and n and m are values between 1 and 8. These compounds do
not suffer from the problem of patient tolerance that is associated
with the use of conventional agents such as organic nitrates.
Inventors: |
Zhang, Zhi; (London, GB)
; Naughton, Declan P.; (Brighton, GB) ; Sumi,
Yoshihiko; (Tokyo, JP) ; Imaizumi, Atsushi;
(Tokyo, JP) |
Correspondence
Address: |
SUGHRUE, MION, ZINN, MACPEAK & SEAS, PLLC
2100 Pennsylvania Avenue, N.W. Suite 800
Washington
DC
20037-3213
US
|
Assignee: |
TEIJIN LIMITED
|
Family ID: |
10852643 |
Appl. No.: |
09/859463 |
Filed: |
May 18, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09859463 |
May 18, 2001 |
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09561910 |
May 1, 2000 |
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6346634 |
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Current U.S.
Class: |
514/509 ;
552/293 |
Current CPC
Class: |
A61K 47/52 20170801;
A61P 9/00 20180101 |
Class at
Publication: |
514/509 ;
552/293 |
International
Class: |
A61K 031/21; C07C
023/04 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 30, 1999 |
GB |
9910104.0 |
Claims
1. A compound comprising a superoxide scavenger and an organic
nitrate or nitrite moiety.
2. A compound according to claim 1, which is represented by formula
(I): (A)n(B)m (I) where A is a superoxide scavenger, B is an
organic nitrate or organic nitrite moiety, n and m are values
between 1 and 8.
3. A compound according to claim 2, wherein in formula (I), n and m
are integers.
4. A compound according to claim 3, wherein the values of n and m
are both 1.
5. A compound according to claim 2 wherein A and B are stably
linked.
6. A compound according to claim 2, wherein said organic nitrate or
nitrite moiety forms nitric oxide in the body of an animal.
7. A compound according to claim 6, wherein the nitric oxide is
formed by enzymatic conversion of said organic nitrate or nitrite
moiety by endogenous enzymes in the body of an animal.
8. A compound according to claim 7, wherein said enzymatic
conversion is by xanthine oxidase.
9. A compound according to claim 6, wherein the superoxide
scavenger remains effective in trapping superoxide upon enzymatic
conversion of the organic nitrate or nitrite moiety to form nitric
oxide
10. A compound according to claim 2, wherein the superoxide
scavenger is a low molecular mass superoxide dismutase analog.
11. A compound according to claim 2, wherein the superoxide
scavenger is a spin trap capable of trapping superoxide.
12. A compound according to claim 2 wherein the superoxide
scavenger contains one or more thiol groups.
13. A compound according to claim 2, wherein said superoxide
scavenger is linked to the organic nitrate or nitrite moiety by a
linkage that is stable S under physiological conditions.
14. A compound according to claim 13, wherein said linkage is a
thiol linkage.
15. A compound having the formula 1
16. A composition comprising a compound according to claim 2 in
conjunction with a pharmaceutically-acceptable excipient.
17. A method of treating heart disease comprising administering a
compound according to claim 2 in a therapeutically effective amount
to a patient in need thereof.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compounds suitable for use
in the treatment of heart disease. These compounds do not suffer
from the problem of patient tolerance that is associated with the
use of conventional agents such as organic nitrates.
BACKGROUND OF THE INVENTION
[0002] Organic nitrates and nitrites have been widely prescribed
for the prophylactic treatment of angina for over 100 years. More
recently, these drugs have been extended to manage coronary artery
disease, acute myocardial infarction and congestive heart failure
(Parker & Parker, (1998) N. Engl. J. Med. 338; 520-531).
Examples of such drugs include glyceryl trinitrate (GTN),
1,2-glyceryl dinitrate, 1,3-glyceryl dinitrate, isosorbide
dinitrate, isosorbide-2-mononitrate and
isosorbide-5-mononitrate.
[0003] The primary action of organic nitrates is vasodilation,
which is attributable primarily to nitrate-induced relaxation of
vascular smooth muscle in veins, arteries, and arterioles. Organic
nitrates are converted in the body to endothelium-derived relaxing
factors (EDRFs), which act to dilate vascular smooth muscle and to
inhibit platelet aggregation by activating guanylyl cyclase and
increasing intracellular cyclic-3',5'-guanosine monophosphate
(cGMP). This represents the cellular basis for the vasodilatory
action of organic nitrates.
[0004] Organic nitrate administration has been used as a means of
providing an exogenous source of EDRF that may help replenish or
restore endogenous EDRF levels that are usually impaired in
patients with coronary artery diseases such as atherosclerosis.
[0005] Discovered to be an EDRF, nitric oxide (NO) is an important
endogenous modulator of vascular tone (Ignarro et al., (1987) Proc.
Natl. Acad Sci USA 84: 9265-9269; Palmer et al, (1987) Nature 327:
524-526). A great deal of interest has been shown in the in vivo
metabolism of organic nitrates to produce NO. However, the cellular
mode of action of organic nitrates, in particular, the details of
nitrate to NO bio-transformation, still remain unclear. It has been
suggested that bio-transformation of organic nitrates to NO is a
thiol-dependent enzymatic denitration process catalyzed by
glutathione-s-transferase and the cytochrome P450-NADPH cytochrome
P450 reductase system (Bennette et al., (1994) Trends Pharmaciol.
Sci. 15; 245-249). However, it has since been discovered that
glutathione-s-transferase catalyzes the reduction of organic
nitrate to nitrite, and does not catalyze the reduction of nitrite
to NO.
[0006] The major problem with nitrate therapy is the rapid
development of tolerance and cross-tolerance during repeated dosing
with these agents (Parker & Parker, 1998). Clinically,
intermittent dosing regimens that allow for a drug-free interval
represent the only practical and effective strategy for avoiding
nitrate tolerance. Clearly, the need to interrupt drug
administration regularly reduces the effectiveness of this form of
therapy.
[0007] Nitrate tolerance is believed to be a complex
multi-factorial phenomenon, and the underlying mechanism of organic
nitrate tolerance is poorly understood. One possible route of
nitrate tolerance is due to a relative depletion of sulfhydryl
groups required for bio-conversion of organic nitrates to NO. More
recently it has been suggested that enhanced vascular superoxide
Production from endothelium plays an important role in this
phenomenon (Munzel et al., (1995) J. Clin. Invest. 95: 187-194;
Rajagopalan et al, (1996) J. Clin. Invest. 97: 1916-1923).
[0008] There thus remains a great need for compounds that are
effective in the body as vasodilators and which may be administered
continuously for a sustained period of time without suffering a
reduction in efficacy due to development of patient tolerance.
SUMMARY OF THE INVENTION
[0009] According to the present invention there is provided a
compound comprising a scavenger of superoxide and an organic
nitrate or nitrite moiety. Such compounds are effective
vasodilators, yet do not exhibit the problems of patient tolerance
to nitrates, from which conventional vasodilatory agents
suffer.
BRIEF DESCRIPTION OF THE DRAWING
[0010] The Figure shows a reaction scheme for preparing a
superoxide scavenger-organic nitrate ester.
DETAILED DESCRIPTION OF THE INVENTION
[0011] The advance that led to the development of the compounds of
the invention is based on the inventors' observation of a novel
molecular mechanism of the bio-conversion of organic nitrate to NO
by xanthine oxidase (XO). The XO enzyme is a homodimer of 150 kDa
subunits, and contains four oxidation and reduction centers, one
molybdenum cofactor, one flavin adenine dinucleotide (FAD) and two
[Fe.sub.2S.sub.2] clusters. XO catalyzes the oxidative
hydroxylation of a range of aromatic heterocyclic compounds of
which the most notable are hypoxanthine and xanthine. During the
process of purine metabolism, XO catalyzes the two-step oxidation
of hypoxanthine, through xanthine, to uric acid. The oxidation of
hypoxanthine or xanthine is concomitantly accompanied by the
reduction of oxygen to form superoxide and H.sub.2O.sub.2 (see
Table 1, reaction scheme I).
1TABLE 1 Reactions catalyzed by XO Xanthine oxidase XH + H.sub.2O +
O.sub.2 .fwdarw. X.dbd.O + H.sub.2O.sub.2 + O.sub.2.sup..cndot.- I
activity NADH oxidase 2NADH + 2O.sub.2 .fwdarw. NAD +
O.sub.2.sup..cndot.- + H.sub.2O.sub.2 II activity Nitrate reductase
2NO.sub.3.sup.- .fwdarw. NO.sub.2.sup..cndot. + O.sub.2 III
activity Nitrite reductase 2NADH + 2NO.sub.2.sup..cndot. .fwdarw.
2NAD + H.sub.2O.sub.2 + 2NO.sup..cndot. IV activity
[0012] Despite being a reducing agent that is itself capable of
causing significant damage to biomolecules, such as by initiating
lipid peroxidation, superoxide is considered to be the most
important source of oxidative stress. It can be rapidly converted
to the highly toxic hydroxyl radical via the Fenton reaction or the
Haber-Weiss reaction. It can also react rapidly with NO to form
deleterious diffusion-controlled peroxynitrate (Beckman et al.,
(1990) Proc. Natl. Acad. Sci. USA 87; 1620-1624). Both hydroxyl
radicals and peroxynitrate have been shown to initiate lipid
peroxidation, protein and enzyme inactivation and DNA
fragmentation. On this basis, the classical pathway of superoxide
production by XO (see Table 1, reaction scheme I) has been
implicated as constituting a major role in a number of pathogenic
conditions, such as atherosclerosis, hypercholesterolaemia,
diabetes mellitus and rheumatoid arthritis.
[0013] In particular, the role of XO in the generation of excess
superoxide during hypoxic reperfusion injury has received a great
deal of attention (McCord J. M. (1985) N Engl. J. Med. 312:
159-163). During ischaemia, endogenous xanthine dehydrogenase is
converted to XO. Concomitantly, hypoxanthine and xanthine are
accumulated as a consequence of ATP breakdown. The reperfusion
phase following ischaemia allows the XO to use accumulated
hypoxanthine or xanthine together with oxygen to produce a burst of
tissue-damaging superoxide and H.sub.2O.sub.2.
[0014] In addition to the above-described classical reaction of XO,
early studies have shown that XO can use NADH as a reducing
substrate, possibly binding at a site different from that at which
xanthine binds. However, this NADH oxidase activity of XO is not
generally recognized and has been little studied over the
years.
[0015] Several recent studies have suggested that endothelium and
vascular smooth muscle contain membrane-bound NADH oxidase enzymes
that use NADH as a substrate to produce superoxide (Sanders et al.,
(1997) Eur. J. Biochem. 289: 523-527). The inventors' previous
research and that of others has demonstrated that human XO can use
not only hypoxanthine or xanthine (Table 1, reaction scheme I) but
also NADH (Table 1, reaction scheme II) as a substrate to generate
superoxide (Zhang et al., (1998) Free Rad. Res. 28; 151-164).
[0016] This NADH-oxidizing activity of XO is blocked by
diphenyleneiodonium (DPI) but is not suppressible by the
conventional xanthine-based inhibitors, such as allopurinol,
oxypurinol, BOF-4272 and Amfiutizole. Therefore, apart from the
xanthine-based free radical-generating pathway, the NADH oxidase
activity of XO may operate as an additional pathway to produce free
radicals. In other words, XO can contribute to tissue damage
depending on which substrate is available in pathological
situations.
[0017] The third and less well-known phenomenon by XO is that this
enzyme is capable of catalyzing the reduction of nitrate to nitrite
under anaerobic conditions (Table 1, reaction scheme III). Although
the nitrate reductase activity of XO has long been known (Fridovich
and Handler (1962) J. Biol. Chem., 237: 916-921), it has been
little studied and is not generally recognized. This property of
the enzyme is shared with the assimilatory nitrate reductase of
bacteria, algae and fungi (Payne et al., (1997) BioFactors 9: 1-6).
Both enzymes are molybdoenzymes containing FAD redox centers and
utilizing NAD(P)H as a reducing substrate to catalyze the reduction
of nitrate to nitrites.
[0018] In a recent paper, the inventors investigated the possible
mechanism of nitrite reductase activity of XO by directly detecting
NO formation (Zhang et al., (1998) Biochem. Biophys. Res. Commun.
249; 767-772). It was found that XO catalyzes the reduction of
nitrite to NO with NADH as a source of electrons (Table 1, reaction
scheme IV). This reductive reaction occurs regardless of
environmental oxygen tension, i.e. XO can reduce nitrite in both
the presence or absence of oxygen once an electron donor is
available. By using two different site-directed XO inhibitors,
allopurinol and DPI, it was found that the reduction of nitrite
takes place at the molybdenum center of XO, while NADH is oxidized
at the FAD center. This reaction pathway may play a very important
role in redistribution of blood flow to ischaemic tissue by virtue
of the vasodilatory effect of NO, since conventional NO synthesis
by nitric oxide synthase (NOS) is impaired under ischaemic
conditions.
[0019] More importantly, the nitrate and nitrite reductase
activities of XO has allowed the inventors to develop a novel
explanation for the bio-transformation of organic nitrates to NO in
organic nitrate and nitrite therapy of angina pectoris. Indeed, it
has been found that XO catalyzes not only the reduction of nitrite
to NO, but also the reduction of organic nitrates such as glyceryl
trinitrate to NO.
[0020] During the catalytic reduction of nitrite at the molybdenum
center of XO, oxidation is concomitantly required at the FAD site
using NADH as its electron donor. In the absence of oxygen, this
reaction will only generate NO from the molybdenum center. However,
in the presence of oxygen, XO exhibits not only nitrite reductase
activity (Table 1, reaction scheme IV) but also NADH oxidase
activity (Table 1, reaction scheme II). Thus, oxygen will act as an
electron acceptor along with nitrite and produce superoxide.
Although NO and superoxide may be generated at different redox
sites, it is proposed that the simultaneous production of both
radicals by the same XO enzyme will result in the formation of
peroxynitrate and decrease the net production of NO formation by
XO. Increased vascular superoxide production during continuous
dosing inactivates NO by forming peroxynitrate, which consequently
inhibits NO-mediated vasorelaxation produced by organic
nitrates.
[0021] Peroxynitrate itself is a non-selective and extremely
reactive ion. Its deleterious function inactivates or modifies not
only large molecules such as metalloenzymes, but also small
molecules, for example, thiols. The formation of peroxynitrate is
thus proposed to lead to both the inactivation of XO and the
depletion of thiols at one specific enzyme site. The inventors
propose that this inactivation, together with the mopping-up effect
of superoxide on NO explains the hitherto unsolved phenomenon of
nitrate tolerance.
[0022] As a result of these observations on XO, novel compounds
have been designed that are generators of organic nitrates. These
compounds are hybrid molecules, comprising both a superoxide
scavenger and a generator of NO. The new agents may be expected to
diminish nitrate tolerance by at least two mechanisms: (1) the
reduction of NO and superoxide interaction and (2) the reduction in
superoxide-mediated thiol depletion, by scavenging superoxide
produced by NADH oxidation.
[0023] In the present invention, the organic nitrate or nitrite
moiety forms nitric oxide in the body of an animal. The organic
nitrate or nitrite moiety should be converted metabolically to NO
by endogenous enzymes in the animal body. By animal is meant any
animal, although mammals, preferably humans, are considered to be
the most appropriate patients for therapy of heart disease. In
accordance with the conclusions discussed above, a principle
mechanism by which the organic nitrate or nitrite moiety is
proposed to be converted to NO is by action of XO in the body.
However, any mechanism of metabolic conversion of organic nitrate
or nitrite to NO is compatible with the method of action of the
compounds of the invention, and any mechanism of metabolic
conversion of organic nitrate or nitrite to NO is compatible with
the conversion of the compounds of the invention to NO. In
addition, the proximity of the hybrid anti-oxidant scavengers can
avert reactive oxygen species-mediated NO consumption or further
production of deleterious species.
[0024] The superoxide scavenger portion of the compounds of the
invention may be any one of a large number of compounds that are
known to be effective scavengers of superoxide. Examples include
spin-traps such as DMPO, low molecular mass superoxide dismutase
(SOD) analogs such as the Cu(salicylate).sub.2 complex, redox
active transition metal complexes such as FeIII EDTA, zinc
carnosine complexes, antioxidants such as desferrioxamine and
substrates for direct redox reactions with superoxide such as
cytochrome-c and vitamin E. Preferably, the superoxide scavenger is
a spin trap that is capable of trapping superoxide. One or more
thiol moieties may form part of the superoxide scavenger.
[0025] Linkage of the superoxide scavenger to the organic nitrate
or nitrite moiety may be through any one of a number of known
chemical bonds including ester, amide and ether bonds. Preferably,
the linkage itself is resistant to enzymatic degradation. Linkage
via a thiol ester is considered to be particularly preferable,
since the use of this linkage will reduce the thiol load that is
necessary for activation of the organic nitrate moiety in the body.
The use of this linkage will thus further minimize tolerance of a
patient to organic nitrate-derived NO.
[0026] According to a further aspect of the invention, there is
provided compound according to formula I:
(A).sub.n(B).sub.m (I)
[0027] where A is a scavenger of superoxide and B is an organic
nitrate or organic nitrite moiety. "m" and "n" may be any number,
although it is envisaged that the values of m and/or n will not
generally exceed about 8. Desirably, m and n are values between 1
and 8. Preferably, m and n are integers. Particularly preferably,
the values of m and n are both 1.
[0028] Preferably, A and B are stably linked. By "stably linked" is
meant that the chemical bond that joins the superoxide scavenger
moiety and the organic nitrate or nitrite is stable to degradation
under physiological conditions. This increases the therapeutic
capacity of the compound, since it will not be inappropriately
broken down by the metabolism of the body to form compounds that
are not functional as either superoxide scavengers or generators of
NO. A preferable linkage is a thiol linkage. Ideally, the linkage
should also be stable at room temperature and pressure, to increase
the shelf-life of the compound. Ideally, the linkage should also be
stable or stabilizable at storage temperature.
[0029] In order that both the superoxide scavenger and
NO-generating functions of the compound are fully exploited, the
superoxide scavenger moiety should remain active in trapping
superoxide after conversion of the organic nitrate or nitrite to
NO.
[0030] A particularly preferred compound according to the invention
is the nitrate ester (5), the structure of which is illustrated in
the Figure. This compound is proposed to be particularly effective
in minimizing superoxide interaction with NO upon the conversion of
the organic nitrate moiety in the compound.
[0031] According to a further aspect of the invention, there is
provided a composition containing one or more compounds as
described above, in conjunction with a pharmaceutically-acceptable
excipient. Suitable excipients will be well known to those of
ordinary skill in the art and may, for example, comprise a
phosphate-buffered saline, a liquid such as water, saline, glycerol
or ethanol, optionally also containing mineral acid salts such as
hydrochlorides, hydrobromides, phosphates, sulphates and the like;
and the salts of organic acids such as acetates, propionates,
malonates and benzoates. Auxiliary substances such as wetting or
emulsifying agents and pH buffering substances may also be present.
A thorough discussion of pharmaceutically acceptable excipients is
available in Remington's Pharmaceutical Sciences (Mack Pub. Co.,
N.J. 1991).
[0032] According to a still further aspect of the invention there
is provided a compound or a composition as described above, for use
as a pharmaceutical. Such compounds or compositions are suitable
for the preventative or curative therapy of a wide variety of
pathological conditions associated with endothelial dysfunction, in
particular coronary artery diseases such as atherosclerosis and
hypertension, and rheumatoid arthritis, diabetes and
neurodegenerative diseases. The invention also embraces methods of
therapy of heart disease comprising administering to a patient an
effective amount of a compound or composition as described
above.
[0033] Various aspects and embodiments of the present invention
will now be described in more detail by way of example, with
particular reference to an antioxidant nitrate ester. It will be
appreciated that modification of detail may be made without
departing from the scope of the invention. All documents mentioned
in the text are incorporated herein by reference.
[0034] The Figure is a reaction scheme illustrating the preparation
of superoxide scavenger-organic nitrate ester (5), and is discussed
in greater detail below.
EXAMPLES
Example 1
Synthesis of Antioxidant Nitrate Ester
[0035] Step 1
[0036] A solution of 2 g (8 mmol) of optically pure
(R)-(+)-3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-carbo-
xylic acid (1) and 0.1 g of p-toluenesulphonic acid monohydrate in
40 ml methanol were stirred and re-fluxed for 4 hours. After
cooling, the solution was diluted with water, extracted three times
into ether that was subsequently washed with brine and aqueous
sodium bicarbonate solution. The ether solution was washed, dried
with MgSO.sub.4 and evaporated to give (R)-(+)-methyl
3,4-dihydro-6-hydroxy-2,5,7,8-tetrameth-
yl-2H-1-benzopyran-2-carboxylate (2).
[0037] Step 2:
[0038] To a stirred solution of 1.5 g (5.68 mmol) of (2) in 22 ml
ether, was added a solution of 4.5 g (16.6 mmol) of ferric
chloridehexahydrate in 17 ml of water and 17 ml of methanol. The
addition was carried out drop-wise over 30 minutes. After 1 hour
the ether layer was separated, and the aqueous phase was further
extracted with ether. The combined ether layers were
chromatographed on silica gel by flash chromatography using
toluene-ethyl acetate as the eluant to provide 1.39 g of the
oxidized quinone (R)-(+)-methyl
2-hydroxy-2-methyl-4-(3,4,5-trimethyl-3,6-
-dioxo-1,4-cyclohexadien-1-yl)butanoate(3).
[0039] Step 3:
[0040] A solution of 0.5 g of the tertiary alcohol (3) in dry
dichloromethane (20 ml) was charged 5 with SOCl.sub.2 (5 ml) and
stirred at room temperature for 30 minutes. The resulting solution
was evaporated in a vacuum and re-dissolved in ethyl acetate. This
procedure was repeated twice to remove residual SOCl.sub.2. The
chlorinated product (R)-(+)-methyl
2-chloro-2-methyl-4-(3,4,5-trimethyl-3,6-dioxo-1,4-cyclohe-
xadien-1-yl)butanoate (4) was used without further
purification.
[0041] Step 4:
[0042] To a stirring solution 0.1 g of (4) in dry acetonitrile at
room temperature was added one equivalent of silver nitrate
(AgNO.sub.3). A precipitate of silver chloride formed. The reaction
mixture was stirred for a further 30 minutes and was then filtered
and evaporated. The nitrate ester (5) was purified on silica gel
chromatography using CHCl.sub.2 and ethyl acetate as the
eluant.
Example 2
Exemplification of Protective Effect of Antioxidant
[0043] Buffered solutions containing an organic nitrate as follows
[all containing vitamin C between 1 and S equivalents]: (i) propyl
nitrate, (ii) propyl nitrate plus one equivalent of compound (3)
and (iii) compound (S) in the range of 1 to 10 .mu.mol were
prepared. These solutions were incubated in sealed glass vessels
with appropriate concentrations of XO and NADH under both hypoxic
and normoxic conditions. After 24 hours levels of NO were measured
by aspirating the glass vessels and monitoring NO concentration
directly. The results demonstrated the ability of superoxide
scavengers to minimize superoxide interaction with NO upon
activation of organic nitrates.
[0044] While the invention has been described in detail with
reference to specific embodiments thereof, it will be apparent to
one of ordinary skill in the art that various changes and
modifications can be made therein without departing from the spirit
and scope thereof.
* * * * *