U.S. patent application number 09/794292 was filed with the patent office on 2001-11-22 for desmethyl tocopherols for protecting cardiovascular tissue.
This patent application is currently assigned to OKLAHOMA MEDICAL RESEARCH FOUNDATION.. Invention is credited to Floyd, Robert A., Hensley, Kenneth L..
Application Number | 20010044462 09/794292 |
Document ID | / |
Family ID | 26882105 |
Filed Date | 2001-11-22 |
United States Patent
Application |
20010044462 |
Kind Code |
A1 |
Hensley, Kenneth L. ; et
al. |
November 22, 2001 |
Desmethyl tocopherols for protecting cardiovascular tissue
Abstract
The present invention involves the use of desmethyl tocopherols
such as gamma tocopherol for the protection of cardiovascular
tissue from nitrative stress. While mechanisms other than
scavenging of reactive nitrogen species may be involved, desmethyl
tocopherols exhibit significant protection and may be utilized to
treat or help prevent cardiovascular particularly arterial vascular
disease. The desmethyl tocopherols may be administered dietarily or
parenterally when a more direct dosage is desired. Both routes may
be utilized together or separately to optimize therapeutic and
prophylactic benefits. The lessening of damage induced by reactive
nitrogen species leads to the lessening of arterial blockage in
thrombosis.
Inventors: |
Hensley, Kenneth L.;
(Oklahoma City, OK) ; Floyd, Robert A.; (Oklahoma
City, OK) |
Correspondence
Address: |
Daniel S. Hodgins
HEAD, JOHNSON & KACHIGIAN
Ste. 3030
204 N. Robinson
Oklahoma City
OK
73102
US
|
Assignee: |
OKLAHOMA MEDICAL RESEARCH
FOUNDATION.
|
Family ID: |
26882105 |
Appl. No.: |
09/794292 |
Filed: |
February 27, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60186455 |
Mar 2, 2000 |
|
|
|
Current U.S.
Class: |
514/458 |
Current CPC
Class: |
A61K 31/355
20130101 |
Class at
Publication: |
514/458 |
International
Class: |
A61K 031/355 |
Claims
What is claimed is:
1. A method of delaying or preventing cardiovascular disease which
comprises the administration of an effective amount of at least one
desmethyl tocopherol.
2. A method of delaying or preventing symptoms and consequences of
cardiovascular disease which comprises the administration of an
effective amount of gamma tocopherol.
3. A method of inhibiting cardiovascular damage resulting from
thrombosis which comprises administration of an effective amount of
at least one desmethyl tocopherol.
4. The method of claim 1, or 2 wherein said cardiovascular disease
is atherosclerosis, coronary artery disease or ischemic injury.
5. A method of inhibiting cardiovascular damage resulting from
thrombosis which comprises the administration of an effective
amount of gamma tocopherol.
6. The method of claim 1, or 3 wherein said desmethyl tocopherol is
in pure form or in mixtures or formulations with drugs or other
antioxidants.
7. A method of preserving mitochondrial function in cardiovascular
tissue which comprises the administration of a safe and effective
amount of gamma tocopherol.
8. The method of claim 7 wherein said gamma tocopherol is
administered orally, intravenously or in cardioplegia fluid.
9. A method of preserving mitochondrial function in cardiovascular
tissue which comprises administration of an effective amount of at
least one desmethyl tocopherol.
10. The method of claim 9 wherein said desmethyl tocopherol is
administered orally, intravenously or incardioplegia fluid.
11. The method of any one of claim 1, 2, or 3 wherein the
tocopherol is used as a mixture of isomers.
12. The method of claim 1, 2, or 3 wherein the tocopherol is
isolated from natural products.
13. The method of claim 1, 2, or 3 wherein the tocopherol is
synthetically prepared.
14. The method of claim 1, 2, or 3 wherein the tocopherol is
administered as a prodrug.
15. The method of claim 1, 2, or 3 wherein the tocopherol is
administered as a water-soluble ester.
16. The method of claim 1, 2, or 3 wherein the tocopherol is orally
administered at a daily dose of from about 100 to about 400 mg.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Priority is claimed from provisional application U.S. Ser.
No. 60/186,455 filed on Mar. 2, 2000, and incorporated by reference
herein.
BACKGROUND
[0002] The present invention relates to concentrated preparations
of desmethyl tocopherols, including but not restricted to gamma
tocopherol (.gamma.T), which localize to lipid environments in
cardiovascular tissue and scavenge reactive nitrogen species (RNS)
by virtue of a phenolic structural element lacking one or more
methyl substituents on the phenolic ring system. The capability to
scavenge RNS imparts cardiovascular protective properties to the
compound.
[0003] Tocopherols are a class of lipophilic, phenolic compounds of
plant origin. The major tocopherol found in mammalian tissue is
alpha tocopherol (.alpha.-tocopherol or .alpha.T or vitamin E) FIG.
1, although significant quantities of demethylated (desmethyl)
forms (particularly .gamma.-tocopherol or .gamma.T) FIG. 1, are
also present. .alpha.-Tocopherol acts as a free radical scavenger
(i.e., a chain-breaking antioxidant) when the phenolic head group
encounters a free radical:
Toc-OH+L..fwdarw.Toc-O.+LH
[0004] Toc-OH=tocopherol
[0005] L.=lipidradical
[0006] The phenoxyl radical Toc-O. is much more stable, and less
reactive, than L.. The aromatic nature of the tocopherol ring
system, combined with steric and electronic influences from the
methyl
[0007] The phenoxyl radical Toc-O.. is much more stable, and less
reactive, than L.. The aromatic nature of the tocopherol ring
system, combined with steric and electronic influences from the
methyl substituents, stabilizes the tocopheroxyl radical and
thereby ends the lipid peroxidation process. Eventually, Toc-O. is
reduced back to Toc-OH by ascorbate acting in conjunction with
NADPH reductase. While .alpha.-tocopherol is the major tocopherol
in the body, other tocopherols exist. The second major tocopherol
in the human body is .gamma.-tocopherol (.gamma.T), which, like
.alpha.-tocopherol, is made by plants and taken into the human diet
with foodstuffs.
[0008] Recently, it has become appreciated that reactive nitrogen
species (RNS) are significant to many diseases including coronary
artery disease (CAD), hypertension, and other forms of
cardiovascular disease where localized inflammatory reactions
occur. RNS are derived from the enzymatic oxidation of arginine via
the intermediate nitric oxide free radical (FIG. 2). Unlike
oxygen-centered free radicals, reactive nitrogen species are not
scavenged effectively by .alpha.-tocopherol. On the other hand,
.gamma.-tocopherol can react easily with RNS because of the
presence of an open space on the chromanol head of the molecule
(FIG. 1). The major product of .gamma.-tocopherol reaction with RNS
is 5-nitro-.gamma.-tocopherol (5N.gamma.T, FIG. 1). Recent
discoveries indicate that: (A) .gamma.T protects biological systems
from RNS much more effectively than .alpha.T; (B) .gamma.T is
extensively nitrated in human plasma, particularly among smokers
and hypertensive individuals; (C) .gamma.T inhibits RNS toxicity to
a critical enzyme (.alpha.-ketoglutarate dehydrogenase, or
.alpha.KGDH) which is severely damaged in injured vascular tissue;
and (D) .gamma.T protects cultured endothelial cells from RNS.
Thus, .gamma.T possesses unique biochemical functions from .alpha.T
that suggest .gamma.T may be a superior dietary supplement,
cardioprotectant, cardioplegia additive, or a preservative in
cardiovascular tissue exposed to RNS. Other desmethyl tocopherols
likewise should be cardioprotective by this mechanism or
another.
[0009] Chemistry of Tocopherol Reaction with Oxidizing Agents.
[0010] .gamma.-Tocopherol is a natural product (a desmethyl
tocopherol) ofplant origin, present in many vegetable oils,
especially soybean oil (1-2). .gamma.-Tocopherol is normally taken
into the body through consumption of foodstuffs. Human plasma
.gamma.T concentration is variously reported as 5-30% of .alpha.cT
(3). The .gamma.T/.alpha.T ratio varies markedly among individuals;
plasma .gamma.T/.alpha.T proportionalities may be as low as 0.2%
and as high as 30% (inventors' observations). Both .alpha.T and
.gamma.T are absorbed equally well by the gut, but .gamma.T is
packaged into lipoproteins less effectively than .alpha.T (4). For
this reason, .alpha.T supplementation decreases systemic .gamma.T
concentration (3-4).
[0011] To date, only three well-disseminated studies have compared
.alpha.T and .gamma.T with respect to their ability to inhibit
nitrative stress specifically (5-7). These studies generally
investigated the in vitro reaction of nitrating equivalents with
target substrates in "pure" chemical systems, and two of the three
studies reached very different conclusions. The first investigation
from Cooney's lab (5) reported that .gamma.T reaction with NO.sub.2
gas was 6 times more rapid than the corresponding reaction of
.alpha.T. Furthermore, exposure of .alpha.T (but not .gamma.T) to
NO.sub.2 caused the formation of a secondary nitrating species
which could nitrate the target compound morpholine (5). In the same
manuscript, Cooney et al. showed that .gamma.T was 4-fold more
effective than .alpha.T at inhibiting neoplastic transformation of
methylcholanthrene-treated C3H/10T1/2 fibroblasts, a process which
the authors suggest might involve nitrative stress (5). The second
study (Christen et al. 1997; reference 6) incorporated either
.alpha.T or .gamma.T, or both, into liposomes which were then
exposed to synthetic peroxynitrite (ONOO.sup.-). Christen and
colleagues found that .gamma.T was twice as effective as .alpha.T
at inhibiting lipid hydroperoxide formation in liposomes exposed to
ONOO.sup.-. Moreover, these researchers found that .gamma.T
nitration rates were not influenced by the presence of .alpha.T.
This latter finding suggests that nitration of .gamma.T may occur
preferentially to reaction with .alpha.T when both tocopherols are
simultaneously exposed to a nitrating species. In the third study
(7), Goss et al. take issue with the findings of Christen et al.
and report that .alpha.T does spare .gamma.T in liposomes exposed
to the superoxide and NO-generating compound SIN-1
[5-amino-2-(4-morpholinyl)-1,2,3-oxadiaz- olium].
[0012] A search of the literature revealed only two studies in
which .alpha.T and .gamma.T were compared for efficacy using in
vivo models of cardiovascular stress (no studies were found
investigating neurological stress). In the first study (c. 1983),
tocopherol-depleted rats were fed .alpha.T or .gamma.T for two
weeks after chronic exposure to iron-dextran as an inducer of
oxyradical stress (8). While both .alpha.T and .gamma.T inhibited
systemic lipid oxidation in the animals, .gamma.T was approximately
35% as effective as .alpha.T. Lipid nitration was not an endpoint
of this investigation, and physiologic parameters were not
recorded. In a second, very recent study (reference 9; Saldeen et
al., J. Am. Coll. Card.,October 1999), rats on an otherwise normal
diet were fed .alpha.T or .gamma.T (100 mg/kg/day) for 10 days
after which the abdominal aorta was exposed to patch soaked in 29%
FeCl.sub.3 (9). This stress induced obstructive thrombus within 20
minutes. Saldeen et al. found that .gamma.T supplementation was
significantly more effective than .alpha.T supplementation at
inhibiting iron-induced lipid peroxidation and occlusive thrombus
(9). Time to occlusive thrombus was delayed by 25% in the .alpha.T
supplemented animals while the same parameter was increased by 65%
in .gamma.T supplemented animals (9). Platelet aggregation kinetics
were similarly inhibited, with .gamma.T supplementation being
2-fold more efficacious than .alpha.T supplementation (9). Most
importantly, the .gamma.T concentration in the plasma of the
.gamma.T supplemented rats never exceeded 10% of the .alpha.T
concentration although the feeding paradigm did increase .gamma.T
levels 6-fold above baseline (9). By comparison, .alpha.T
supplementation increased .alpha.T plasma concentration only 2-fold
(9). When treatment effects were considered in reference to plasma
tocopherol concentrations, the Saldeen study found .gamma.T to be
20-30 times more potent than .alpha.T at inhibition of throbogenic
correlates. No conclusive explanation for the .gamma.T effect was
offered by the Saldeen study, though superoxide dismutase activity
increased significantly in the aortas of .gamma.T treated animals
as compared to the .alpha.T treated group (9). The unexpected
efficacy of .gamma.T might also stem from a differential vascular
partitioning of .gamma.T, since .gamma.T is reportedly incorporated
into endothelial cells more rapidly than is .alpha.T (10). In any
case, the efficacy of .gamma.T as a vascular or neuroprotectant
cannot be predicted from its bioactivity in traditional fertility
assays, or from its oxyradical scavenging capacity as measured in
vitro.
[0013] Role of Oxidative and Nitrative Stress in
Atherosclerosis.
[0014] Oxidative stress is centrally involved with both the
initiation and the progression of atherosclerosis. In normal
vasculature, low density lipoprotein (LDL) crosses the endothelium
to provide lipids and cholesterol to the vascular intima. Normal
LDL is taken up in by specific cell-surface receptors whose
expression is tightly regulated so as to preclude intracellular
accumulation of cholesterol. Chemically-modified LDL, including
oxidized LDL (oxLDL), is taken up more rapidly and less
specifically, particularly by macrophages (11-22). The accumulation
of excessive oxLDL converts these into "foam cells", a hallmark of
early atherosclerosis (12). Oxidized lipids and proteins are
abundant in atherosclerotic lesions, though the specific nature of
the oxidative modifications is unclear (13-16). LDL can be oxidized
in vitro by exposure to metal-catalyzed oxyradical generating
systems, and this oxLDL will convert macrophages into foam cells
(13,16); however, this "synthetic" oxLDL differs from natural oxLDL
in several respects. Synthetic oxLDL is taken up by macrophage
scavenger receptors only after complete depletion of .alpha.-T
resident within the LDL particle (18). Natural oxLDL is not
recognized by the scavenger receptor, indicating that the chemical
modification of natural oxLDL is different from that of in vitro
modified LDL (17). Moreover, .alpha.-T content of native oxLDL is
not substantially depleted, even in extracts taken from severe
lesions (19). OxLDL is chemotactic and stimulates expression of
vascular adhesion molecules, thereby recruiting leukocytes to the
subendothelial space (14). Neutrophils and macrophages may become
activated in this milieu, releasing pro-inflammatory cytokines and
generating more ROS and reactive nitrogen species. Chronic exposure
to oxLDL causes macrophage and endothelial death and release of
lipids from the dying cells (reviewed in 20). Further leukocyte
recruitment to the necrotic focus accelerates the atherogenic
process. An additional consequence of subendothelial inflammation
is proliferation of VSM cells in response to cytokine exposure,
which further decreases perfusion through the affected vessel (20).
End-stage disease is characterized by ischemic damage to the heart
and major perfused organs, and with increased risk of occlusive
thrombus as portions of plaque disintegrate and initiate
coagulation cascades.
[0015] As previously discussed, the combination of NO with
superoxide or other leukocyte-derived oxidants yields peroxynitrite
and othernitrating agents. Furthermore, activated macrophages
produce profligate quantities of NO via iNOS (inducible nitric
oxide synthate). It therefore appears that NO-- derived products
play a role in vascular modification during atherosclerosis. iNOS
and nitrotyrosine have been immunochemically detected in human
atherosclerotic plaques, where most staining occurs in foam cells
and VSM cells (21-24). Some iNOS is present in VSM even in normal
vessel walls (24). Similar iNOS immunoreactivity is found in
experimental atheroscerotic lesions of hypercholesterolemic rabbits
(25). Endothelial cells express very little iNOS in vivo or in
vitro; however, the endothelium is likely to encounter nitrating
agents derived from other cell types. Combination of eNOS
(endogenous nitric oxide synthate)-derived NO with
leukocyte-derived ROS might also form peroxynitrite in the
subendothelial space. Quantitative mass spectrometric studies
indicate that LDL isolated from human atherosclerotic plaques
contains 100 times more nitrotyrosine than LDL from normal plasma
(26). Similar LDL protein nitration is observed in rabbits fed a
high cholesterol diet (27). Lipid nitration in atherosclerosis has
not been investigated.
[0016] .alpha.-Tocopherol in Human Cardiovascular Disease.
[0017] Considering the importance of lipoprotein oxidation in the
pathogenesis of atherosclerosis, it seems logical that
.alpha.-tocopherol should decrease the incidence or severity of
CAD. In the period from 1985-1995, numerous epidemiological,
cross-sectional and observational studies were undertaken to
determine if this might be the case. Initial studies using
relatively small populations (<100 subjects) failed to find a
correlation between .alpha.-T and vascular disease, although these
studies have been criticized for failure to normalize .alpha.T to
lipid content, which might confound the interpretation of the data
(28). A 1991 study by Gey et al. correlated ischemic heart disease
(IHD) rates with lipid-standardized .alpha.T concentrations using
mean values obtained from male populations in 16 European nations
(29). A highly significant negative correlation .RTM.=0.79) was
found between these parameters, indicating a beneficial role for
.alpha.T in IHD. The authors conclude that a 40% increase in plasma
.alpha.T was associated with an 84% lower mortality rate.
[0018] Data from cross-sectional and epidemiological human studies
generally support the contention that .alpha.T is protective
against vascular pathology, though perhaps not in all human
populations. In the hope of overcoming limitations inherent to
cross-sectional studies, several large-scale, longitudinal
investigations were undertaken in the early 1990s to formally test
the importance of .alpha.-T as a vasoprotectant. The "US Nurses'
Health Study" analyzed self-reported vitamin E intake among 87,425
American nurses over 8 years (30) and found a 34% diminished risk
of coronary disease among subjects within the upper quintile of
.alpha.T consumption compared to subjects within the lowest
quintile. In a similar study (the Health Professions Follow-up
Study) involving 39,910 men, the risk of CAD was diminished by 39%
for men with a median tocopherol intake of 419 vs. 6.4 IU/day (1
IU=1 mg d-.alpha.-tocopherol acetate; 31). From these studies, a
daily intake of 100 IU of .alpha.T is most consistently associated
with benefit (28) while the US Reference Daily Intake for vitamin E
is 15 IU/day (34). These several investigations did not
discriminate thoroughly between "dietary" versus "supplementary"
sources of .alpha.T, and no specific consideration was made of
.gamma.T or other co-antioxidants. In the one large study which has
attempted to discriminate between dietary vitamin E and vitamin
supplements, 35,000 postmenopausal women were followed for 7 years
(the "Women's Health Study", ref. 36). Cardiovascular death was
negatively associated with high intake of vitamin E from food,
while no benefit was apparent when vitamin E supplements were
evaluated alone or in combination with dietary vitamin E intake
(36).
[0019] Data from large-scale, prospective, controlled tocopherol
supplementation trials is currently being analyzed and published
with somewhat paradoxical results. Despite epidemiological evidence
that .alpha.-T correlates inversely with vascular disease,
controlled .alpha.T supplementation has a relatively subtle
protective effect against CAD and possibly a detrimental effect on
hemorrhagic pathology. In the Cambridge Heart Antioxidant Study
(CHAOS), 2002 male smokers with angiographically proved CAD
received vitamin E supplements of 400-800 IU/day and were followed
for 18 months (32). In this study, vitamin E supplements caused a
significant 77% reduction in nonfatal myocardial infarction but a
29% increase in all-cause mortality. In a similar study involving
1,862 male smokers with previous myocardial infarction, a 50 mg/kg
supplement of vitamin E had no effect on MI or mortality after 5.3
years of follow-up (33). An independent study reports that 50
IU/day of .alpha.T does not decrease total mortality of smokers but
increases death from hemorrhagic stroke after 5-8 years (35).
Interestingly, plasma .alpha.T increased 50% in this latter
supplementation paradigm, a quantity previously associated with an
80% reduction of ischemic heart disease in the cross-cultural
epidemiological study by Gey et al. (29). In the most recent
evaluation of .alpha.T, the Heart Outcomes Prevention Evaluation
(HOPE), Canadians at risk for heart disease were studied (38). In a
total population of 9541 subjects, 400 IU/day of "natural" .alpha.T
had no effect on primary or secondary cardiovascular outcomes or
death over a 4.5 year period (38). The quantitative discrepancies
between epidemiological data and intervention studies are
disturbing. The disparity may indicate that .alpha.T can inhibit
the development of CAD in the early stages but not in more advanced
clinical conditions. Alternatively, it has been suggested that
intake of .alpha.T from food is correlated with the intake of other
co-antioxidants which are required for maximal cardiovascular
benefit, and that current .alpha.T supplementation paradigms fail
to take into account these necessary "cofactors" (36-37). The
identity (identities) of these putative "cofactors" has not been
suggested.
[0020] .gamma.-Tocopherol in Human Biology and Cardiovascular
Disease.
[0021] Relative to .alpha.T, a dearth of epidemiological data
exists for .gamma.T. Human plasma.gamma.T concentration is
variously reported as between 5-30% that of .alpha.T (41). In
platelet-poor plasma, we find that 7% is very close to the correct
value in young healthy subjects. The .gamma.T/.alpha.T ratio varies
markedly among individuals; we have observed plasma
.gamma.T/.alpha.T proportionalities as low as 0.2% and as high as
30%. .gamma.T is now a major tocopherol in the US diet, due to the
high intake of soybean and vegetable oils that are abundant sources
of .sub.7T (40). Both .alpha.T and .gamma.T are absorbed equally
well by the gut, but .gamma.T is packaged into lipoproteins less
effectively than .alpha.T (39). For this reason, .alpha.T
supplementation decreases systemic .gamma.T concentration (41-42).
.delta.-Tocopherol, .beta.-tocopherol and tocol (other demethylated
tocopherol homologs) exist in human plasma at approximately 1:10
ratios relative to .gamma.T (41). Detailed demographic data
regarding plasma and tissue levels of desmethylated tocopherols and
their oxidation products have never been published. While extensive
data has been collected on .alpha.-tocopherol as a possibly
beneficial molecule in cardiovascular disease, very little data has
been collected on .gamma.T.
[0022] The small amount of published clinical data regarding
.gamma.-tocopherol is provocative. Two small studies have
investigated .gamma.T in CAD. A 1999 study reports a 40% decrease
in plasma .gamma.T in patients (N=34) with atherosclerosis while
.alpha.T increased by 30% (43). An earlier 1996 study by Ohrvall et
al. found that CAD patients (N=69) had a significant 25% reduction
in lipid-normalized plasma .gamma.T concentration while .alpha.T
was statistically unaffected (44). In the latter study, the ratio
of.gamma.T/.alpha.T in the CAD patients was decreased by 35% (44).
Importantly, Ohrvall et al. note that very few of the CAD patients
had supplemented their diet with vitamin preparations. Tocopherol
oxidation and nitration products were not measured in either study.
In a separate but very remarkable study of smokers (a group at high
risk for vascular disease), plasma .gamma.T levels were reduced by
more than 50% in chronic smokers while plasma .alpha.T
concentration was diminished by only 20-25% (45). Moreover,
cessation of smoking for 84 hours resulted in a 35% recovery of
.gamma.T in plasma and a 65% recovery of .gamma.T in low density
lipoprotein (LDL) while .alpha.T recovery was not significant.
Interestingly, the magnitude of .gamma.T rebound following
cessation of tobacco use correlated very strongly with the extent
of tobacco use preceding the period of voluntary abstinence (45).
Again, tocopherol oxidation and nitration products were not
measured. While several high-profile studies have shown .alpha.T
intake somewhat protective against CAD in smokers (45), no similar
studies have been undertaken using .gamma.T as an independent
variable.
[0023] While chronic .alpha.T supplementation can increase plasma
levels of .alpha.T by 300-400%, very little data exists regarding
the effect of dietary .gamma.T. To the knowledge of the P.I., no
serious attempt has been made to increase plasma .gamma.T in humans
in the context of a formal scientific study. Several small studies
using very small study populations have indicated that dietary
supplementation with .alpha.T decreases plasma .gamma.T in humans
and rodents (41-42), while chronic dietary supplementation of
.gamma.T might conceivably increase plasma and tissue .gamma.T
concentration. The human biology of other, less common
desmethylated tocopherols is essentially uninvestigated. It cannot
be assumed, however, that the relative importance of the various
tocopherols can be anticipated solely on the basis of their
relative tissue concentrations, independent of other biochemical
variables.
[0024] The present invention is intended to solve the problems
described above, namely, the inefficacy of .alpha.-tocopherol
(vitamin E) to adequately protect against cardiovascular disease in
clinical investigations, and to improve the ability of the
tocopherol to inhibit the progression of cardiovascular diseases
including but not limited to atherosclerosis. The mechanism of the
invention at least in part involves the improved ability of a
tocopherol desmethyl homolog to scavenge reactive nitrogen species
(RNS).
SUMMARY OF INVENTION
[0025] The present invention involves the use of .gamma.-tocopherol
and other desmethyl tocopherols as scavengers of reactive nitrogen
or other reactive species in tissue exposed to an inflammatory
stress, particularly in cardiovascular tissue exposed to nitrative
stress. The preferred desmethyl tocopherols of the present
invention have the following structures: 1
[0026] The only constraint placed on the structure above is that at
least one of the set R.sub.1, R.sub.2 and R.sub.3 must be a H atom.
Additionally, the alkyl (linear, branched, or cyclic) tail of the
molecule may include either saturated or unsaturated variants
(unsaturated variants comprising the chemical subclass of
tocotrienol tocopherols). Since the main bioactive function of the
above structure is the phenolic head group, any stereoisomer of the
tocopherol may be used. Furthermore, since the main bioactive
function of the above structure is the phenolic head group, any
carbon can be eliminated from the carbon centers labeled 2-4 in the
structure above. Furthermore, the --OH group can be esterified or
otherwise modified to form a prodrug or a more water-soluble
derivative such as an ester, for example, which would regenerate
the --OH group in vivo.
[0027] These and other homologs of the tocopherols can be
chemically synthesized or isolated from natural products. In the
method of the present invention, the tocopherols are administered
in a safe and effective amount to scavenge reactive nitrogen or
other species and slow the progression of nitrative stress in
tissue undergoing progressive degeneration. These and other
advantages and objects of the invention will be apparent to those
skilled in the art.
[0028] The present invention also involves a method for protecting
or delaying cardiovascular disease, its symptoms, consequences, or
related damage. Cardiovascular disease includes ischemia disease
(including thrombosis). Mitochondrial function of the myocardial is
likewise protected.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 shows tocopherol structures. Arrows indicate the 5
position of the chromanol ring system, which is methylated in
.alpha.-tocopherol (vitamin E) but not in .gamma.-tocopherol. This
structure difference allows .gamma.-tocopherol to scavenge RNS in a
manner that .alpha.-tocopherol cannot. The product of the
scavenging reaction is 5-nitro-.gamma.-tocopherol.
[0030] FIG. 2 shows pathways for generation of nitrating agents and
their subsequent reaction with phenolic substrates such as tyrosine
or .gamma.-tocopherol.
[0031] FIG. 3 is a graph showing rat brain mitochondria exposed to
0.4 mM SIN-1 for 1 H after addition of tocopherol.
(.circle-solid.).alpha.-tocop- herol; (.box-solid.)
.gamma.-tocopherol. The scale bars labeled [.alpha.].sub.norm and
[.gamma.].sub.norm. indicate the normal endogenous quantities of
.alpha.-tocopherol and .gamma.-tocopherol, respectively, in human
brain.
[0032] FIG. 4 is a graph showing tocopherol effects on .alpha.XKGDH
in vivo. Rats were injected with 15 mg of tocopherol over the first
1 month of life; specific groups were then challenged with a septic
dose of LPS and heart .alpha.KGDH activity was assayed 24 H later.
*p<0.1 relative to vehicle; .dagger.p<0.05 relative to
vehicle. N=5/group.
[0033] FIG. 5 is a graph showing influence of .alpha.-toc and
.gamma.-toc on viability of ECV034 endothelial cells exposed to
SIN-1 as assessed by ability of viable cells to reduce the
tetrazolium dye MTT>N=5 experiments. *P<0.5 relative to cells
which had received SIN-1 only.
[0034] FIG. 6 is a graph showing nitration of plasma
.gamma.-tocopherol in vivo during FeCl.sub.3-induced thrombosis in
the rat. *Significant increase above baseline values (i.e., before
initiation of thrombosis; p<0.05, N=5).
DETAILED DESCRIPTION OF THE INVENTION
[0035] The present application demonstrates the superiority of
desmethyl tocopherols, exemplified by gamma tocopherol, as
protectors against nitrative damage to biological systems. The
results described here are novel in several respects. Particularly,
the results demonstrate that gamma tocopherol (.gamma.-tocopherol
or .gamma.T) is superior to alpha tocopherol (.alpha.-tocopherol or
.alpha.T) (i.e., vitamin E, a fully alkylated tocopherol) in
systems where nitrative stress is a relevant phenomenon. The
invention of this utility for .gamma.-tocopherol (and other
desmethyl tocopherols) is not obvious to ordinarily skilled
practitioners of the art of antioxidant therapy. This contention is
demonstrated by the fact that only .alpha.-tocopherol is currently
being studied as a clinically relevant antioxidant in the treatment
ofcardiovascular or neurodegenerative disease (15). In clinical
studies performed to date, .alpha.-tocopherol has failed to provide
a consistent beneficial effect on outcome parameters (32-33,25-38).
In point of fact, oral supplementation of humans with
.alpha.-tocopherol actually depletes the human body of
.gamma.-tocopherol (reference 3 and the present inventors'
observations).
[0036] .gamma.-Tocopherol and other desmethyl tocopherols are
present in natural foods (particularly soy and wheat) in small
amounts and are generally regarded as safe for human subjects. The
biological activity of desmethyl tocopherols is associated with the
chromanol head group of the molecule (indicated by arabic numbers
in the structure above). This is to distinguish the tocopherols
from tocotrienols, which inhibit cholesterol biosynthesis but whose
activity is resident in the unsaturated lipid tail of the
tocotrienol molecule. Gamma tocopherol (and other desmethyl
tocopherols) may be chemically synthesized or isolated from natural
products.
[0037] In practice, the .gamma.-tocopherol (or other desmethyl
tocopherols) would be formulated in a manner allowing safe delivery
of effective doses to humans. The .gamma.-tocopherol (or other
desmethyl tocopherols) can be absorbed orally by mammals and could
be used by oral administration. The .gamma.-tocopherol (or other
desmethyl tocopherols) could be administered topically to inflamed
skin or gum/mouth or other mucosal tissue as a cream or gel, or
could be inhaled as an aerosol. The relative stability and
lipophilicity of .gamma.-tocopherol (and other desmethyl
tocopherols) make these compounds amenable to delivery in numerous
possible formulations. Derivatives of .gamma.-tocopherol (or other
desmethyl tocopherols) which retain the structure of a phenolic
ring lacking a H atom near the --OH group would also be useful as a
protectant against nitrative stress in neurodegenerative
conditions; intraperitioneal or intravascular administration in
appropriate media may also be used when desired.
[0038] As a cardiprotectant or neuroproctectant, oral
.gamma.-tocopherol supplements could be taken at a dose of 100-4000
mg/day by individuals suffering from or at risk for cardiovascular
or neurological diseases. The .gamma.-tocopherol supplements would
consist of .gamma.-tocopherol alone or as a predominant component
mixed with other tocopherols, medications or nutritive supplements.
As a component of topical products or for intravenous
administration, .gamma.-tocopherol could be used alone or in
combination with .alpha.-ketoglutarate and/or other tocopherols. In
these applications, effective in vivo concentrations would likely
be from about 1 .mu.M to about 10 mM, more preferably from about 1
nM to about 10 mM.
[0039] After consideration of the data described below, these and
other advantages and objects of the invention will be apparent to
those skilled in the art. The following examples are intended for
illustrative purpose only and are not to be construed as limiting
the invention in sphere or scope.
EXAMPLE 1
Demonstration of .alpha.KGDH Protection Against Nitrative Stress by
Gamma Tocopherol
[0040] .alpha.KGDH is a rate-limiting enzyme of mitochondrial
energy production which is diminished in conditions of vascular
ischemia (53-58); augmentation of the enzyme with
.alpha.-ketoglutarate has been found beneficial and
.alpha.-ketoglutarate is now included as a component in
cardioplegia fluid to improve recovery after heart surgery (59). We
undertook to determine whether .gamma.T protects .alpha.KGDH
against nitrative stress in vitro. Mitochondria were isolated from
adult rat brain then sonicated briefly in the presence of either
.alpha.T or .gamma.T, or an ethanol vehicle. Mitochondria were then
exposed to SIN-1, which generates NO and superoxide simultaneously
at a known rate (7). Combination of NO and superoxide yields
ONOO.sup.-. in situ (discussed above). FIG. 3 illustrates the
protection of .alpha.KGDH by .alpha.T and .gamma.T present during
exposure to the peroxynitrite (RNS)-generating compound SIN-1. A
400 .mu.M concentration of SIN-1I was sufficient to diminish
.alpha.KGDH activity by approximately 50% in one hour. Under these
conditions of nitrative stress, the .alpha.KGDH activity varied in
a biphasic manner with respect to tocopherol concentration. At
higher tocopherol concentrations, the reaction medium became
grossly turbid so that the apparent loss of enzyme activity might
reflect a nonspecific physical consequence of the extreme lipid
content. At all concentrations tested, .gamma.T was more protective
than .alpha.T when tested in side-by-side comparisons. Maximal
protection was observed at 1 .mu.M tocopherol in the case of both
.alpha.T and .gamma.T (FIG. 3). The maximal protection by .gamma.T
was approximately 2.5 times greater than the maximal protection
afforded by .alpha.T. At concentrations near 100 nM, .gamma.T was
approximately 5 times more protective than the corresponding
concentration of .alpha.T. Moreover, 50-100 nM of .gamma.T offered
as much protection as 1-10 .mu.M .alpha.T. Thus, .gamma.T may be as
important (or more important) an antioxidant as .alpha.T during
nitrative stress, despite the lower intrinsic concentration of
.gamma.T in most mammalian tissue.
EXAMPLE 2
Demonstration of .alpha.KGDH Protection Against Nitrative Stress In
Vivo by Gamma Tocopherol
[0041] Rat pups were injected intraperitoneally (I.P.) with
.alpha.T or .gamma.T in an olive oil vehicle every other day for 30
days beginning 2 days after birth; control animals received vehicle
only. Olive oil was chosen as a vehicle because of the low
tocopherol content in this particular vegetable oil. A total of 15
mg tocopherol was delivered to each animal over the 30 day period;
animal weight at the end of the period was approximately 90 g for
all three groups. One half of each animal group was injected with a
septic dose of LPS (2.5 mg/kg, I.P). After 24 H, animals were
killed and organs collected. .alpha.KGDH was assayed in heart
tissue and tocopherols were measured by HPLC-ECD/PDA. The
supplementation paradigm was sufficient to approximately double the
heart tissue level of both .alpha.T and .gamma.T [.alpha.T
concentration=23.+-.12 ng/mg protein in controls vs. 48.+-.13 ng/mg
in .alpha.T supplemented animals (N=5); .gamma.T
concentration=1.8.+-.0.4 ng/mg protein in controls vs. 3.9.+-.0.7
ng/mg in .gamma.T supplemented animals (N=5)]. Similar changes were
noted in plasma tocopherol concentrations (not shown). The effect
of tocopherol supplementation on cardiac .alpha.KGDH activity is
illustrated in FIG. 4. Both .alpha.T and .gamma.T supplementation
increased mean .alpha.KGDH activity by 40% in animals not subjected
to LPS stress. This increase was significant at the 90% confidence
level (p<0.1 by Student's t-test). In LPS-treated animals,
.alpha.KGDH activity decreased by 20-40% in all groups. Notably,
the .gamma.T supplemented animals maintained the highest heart
.alpha.KGDH activity among the several groups after LPS challenge
(FIG. 4). These data indicate that .gamma.T supplementation is at
least as effective and probably more effective at maintaining
mitochondrial homeostasis under a condition where nitrative stress
is known to be relevant.
EXAMPLE 3
Demonstration of Endothelial Cell Protection by
.gamma.-Tocopherol
[0042] Cultured ECV304 human endothelial cells were exposed to
SIN-1 in the presence of 10 .mu.M .alpha.-T, 10 M .gamma.-T or 5
.mu.M of each (FIG. 5). Tocopherols were incubated with the cells
for 19 hours prior to addition of SIN-1. Viability was assayed 24
hours later using a standard tetrazolium (MTT) reduction assay.
These cells proved very resistant to damage by SIN-1; however, a 5
mM initial concentration of SIN-1 produced approximately 26%
toxicity within 24 hours (N=5; FIG. 5). While .alpha.-T had no
apparent effect on SIN-1 toxicity, .gamma.-T promoted viability
somewhat and the combination of .gamma.-T with .alpha.-T (1:1 molar
ratio) completely prevented SIN-1 toxicity (FIG. 5). The data
suggest that .gamma.-T may protect cells in a way that .alpha.-T
does not.
EXAMPLE 4
.gamma.T Scavenges Reactive Nitrogen Species in Smokers and
Hypertensive Individuals
[0043] Smoking is recognized as a major contributing factor to
heart disease and .gamma.-T reportedly decreases more than
.alpha.-T in smokers. Hypertension is also a strong risk factor for
heart disease. We have begun collection of data from "normal"
subjects who do not currently have CAD, including smokers and
nonsmokers, and hypertensive subjects. 11 of 54 volunteers from the
Oklahoma City Veteran's Administration Hospital and the Oklahoma
Medical Research Foundation indicated a current smoking habit.
Seven subjects indicated a chronic hypertensive condition. As
outlined in Table I, .gamma.-tocopherol tended to decrease in
hypertensive subjects while nitration products tended to increase
in both smokers and hypertensive subjects. This was the trend
regardless of whether the tocopherol concentrations were normalized
to plasma triglycerides (Table I).
1TABLE I Preliminary statistical data regarding plasma
concentrations of .alpha.-tocopherol, .gamma.-tocopherol and
5-NO.sub.2 .gamma.-tocopherol in a population of random volunteers
from Oklahoma City. population mean .+-. SEM smokers mean .+-. SEM
hypertensives mean .+-. SEM N = 54 N = 7 N = 7 .alpha. - tocopherol
.mu.g/mL plasma 18.3 .+-. 2.6 24.9 .+-. 9.2 23.1 .+-. 12.2 .mu.g/mg
triglyceride 34.2 .+-. 14.2 21.5 .+-. 4.5 21.9 .+-. 5.9 .gamma. -
tocopherol .mu.g/mL plasma 0.713 .+-. 0.067 0.680 .+-. 0.153 0.463
.+-. 0.165 .mu.g/mg triglyceride 0.813 .+-. 0.088 0.810 .+-. 0.213
0.606 .+-. 0.153 .gamma.-toc/.alpha.-toc .times. 100 6.78 .+-. 1.13
5.54 .+-. 1.26 4.29 .+-. 1.40 5-NO.sub.2-.gamma.-tocopherol ng/mL
plasma 11.3 .+-. 1.6 20.0 .+-. 3.3 20.0 .+-. 3.7 ng/mg triglyceride
14.0 .+-. 2.5 23.5 .+-. 7.2 34.9 .+-. 10.9
5-NO.sub.2-.gamma.-toc/.ga- mma.-toc .times. 1000 3.49 .+-. 0.74
5.47 .+-. 1.96 8.20 .+-. 2.21
EXAMPLE 5
.gamma.T Scavenges Reactive Nitrogen Species During Thrombosis in
an Animal Model
[0044] As discussed above, .gamma.T has been found much more
effective than .alpha.T in the inhibition of thrombosis in a rat
model (Saldeen et al. reference 9). Consequently, we set forth to
determine whether 5-NO.sub.2-.gamma.-toc might be formed during
thrombosis in vivo. In a first experiment, the Saldeen model was
used to induce occlusive thrombosis by application of an
FeCl.sub.3-laden patch to the descending aorta of anesthetized
rats. 0.2 mL samples of blood were withdrawn into a 2 mM
EDTA/saline solution by heart puncture at various time points after
application of the FeCl.sub.3-laden patch. Control experiments were
conducted using a saline-soaked patch, which does not induce
thrombosis. Plasma was isolated by centrifugation of the blood and
was analyzed by HPLC-ECD. As illustrated in FIG. 6,
5-NO.sub.2-.gamma.-tocopherol was formed within minutes after
initiation of thrombosis and increased steadily until the animal
died (approximately 20 min. after initiation of thrombosis). Plasma
nitrite concentrations were not statistically altered in this model
of thrombogenesis. The increase in 5-NO.sub.2-.gamma.-tocop-
herol/.gamma.-tocopherol was due both to a decrease in
.gamma.-tocopherol and an increase in
5-NO.sub.2-.gamma.-tocopherol; .alpha.-tocopherol was not
substantially affected by FeCl.sub.3-induced thrombosis (data not
illustrated).
EXAMPLE 6
.gamma.-Tocopherol Inhibits Platelet Aggregation
[0045] .gamma.T inhibits platelet aggregation in vitro and in vivo,
apparently through antagonism of protein kinase C (PKC) (60-63).
Concentrations of .alpha.T necessary to inhibit aggregation in
vitro are on the order of 500 .mu.M (60-63), or approximately
10-fold higher than achieved in vivo. The concentration difference
between in vitro and in vivo efficacy has been rationalized on the
basis of incomplete tocopherol incorporation into membranes during
in vitro incubation (63). Nonetheless, subjects supplemented with
oral .alpha.T have diminished platelet aggregation (63). We
therefore sought to investigate the ability of .gamma.-tocopherol
to inhibit platelet aggregation. PRP was obtained from two
volunteers. Platelets were stimulated to aggregate with ADP,
thrombin receptor activating peptide (TRAP) or the PKC agonist
phorbol myristyl acetate (PMA) and aggregation was measured using a
4-channel clinical aggregometer. Specific platelet samples were
preincubated with vehicle, .alpha.T, .gamma.T or
5-NO.sub.2-.gamma.-tocopherol for 1 H prior to stimulation.
Tocopherol concentrations were set at 10-fold excess to average PRP
levels based on the precedent of previous in vitro aggregation
experiments that used .alpha.T as an inhibitor (60-63). As shown in
Table II, .gamma.T was similarly efficacious to .alpha.T when
tested for ability to inhibit PKC-linked platelet aggregation
(e.g., when TRAP or PMA was used as the agonist). This data
suggests that .gamma.T has an activity similar to .alpha.T with
respect to antagonism of PKC-dependent platelet aggregation and
corresponding thrombogenic events, while other data (see above)
indicates specifically enhanced RNS scavenging ability inherent to
the .gamma.T structure.
2TABLE II effect of tocopherols on platelet aggregation in vitro. %
maximum = 100% .times. (aggregability with added
tocopherol/aggregability without added tocopherol). Platelet
Aggregation (% maximum) ADP TRAP PMA 20 .mu.M 20 .mu.M 200 nM
Subject 1 vehicle (ethanol) 100 100 100 500 .mu.M
.alpha.-tocopherol 85 44 74 50 .mu.M .gamma.-tocopherol 117 73 57
Subject 2 vehicle (ethanol) 100 100 100 500 .mu.M
.alpha.-tocopherol 72 75 34 50 .mu.M .gamma.-tocopherol 90 88
29
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