U.S. patent application number 10/132231 was filed with the patent office on 2002-12-05 for antioxidant enzyme compositions for use with cigarettes.
Invention is credited to Hersh, Rebecca, Hersh, Theodore.
Application Number | 20020179103 10/132231 |
Document ID | / |
Family ID | 24436760 |
Filed Date | 2002-12-05 |
United States Patent
Application |
20020179103 |
Kind Code |
A1 |
Hersh, Theodore ; et
al. |
December 5, 2002 |
Antioxidant enzyme compositions for use with cigarettes
Abstract
A composition for inclusion within a cigarette, cigar, pipe or
smokeless tobacco. The composition can be included within the
tobacco itself, a filter for filtering tobacco smoke once burned or
even within the paper or wrapper surrounding the tobacco product.
In the cigarette filter, be it internal or external filters, the
antioxidant complex is capable of scavenging and neutralizing the
free radicals emanating from the burning or heated tobacco and
passing through the filter as the smoker inhales. The composition
is also capable of reducing free radical damage to the
oro-pharyngeal cavity, respiratory tract and lungs resulting from
tobacco smoke. The composition includes glutathione and preferaby
L-glutathione and sources of the antioxidant enzymes catalase and
superoxide dismutase.
Inventors: |
Hersh, Theodore; (Atlanta,
GA) ; Hersh, Rebecca; (Atlanta, GA) |
Correspondence
Address: |
Nathan P. Koenig
Crosby, Heafey, Roach & May
P.O. Box 7936
San Francisco
CA
94120-7936
US
|
Family ID: |
24436760 |
Appl. No.: |
10/132231 |
Filed: |
April 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10132231 |
Apr 25, 2002 |
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09608500 |
Jun 30, 2000 |
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6415798 |
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09608500 |
Jun 30, 2000 |
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09185172 |
Nov 3, 1998 |
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6138683 |
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09185172 |
Nov 3, 1998 |
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08933696 |
Sep 19, 1997 |
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5829449 |
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Current U.S.
Class: |
131/352 ;
131/332; 131/360 |
Current CPC
Class: |
A24B 15/308 20130101;
A24B 15/30 20130101; A24B 15/16 20130101; A24B 15/32 20130101; A24B
15/42 20130101; A24D 3/16 20130101; A24B 15/301 20130101; A24D 3/14
20130101; A24B 15/302 20130101; A24D 1/002 20130101 |
Class at
Publication: |
131/352 ;
131/332; 131/360 |
International
Class: |
A24D 001/04; A24F
007/04 |
Claims
I claim:
1. A composition for inclusion within a cigarette, cigar, pipe
tobacco or smokeless tobacco for reducing free radical damage to
the oro-pharyngeal cavity, respiratory tract and lungs from tobacco
smoke, said composition comprising glutathione and enzymes catalase
and superoxide dismutase.
2. The composition of claim 1 further comprising a source of
selenium as a member selected from the group consisting of
selenomethionine and L-selenocysteine.
3. The composition of claim 2 wherein said glutathione is
L-glutathione.
4. The composition of claim 1 further comprising a member selected
from the group consisting of L-cysteine and
N-acetyl-L-cysteine.
5. The composition of claim 1 further comprising vitamin C as a
member selected from the group consisting of ascorbyl palmitate and
ascorbic acid esters.
6. The composition of claim 1 wherein said composition is included
within a cigarette wherein said glutathione is contained in an
amount between at least 0.01 to 20 mgs and the catalase in an
amount between 0.1 to 1,000 ppm and superoxide dismutase in amounts
between 0.1 to 1,000 units ppm.
7. The composition of claim 3 wherein said composition is included
within a cigarette wherein said glutathione is contained in an
amount between at least 0.01 to 20 mgs and the selenium is
contained in an amount between approximately 0.01 to 10 mcgm.
8. The composition of claim 1 for inclusion within a cigarette
further comprising vitamin C which is contained in an amount
between approximately 0.1 mgs to 60 mgs.
9. The composition of claim 4 for inclusion within a cigarette
wherein said L-cysteine or N-acetyl-L-cysteine is contained in an
amount between approximately 0.1 mgs to 10 mgs.
10. The composition of claim 1 for inclusion within a cigarette
further comprising a member selected from the group consisting of
methionine and taurine which is included in an amount between
approximately 0.5 mgs to 20 mgs.
11. A cigarette comprising a paper wrapper surrounding a charge of
tobacco, said cigarette further comprising a composition for
reducing free radical damage to the oro-pharyngeal cavity,
respiratory tract and lungs from tobacco smoke generated by said
cigarette, said composition comprising glutathione, and enzymes
catalase and superoxide dismustase.
12. The cigarette of claim 11 wherein said glutathione is
L-glutathione, and said composition further comprises a source of
selenium selected from the group consisting of L-selenomethionine
and L-selenocysteine.
13. The cigarette of claim 11 further comprising vitamin C as a
member selected from the group consisting of ascorbyl palmitate and
ascorbic acid esters.
14. The cigarette of claim 11 further comprising a member selected
from the group consisting of L-cysteine and
N-acetyl-L-cysteine.
15. The cigarette of claim 11 further comprising vitamin E as a
member selected from the group consisting of tocopherol acetate and
tocopherol succinate.
16. The cigarette of claim 11 wherein said composition is
incorporated in a liposome.
17. The cigarette of claim 12 wherein said composition of
L-glutathione is contained with an amount between at least 0.01 to
20 mgs and the source of selenium is contained in an amount between
approximately 0.01 to 10 mcgm.
18. The cigarette of claim 13 wherein said vitamin C is contained
in an amount between approximately 0.1 mgs to 60 mgs.
19. The cigarette of claim 14 wherein said L-cysteine or its ester
N-acetyl-L-cysteine is contained in an amount between approximately
0.1 mgs to 10 mgs.
20. The cigarette of claim 15 wherein said vitamin E is contained
in an amount between approximately 0.01 I.U. to 10.0 I.U.
21. A filter for filtering smoke generated by a tobacco product,
said filter comprising a filtration material for filtering the
smoke from burning tobacco which passes through said filtration
material and an antioxidant composition which is dispensed into
said smoke as it passes through said filtration material, said
composition comprising glutathione and enzymes catalase and
superoxide dismutase.
22. The filter of claim 21 wherein said glutathione is
L-glutathione and said composition further comprises a source of
selenium selected from the group consisting of L-selenomethionine
and L-selenocysteine.
23. The filter of claim 21 further comprising vitamin C as a member
selected from the group consisting of ascorbyl palmitate and
ascorbic acid esters.
24. The filter of claim 21 further comprising a member selected
from the group consisting of L-cysteine and
N-acetyl-L-cysteine.
25. The filter of claim 21 further comprising vitamin A.
26. The filter of claim 22 wherein said composition of
L-glutathione is contained with an amount between at least 0.01 to
20 mgs and the source of selenium is contained in an amount between
approximately 0.01 to 10 mcgm.
27. The filter of claim 23 wherein said vitamin C is contained in
an amount between approximately 0.1 mgs to 60 mgs.
28. The filter of claim 24 for inclusion within a cigarette wherein
said L-cysteine or N-acetyl-L-cysteine is contained in an amount
between approximately 0.1 mgs to 10 mgs.
29. The filter of claim 21 for inclusion within a cigarette further
comprising vitamin E which is contained in an amount between
approximately 0.01 I.U. to 10.0 I.U.
30. The filter of claim 21 wherein said antioxidant composition is
encapsulated as a member selected from the group consisting of
liposomes, glycospheres and nanospheres.
31. The filter of claim 22 wherein said antioxidant composition is
encapsulated as a member selected from the group consisting of
liposomes, glycospheres and nanospheres.
32. The filter of claim 21 wherein said antioxidant composition is
incorporated within said filter as a powder.
33. The filter of claim 21 wherein said antioxidant composition is
incorporated within said filter as a gel.
34. The filter of claim 21 wherein said antioxidant composition is
contained within an aqueous solution in the form of a rupturable
capsule.
Description
RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of U.S.
application Ser. No. 09/185,172 filed Nov. 3, 1998 which, in turn,
is a continuation-in-part of U.S. application Ser. No. 08/933,696,
now U.S. Pat. No. 5,829,449.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention deals with the combination of various
synergistic antioxidants, enzymatic co-factors and amino acids in
appropriate delivery vehicles employed in cigarette filters and in
external filters such as cigarette and cigar "holders," in "pipe
filters" and in tobacco, wrappers and papers and in so-called
smokeless tobacco as a means of preventing or ameliorating signs
and symptoms and complications to the oro-pharyngeal cavity,
respiratory tract and lungs from damage by tobacco smoke and
tobacco chewed induced free radical species. The present invention
can be employed in filter cigarettes, unfiltered cigarettes,
cigars, pipes, and smokeless tobacco products.
BACKGROUND OF THE INVENTION
[0003] The deleterious effects of tobacco abuse are well known and
regulatory agencies as well as the public constantly react to these
scientific and epidemiologic evidences. Tobacco is indeed a
worldwide public health hazard accounting for significant morbidity
and mortality. Although smoking places an abundant oxidant insult
to the oral cavity, respiratory tract and lungs, evidence supports
the notion that the oxidant burden is on the entire organism of the
smoker. Smoking promotes development or enhancement of
atherosclerosis, causing cardiovascular disease, chronic
obstructive pulmonary disease, recently labeled "smoker's lung,"
cutaneous damage, especially to the face, called "smoker's face,"
and various forms of cancer, including carcinomas of the mouth,
pharynx, esophagus and lung.
[0004] Tobacco is a substance consisting of the dried leaves and
stems of the plant Nicotiana tabacum. Tobacco contains the drug
nicotine, which is very addictive. The plant is native to North
America and now is grown worldwide. Tobacco abuse has been
identified as the single most preventable cause of disease,
morbidity and mortality, for tobacco smoke contains many toxic
chemicals, in tar and gas phase smoke.
[0005] There are three principal ways to consume tobacco: 1)
smoking, 2) chewing and dipping, and 3) snuffing. Fifty million
Americans smoke, and countless others are affected by tobacco
smoke, the so called secondary or environmental smokers. Children
of smokers also breathe this second-hand smoke and have more
respiratory problems than children of non-smokers. Smokeless
tobacco is used by as many as 1 2 million individuals and has
detrimental effects on the oral cavity plus systemic effects
derived from buccal absorption of nicotine and other chemicals.
[0006] Cigarette smoke is divided into two phases, tar and
gas-phase smoke. Cigarette tar contains high concentrations of free
radicals. Common oxidants include semiquinone which is in
equilibrium with hydroquinones and quinones, particularly in the
viscous tar matrix. Many tar extracts and oxidants, including the
latter mentioned, are water soluble and reduce oxygen to its
superoxide radical which can dismutate to form H.sub.2O.sub.2.
Importantly, glass-fiber type cigarette filters retain almost all
of the tar particles that are larger than 0.1 micron. Thus, the
filter acts as a trap for tars in cigarette smoke. There are an
inordinately large number of free radicals, greater than 10.sup.15,
in each puff in the gas-phase of cigarette smoke. While oxidants in
tar are stable, those organic radicals in gas phase smoke are
reactive carbon and oxygen centered radicals with extremely short
half lives. Other free radical species, such as the aldehyde
species have longer half-lives and may be more deleterious,
resulting from lipid peroxidation. Interestingly, concentrations of
free radicals from tobacco are maintained at high levels for more
than 10 minutes and tend to increase as tobacco smoke is aged. It
is thus considered that these gas phase smoke oxidants are in a
steady state as they are both continuously formed and destroyed.
The latter reactions are similar to those noted to occur in smog,
pointing to the extra noxious stimuli to primary and secondary
smokers in atmospheric polluted environments.
[0007] Various studies have correlated the importance of oxidant
stress to various organs resulting from tobacco smoke and other
noxious environmental factors and thus continue to exert a toll on
the public health of all countries. Significant morbidity and
mortality result from smoking tobacco from cigarettes, cigars, and
pipes and local oral pathology from both smoking and chewing
tobacco. Epidemiologic studies have strongly implicated tobacco in
the pathogenesis of atherosclerosis and coronary artery disease,
emphysema and various malignancies, including oro-pharyngeal and
pulmonary neoplasias. Chronic cigarette smoking is associated with
appearance of free radicals inducing oxidative damage. Measurement
in blood, urine and tissues of various antioxidants or of
by-products of free radical metabolic processes are supportive of
tissue oxidant damage in the pathogenesis of various diseases
associated with tobacco smoking and environmental pollutants.
[0008] Studies have estimated that tobacco smoke has over 3,000
different constituents, of which a number are toxic, some are
carcinogenic and many generate free radical species. Most of these
compounds have been identified in so-called mainstream and
sidestream tobacco smoke. The former is that volume of smoke drawn
through the mouthpiece of the tobacco product during puffing while
sidestream smoke is that smoke emitted from the smoldering
cigarette in between puffs. Although tar and nicotine are retained
in the filter of cigarettes, the present invention applies mainly
to mainstream smoke, be it drawn through filtered and non-filtered
cigarettes. It is noted that the emissions of toxic and
carcinogenic components in sidestream smoke are not significantly
reduced in filter cigarettes when compared to their non-filter
counterparts. Thus, sidestream smoke is a major contributor to
environmental smoke, affecting both the smoker and their
non-smoking counterparts, so called secondary smokers. The lower
rates of consumption of cigarettes with high smoke yields has not
reduced the indoor pollutants of carcinogenic substances and free
radicals generating potential of tobacco smoke produced in
sidestream smoke, albeit their diminished levels in mainstream
smoke by smoking low yield tobaccos and filtered cigarettes.
[0009] Leukoplakia, a tobacco induced white patch on the buccal
mucosa, as found in smokers, is a localized irritation due to
direct contact of smoked tobacco and it is directly related to the
frequency and years of tobacco abuse. Although leukoplakia is a
benign oral lesion, these have a malignant potential, requiring a
biopsy of the lesion to rule out cancer. Leukoplakia may regress or
resolve completely when use of tobacco products is
discontinued.
[0010] Over 30,000 new cases of cancer of the oral cavity are
diagnosed annually, accounting for two to four percent of all new
cancers. Oral cancer kills 8,000 patients each year and only half
of cases diagnosed annually have a five year survival. The great
majority of these patients are users of tobacco products. Other
risk factors include alcohol abuse, nutritional deficiencies and
poor oral hygiene.
[0011] Tobacco contributes to other oral symptoms or pathologies of
the mouth and teeth. Tobacco may cause halitosis, may numb the
taste buds, interfere with the smell and the taste of food and may
stain teeth and contribute to dental caries. For example, smokers
have more dental tartar (calculus) than non-smokers. Tobacco is
also associated with destructive periodontal (gum) disease and
tooth loss. Acute necrotizing ulcerative gingivitis ("trench
mouth") is a destructive, painful inflammatory condition occurring
mainly in cigarette smokers. Swelling of the nasal and sinus
membranes have also been associated, purportedly, in individuals
who are "allergic" to tobacco smoke.
[0012] Tobacco, whether smoked as cigarettes, cigars or pipes
causes common untoward effects in the oral cavity. Tobacco smoke
has two chances to exert its deleterious effects in the mouth -
when it is inhaled by the smoker and on its exit during
exhalation.
[0013] Like cigarettes, evidence shows that cigars are also toxic
and addictive. Cigar and cigarette smokers have a similar increased
risk for oral and laryngeal cancers but the latter smokers are more
prone to contract cancer of the lung, emphysema and cardiovascular
disease. While cigarette tobacco is generally flue cured with a
resulting mildly acidic product, the slower curing methods for
cigars render these mildly alkaline. At this pH, nicotine is more
readily absorbed. Unlike cigarettes, cigars are less homogenous and
vary in size and nicotine content. Cigar smokers may spend an hour
smoking a single large "Havana" although some actively inhale very
little of this smoke; however, in non-inhalers, their nicotine
levels may be elevated with no toxic co-absorption, as occurs in
cigarette smokers. Cigar smokers also commonly hold an unlit cigar
in the mouth, exposing the oral cavity to further nicotine by local
absorption. Thus, consumption of cigars may produce an equal or
greater smoke burden of exposure and locally generated free
radicals in the oral cavity which create deleterious effects and a
risk of oro-pharyngeal cancer.
[0014] Carcinoma of the lung and chronic lung disease have been
known to be end stage complications of cigarette abuse. Nicotine
tars contain carcinogens and smoking also induces a free radical
reaction in the respiratory tract, both putative to the
oro-pharyngeal and pulmonary diseases and neoplasias induced by
tobacco abuse. Cigarette filters "trap" nicotine tars but not the
gas-phase compounds. Epidemiologic studies have been done in
various countries to show the differential effects of tar content,
amount of cigarettes smoked, type of tobacco smoked, and use of
filters on oro-pharyngeal and lung cancer risk in cigarette
smokers.
[0015] Under the epithelial lining along the respiratory airways
there is a rich network of micro vessels which carry systemic blood
from the nasal and tracheobronchial arteries. These vessels provide
nutrition to the mucosa to enable it to maintain the protective
functions. This first line of defense initially is non-injurious
and reversible, but overwhelming or chronic and persistent stimuli,
as tobacco smoke and other environmental pollutants, may cause
pulmonary damage from the oxidative damage of the leucocytes, other
free radicals and noxious agents.
[0016] In other in vitro studies, gas-phase cigarette smoke was
assessed in its filtered and whole (unfiltered) states for
oxidative effects on human plasma. Investigators noted the
prevalence of lipid peroxidation in plasma after exposure to gas
phase smoke, but not to whole cigarette smoke. The reaction of
lipid peroxidation did not commence until the endogenous ascorbic
acid had been consumed, that is, vitamin C was oxidized completely.
They also noted that cigarette smoke exposure caused oxidation of
plasma protein thiols (methionine and cysteine amino acid linkages)
and low density lipo-proteins. They concluded that lipid
peroxidation induced by the oxidants of gas-phase smoke leads to
changes in lipoproteins associated with atherogenesis. As noted
herein, the synergistic effect of reduced glutathione and ascorbic
acid or ascorbic acid derivatives such as their esters, are
beneficial in combating tobacco oxidants and in both ameliorating
and delaying the effects of tobacco smoke on oral, pharyngeal and
respiratory epithelia, on bronchoalveolar fluids and on lung
parenchyma.
[0017] Cells subjected to oxidative stress may severely affect
cellular function and cause damage to membrane lipids, to proteins,
to cytoskeletal structures and to DNA. Free radical damage to DNA
has been measured as formation of single-strand breaks,
double-strand breaks and chromosomal aberrations. Cells exposed to
ionizing radiation and cigarette smoke have also been demonstrated
to have an increased intracellular DNA damage, hence the frequency
of oro-pharyngeal, esophageal, and pulmonary carcinomas in tobacco
users.
[0018] The lungs have adapted biochemical enzymatic and
non-enzymatic antioxidant systems as prevention, limitation or
reversal of oxidant damage to the lungs. This is a protective
feature to maintain normal pulmonary function, as the respiratory
tissues operate in an environment of high partial pressure of
oxygen and are continuously exposed to airborne pollutants. Because
of their access to the environment, like the skin to oxygen and
ultraviolet radiation, the lungs may be damaged by inhaled gaseous
and particulate matter, particularly in both active and passive
smokers.
[0019] Reactive oxidizing species, as induced by inhaled tobacco,
smoke, ozone smog and others are important factors in bronchial
hyperresponsiveness and inflammatory lung injury. As in other
tissues, antioxidant enzymes in the lung include superoxide
dismutase (SOD), which converts superoxide to hydrogen peroxide and
catalase which reduces hydrogen peroxide to water. This reaction
may also be catalyzed by the selenium cofactor enzyme glutathione
peroxidase using reduced glutathione (GSH) as a substrate.
Glutathione peroxidase may also reduce lipid peroxide to the
corresponding alcohols also using reduced glutathione.
[0020] The ubiquitous non-enzymatic thiol tripeptide, glutathione
(GSH), plays a vital function in maintaining the integrity of the
reactive oxygen species-free radical sensitive cellular components.
This is accomplished through its direct role as an antioxidant, in
its reduced (GSH) form, as well as a cofactor, as aforementioned.
GSH has been detected in bronchoalveolar lavage fluid. In cells,
GSH is oxidized in this process to GSSG, but its cellular
concentrations for antioxidant activity is maintained in
equilibrium by the enzyme glutathione reductase, consuming NADPH as
the source of reducing equivalents. Under states of GSH depletion,
including malnutrition and severe oxidative stress, as in smoking,
cells may become injured and die.
[0021] The solid phase (tar) of tobacco contains high
concentrations of stable free radicals. These have been identified
as semiquinones which are in equilibrium with quinones and
hydroxyquinones. These free radicals are capable then of reducing
molecular oxygen to form the toxic free radical called superoxide.
Superoxide, upon dismutation, can form the injurious molecule
hydrogen peroxide (H.sub.2O.sub.2). The typical Cambridge
glass-fiber filter is able to withhold over 99% of particles
greater than 0.1 um, but this filter does not trap superoxide or
H.sub.2O.sub.2, which are thus inhaled by the smoker. However,
gas-phase smoke contains over 10.sup.15 organic radicals per each
puff. In contrast to stable free radicals, these have a half-life
of 1 second yet are capable of maintaining their high levels of
activity in the gas phase smoke for over 10 minutes. This smoke
also results in the creation of the H.sub.2O.sub.2 through the
smoke's production of the toxic hydroxyl radical. The present
invention recognizes that the enzyme superoxide dismutase reduces
the toxicity of the hydroxyl radical by the dismutation reaction to
make the relatively less toxic H.sub.2O.sub.2. However, to reduce
H.sub.2O.sub.2 and other peroxide molecules, the enzymes catalase
and glutathione peroxidase are required. The former, catalase,
reduces H.sub.2O.sub.2 to water and O.sub.2.
[0022] Hydrogen peroxide, like other tobacco generated free
radicals, have been implicated in the etiology of oro-pharyngeal
malignancy and pulmonary neoplasms, in smokers. H.sub.2O.sub.2
reacts with the DNA in cells and causes breaks in the double strand
which lead to mutations, precursors of malignant cells.
[0023] Cigarette smoke also contains aldehydes which are capable of
altering protein function by increasing the rate of catabolism.
Aldehydes also cause oxidation of the thiol groups of the plasma
proteins, thereby oxidizing particularly the low density
lipoproteins (LDLs) which transport the "bad" cholesterol. High
serum cholesterol levels and/or rapid oxidation of LDLs in plasma
are the initial putative steps in the development of
atherosclerosis. This is the hallmark lesion that results in
coronary heart, cerebrovascular and peripheral vascular diseases.
The aldehydes cause these alterations in proteins by their carbonyl
group reacting with the thiols and NH.sub.2 moieties of the plasma
proteins.
SUMMARY OF THE INVENTION
[0024] The present invention involves the inclusion of an
antioxidant defense system within a filter to be used with tobacco
products or within tobacco or within a wrapper for such tobacco
products or as applied to smokeless tobacco. The present
application utilizes synergistic antioxidants delivered, for
example, in tobacco filters such as those for cigarettes or
external filters to prevent and ameliorate free radical damage
induced by smoking to the oro-pharynx, respiratory tract and lungs.
The composition is supplied by inhalation through various state of
the art filters. The invention in its broadest terms comprises
glutathione plus the antioxidant enzymes catalase and superoxide
dismutase. The composition also may incorporate glutathione in its
reduced form and a co-ingredient for regenerating the reduced form
of the glutathione, the co-ingredient comprising selenium as a
selenoamino acid such as selenomethionine or selenocysteine. As
further optional ingredients, it is contemplated that the
composition may include ascorbic acid and/or one of its
derivatives, a sulfur containing amino acid such as L-cysteine,
L-taurine and/or L-methionine.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The lungs are very susceptible to damage caused by inhaled
noxious agents rendering a response to this injury by respiratory
epithelial cells and pulmonary vascular endothelium. Bacteria,
fungi and viruses may also induce pulmonary infections. All of the
aforementioned evoke respiratory tissue free radical reactions and
antioxidant-inflammatory responses. Teleologically, the present
invention involves, as a front line defense mechanism to inhaled
particles and gases and their impact upon the respiratory tract and
lungs, the use of active enzymatic and non-enzymatic antioxidants
to prevent, minimize, reverse and even repair this oxidant damage.
The former includes superoxide dismutase, which converts
deleterious superoxide radical to hydrogen peroxide and catalase
which reduces H.sub.2O.sub.2 to water. This latter reaction may
also be catalyzed by selenium containing glutathione peroxidase
which may also reduce lipid hydroperoxides, products of oxidant
induced lipid peroxidation, to alcohols, also using glutathione as
the source of reducing radicals. Thus, the thiol tripeptide,
glutathione, (GSH) acts as a direct antioxidant and as a cofactor
in reactive oxygen species defense mechanisms. In this process,
glutathione becomes oxidized but its cellular concentration as a
reduced compound is maintained by the related enzyme glutathione
reductase.
[0026] The minerals iron and copper of the tar phase contribute to
the generation of hydroxyl radicals as well and to the generation
of peroxy and alkoxy free radicals and other toxic cellular
aldehydes. These radical species are scavenged and neutralized
primarily by glutathione peroxidase with its selenium co-factor in
the presence of the ubiquitous tripeptide reduced glutathione (GSH)
antioxidant. Glutathione, which participates in many detoxifying
defensive cellular and body fluid functions, also serves as a
substrate in the removal of various metabolic intermediates such as
H.sub.2O.sub.2, lipid peroxides and organic hydroperoxides by
action of the enzyme, glutathione peroxidase. This is the synergy
in reducing and neutralizing toxic cigarette free radicals by
superoxide dismutase, catalase, glutathione and selenium. The
adminstration of the latter, selenium, has been shown not only to
itself possess anticarcinogenic properties but it also induces the
production of the vital enzyme, glutathione peroxidase. In
addition, GSH forms conjugates with smoke's organic free radicals
through the action of the glutathione transferases. This GSH
mechanism also works in excreting ingested environmental
pollutants.
[0027] During the process of smoking, pulmonary alveolar
macrophages release both superoxide and nitrous oxide. The reaction
of these two molecules results in the formation of the toxic
radical peroxynitrite (ONOO.sup.-). Peroxynitrite appears in
greater amounts in the exhaled smoke than in the inhaled smoke.
Thus, it is important to reduce the inhalation of superoxide and NO
by antioxidants in tobacco products, particularly in the filter of
the cigarette, as well as to provide the smoker's body with
supplemental antioxidants to help neutralize these oxygen
intermediates and the other free radical species inhaled and
created by tobacco products. Superoxide dismutase, importantly,
catalyzes nitration by peroxynitrite and also catalyzes phenolic
compounds, including tyrosine, in protein molecules. Thus, SOD
helps reduce the body's oxidative stress induced by smoking,
particularly that caused by superoxide and peroxynitrites.
[0028] Fractionation of aqueous cigarette tar extracts contain tar
radicals that cause damage to DNA. By special analysis, these tar
extracts have been identified as catechols and hydroquinones.
Aqueous tar extracts that cause damage to DNA produce the reactive
oxygen intermediates including superoxide, H.sub.2O.sub.2 and
hydroxyl radicals. The enzyme catalase inhibits some of this
damage, indicating that H.sub.2O.sub.2 is the precursor of the
hydroxyl radical emanating from tar extracts, responsible at least
in part for cigarette smoke's damage to DNA and thereby etiologic
of malignancy. See Pryor, et al, Chem Reseach in Toxicology 11:441.
1998.
[0029] Other non-enzymatic molecules playing an antioxidant role in
the lung include the ascorbates (vitamin C); particularly in the
extracellular defenses of the lung, as teleologically, it is
present in high concentrations in the pulmonary airway lining
fluid. Ascorbates as free radical scavengers also react with
oxidized glutathione (GSSG) to reduce it to GSH. Also, in the lipid
membrane of the cells, the hydrophobic alpha-tocopherols (vitamin
E), act synergistically with vitamin C to inhibit lipid
peroxidation, as may be induced by cigarette smoke, by actively
scavenging lipid peroxides and other free radicals.
[0030] The compositions of the present invention can be
incorporated in various smoking products. Examples include, but are
not limited to, U.S. Pat. No. 3,667,478, dated Jun. 6, 1972, which
is herein incorporated by reference and which discloses a filtered
cigarette incorporating a stabilized form of an aqueous emulsion of
an active vitamin A preparation. This patent teaches that the
method provides stability over the length of time before the
cigarette is smoked. As taught in U.S. Pat. No. 3,339,558, the
cigarette can contain, in front of the filter, a rupturable capsule
with a specified amount of Vitamin A as a method of introducing
this vitamin into the mouth and respiratory tract of the smoker.
Prior to lighting up, pressure is applied to the putative capsule,
so that the released active materials are dispersed within the
filter, thereby the Vitamin A is accessible to the cigarette smoke
passing through. The '478 patent further teaches that stabilized
Vitamin A may also be dispersed, impregnated in the tobacco or
provided throughout in droplets or beadlets through the employment
of gelatin or other colloidal materials, so that the stabilized
Vitamin A can be easily entrained by the smoke passing through the
filtering elements. Thus, dispersed and random distribution of the
small liquid droplets or tiny particulate matter of the Vitamin A
preparation is located throughout the tobacco proper or throughout
the filtering medium of a filter cigarette. The Vitamin A is
surrounded and protected in a method akin to
micro-encapsulation.
[0031] Irimi and coworkers taught in U.S. Pat. No. 5,060,672, dated
Oct. 29, 1991, which is herein incorporated by reference, a highly
efficient tobacco smoke filter. They disclosed a composition with
mechanical and/or adsorptive filtering materials including a
compound being chemically reactive with aldehydes that are not
filtered out of the smoke. One such component contains an enediol
structure. The '672 patent points out that the synergistic
compositions eliminate the excited formaldehyde radical from the
tobacco smoke.
[0032] It has been noted that tar in smoke may be reduced by using
low tar tobaccos and cigarette filters. Other efforts have been
directed to reducing toxic and harmful substances in the tobacco
itself or by adding these modifications of filters or by adding
chemicals to the filters. Caseley taught a method to further reduce
aldehydes in tobacco by using non-toxic salts of
w-mercapto-alkalene-sulphonates, as well as cysteine and
acetylcysteine in U.S. Pat. No. 4,532,947, dated Aug. 6, 1985,
which is herein incorporated by reference. These compositions were
to be added to cigarette filters or cigarette holders comprising a
filter for the purposes of reducing toxic tobacco substances in
situ, while smoking cigarettes.
[0033] In U.S. Pat. No. 3,972,335, dated Aug. 3, 1976, which is
herein incorporated by reference, Tiggelbeck and Mannes disclose a
cigarette filter comprising menthol or other smoke-flavoring
agents. They taught the use of impregnating a granular activated
carbon with a pore modifying agent, like sucrose, and thereby
improve the shelf life and delivery of the smoke flavoring agent.
Part of the activated carbon is available for adsorption of menthol
or other flavors.
[0034] In U.S. Pat. No. 5,472,002, dated Dec. 5, 1995, which is
herein incorporated by reference, a cigarette filter is taught for
administering taurine by inhalation. The patent discloses three
methods or devices to administer amino acid to smokers. The
disclosure involves a cigarette filter which comprises a filtration
material for filtering the smoke from burning tobacco and various
means for incorporating taurine therein so that it is introduced
into the smoke as it passes through the filter while the cigarette
is puffed. Taurine by inhalation has been shown to have preventive
and beneficial effects to afflictions of the respiratory tract,
including an important mucolytic property. The latter is similar to
the action of cysteine, as taught by Puracelli, in U.S. Pat. No.
4,910,222, dated Mar. 20, 1990, also incorporated by reference
herein.
[0035] A number of investigators have taught further cigarette
filtering systems to aid in retention of tobacco smoke tars,
nicotine and other toxic chemicals. Choen and Luzio in U.S. Pat.
No. 5,009,239 dated Apr. 23, 1991, which is herein incorporated by
reference, demonstrated a process for improving selective filter
retention and pass through properties of cigarette filter elements.
They used a polyethylene imine buffered with organic acids such as
formic, propionic, lactic, etc. to a pH range of about 8 to 9.5. In
this fashion there was retention of aldehyde and nicotine and
by-products by the filter from cigarette smoke.
[0036] Brown and co-workers in U.S. Pat. No. 5,249,588, dated Oct.
5, 1993, which is incorporated herein by reference, developed a
smoking article which comprised tobacco treated with a high level
humectant of 4% to 15% by weight. This smoking article comprised a
tobacco rod whereby the rod included cut expanded tobacco and a
paper wrapper, with the tobacco having been loaded with the
humectant. Von Borstel and Craig also teach a cigarette filter with
a humectant in U.S. Pat. No. 5,501,238 dated Mar. 26, 1996, which
also is herein incorporated by reference. They disclose sodium
pyroglutamate as a humectant plus a surfactant such as an
ethoxylate in order to absorb moisture from the tobacco smoke to
promote its wet filtration. They also disclose that antioxidants
and anti-carcinogenic agents that serve to filter or inactivate the
toxic component of smoke may be added. The '238 patent discloses
three types of filters to effectively remove tar from smoke: a)
conventional cellulose acetate filter, b) cellulose acetate with
sodium pyroglutate and c) a commercial wet filtration system.
[0037] Lee and Harris disclosed in U.S. Pat. No. 4,964,426 dated
Oct. 23, 1993, which is herein incorporated by reference, both
tobacco smoke filters and processes for their production.
[0038] Applicant's parent applications deal with the synergistic
combination of glutathione and a source of selenium. By contrast,
the present application contemplates the incorporation of the
synergistic combination of glutathione with the antioxidant enzymes
catalase and superoxide dismutase in a tobacco product. The
antioxidant complex may be incorporated in the internal filters of
cigarettes or in external filters, such as those incorporated in
cigarette holders. The antioxidant complex can be placed within the
tobacco itself in cigarettes, cigars, pipe tobacco and smokeless
tobacco or in cigarette papers and cigar leafs.
[0039] Without being bound by any theories, it is noted that
studies have shown that aqueous extracts of cigarette smoke contain
stable oxidants, produced by the interaction of oxygen
intermediates and hydrogen peroxide of the gas phase smoke with
components of the tar phase during the burning of the tobacco.
These oxidants are capable of oxidizing plasma proteins and cause
further protein degradation by proteolytic damage from the enzymes
present in mitochondria. Glutathione and ascorbic acid do prevent
smoke induced protein oxidation. Further protection to other body
proteins like albumia from oxidation by tobacco smoke is provided
additionally by superoxide dismutase and catalase.
[0040] As taught in applicant's parent applications, reduced
glutathione is employed in protecting cells against oxidative
stress by itself being oxidized. Thus, L-glutathione acts in
combination with other enzyme systems in order to be reduced so
that it may renew its role as a free radical scavenger. GSH
functions also coordinately with the enzyme glutathione peroxidase
which requires selenium as a cofactor to exert its biologic
antioxidant function. Selenium compounds have been shown to
scavenge oxygen-centered radicals in vivo with reduced glutathione
through glutathione peroxidase. It is believed that selenium-GSH
peroxidase catalyzes toxic hydrogen peroxidase in the presence of
reduced glutathione. This reaction reduces glutathione to oxidized
glutathione GSSG. In turn, the GSSG is reduced back to GSH by the
enzyme glutathione reductase thereby maintaining abundant cellular
GSH to scavenge free radicals anew. The preferred version of this
invention also takes advantage of this mechanism.
[0041] Further, glutathione and selenium act synergistically in
vivo as they are both constituents of the same enzymatic system.
GSH serves as a specific donor substrate while selenium, provided
from alimentary sources or locally from topically applied
preparations of selenium, or selenoaminio acids, provides the
prosthetic group of GSH peroxidase. The glutathione and selenium
antioxidant functions are intrinsically related since by keeping a
peroxidase in action, the GSH and selenium, contribute to the
removal of the dismutation product of free oxygen radicals, namely,
hydrogen peroxide. Herein lies the synergy between glutathione and
the enzymes catalase & superoxide dismutase. In a broad sense,
GSH and selenium modulate free radical chains initiated or
sustained by hydroperoxides. Selenium is used in the present
invention for its role as an antioxidant as well as its
anticarcinogenic and antimutagenic properties.
[0042] It has now been determined that glutathione can prove to be
an effective antioxidant remediating the harmful free radical
induced disease species resulting from tobacco consumption by
combining glutathione with the enzyme catalase and its synergistic
antioxidant partner superoxide dismutase. Optionally, this complex
can include the reduced form of glutathione and a selenoamino acid
as its cofactor.
[0043] The aforementioned compositions may be particularly useful
in the prevention and treatment of tobacco smoke or other gaseous
or particulate matter exposure. They represent a delicate balance
of ingredients which serve not only to reduce the number of free
radicals but also to inhibit metabolic oxidation in tissues. The
more preferred formulations in accordance with the present
invention also enhance the performance of the composition by
recycling certain antioxidant ingredients in the formulation after
these are absorbed.
[0044] In the preferred embodiment of this invention, the
synergistic antioxidant complex is a dispersion of active materials
throughout the filtering medium of a tobacco filter, although, as
noted previously, the complex can also be incorporated in the
tobacco itself or in the paper wrapper. The antioxidant complex
would be dispersed in the filter as a powder, as a stable solution,
or as an aqueous emulsion, which may include the
micro-encapsulation of these actives, such as in liposomes. The
actives may also be in tiny droplets so that when the smoke
produced by the burning tobacco passes through the filter, the
smoke will pick up or entrain the powdered complex or the tiny
droplets containing the putative antioxidant ingredients. Thus the
smoke with the actives is inhaled by the smoker as the smoke enters
the oral cavity and then inhaled into the respiratory tract and
lungs of the individual. The antioxidants will then be able to
neutralize and scavenge the free radicals both in the tobacco smoke
itself and those generated by the deleterious tobacco smoke in the
oral cavity and respiratory tract, and thereby the complex will
exert its beneficial effects locally in the mucosa and tissues of
the smoker.
[0045] As noted above as an alternative in both filtered and
unfiltered cigarettes, it is contemplated that the present
antioxidant complex be dispersed throughout the tobacco charge of
the product. Although these active ingredients can be localized
near the distal end of the filter tip or the proximal opening of
the unfiltered tobacco product, the antioxidant complex may also be
uniformly and evenly distributed throughout the entire product.
Thus, particularly by employing micro-encapsulation techniques such
as oral liposomes, these active ingredients may be administered in
the filtering medium of a filtered cigarette and within the tobacco
charge of these, or of non-filtered cigarettes and cigars and in
smokeless tobacco.
[0046] In order to protect the active ingredients of this
invention, various encapsulating or chemically protective
techniques are available such as are well known in the art. The
actives may be incorporated in micro-encapsulation vehicles such as
liposomes, glycospheres and nonospheres. Such vehicles for oral use
are well known to the cosmeceutical industry. Liposomes are
lecithin spheres that form an oil protective membrane around the
active ingredient compositions of this invention. The liposome
entrapped active ingredients travel from the tobacco product and
are delivered to the oral cavity where locally they exert both
their preventative and therapeutic functions to neutralize the
various free radical species. In addition, the antioxidants may
also be absorbed as usual by the buccal mucosa for systemic use. It
is noted that Unger and co-workers have taught therapeutic drug
delivery systems comprising gas filled liposomes which encapsulate
the active preparation in U.S. Pat. No. 5,580,573 dated Dec. 3,
1996 which is herein incorporated by reference. Earlier,
Chakrabarti disclosed preparations comprising a lipid and a
modified peptide using liposomes as delivery vehicles. See U.S.
Pat. No. 5,380,531 dated Jan. 10, 1995 which is also herein
incorporated by reference. Knight and co-workers in U.S. Pat. No.
5,049,388 dated Sep. 17, 1991 which is also herein incorporated by
reference, discloses small particle aqueous aerosol droplets
containing liposomes. The patentees taught the inclusion of a drug
or medication interacted within the liposome membrane so that when
the latter ruptures the active ingredient is not lost from the
liposome. The inventors teach various method of preparation of the
aerosol particles containing liposomes.
[0047] Liposome particles as contemplated herein have a diameter of
less than five microns and can easily be prepared in uniform size
with the active ingredients for dispersion in the filtering
material of a cigarette filter or in a rupturable aqueous capsule
which contains the liposome encapsulating the antioxidants. In each
case, the active composition in the liposomes would be inhaled by
the smoker with each puff, thereby neutralizing free radicals in
the oro-pharynx and respiratory tract and lungs generated by the
tobacco smoke.
[0048] Alternatives to placing the antioxidants of this invention
in the filter, tobacco or in encapsulations in front of the filter
is to affix these in a treated cigarette paper. This would reduce
particularly the free radicals in the sidestream smoke which are
particularly injurious to those exposed to secondary smoke as well
as to the primary smoker in both main stream and side stream smoke.
Chad and co-workers disclosed in U.S. Pat. No. 5,540,242, dated
Jul. 30, 1996, which is herein incorporated by reference, a method
for reducing side-stream smoke by incorporating additives in the
cigarette smoke. Their paper includes an alginate as a film forming
agent in combination with a burn additives in the form of alkali
metal salts such as potassium succinate, citrate or acetate to form
a coating that will reduce sidestream smoke. The synergistic group
of antioxidants of this invention may be incorporated in the
cigarette paper to not only reduce sidestream smoke, but also to
neutralize free radicals in inhaled tobacco smoke. The paper so
treated will not produce an off-taste, modify ash appearance, or
reduce the cigarette's puff count. The filter, may as well contain
powdered antioxidant complex to be inhaled by the smoker and may or
may not contain a menthol flavor, as is known in the art.
[0049] In a preferred embodiment of this invention, the active
ingredients comprise a group of synergistic antioxidants employed
in the following dosages in the filter of each cigarette. It must
be recognized that to express the amount per pack of cigarettes,
each value will be multiplied by 20, the usual numbers of
cigarettes sold in one pack, with 10 packs in one carton. The
ranges of each ingredient are expressed whether each is dispersed
in the filtering material of each cigarette, as a powder or a gel
or encapsulated in beads or admixed with a super absorber such as
any acrylamide co-polymer or as polyvinyl alcohol engrafted with
maleic anhydride. In the latter case, the actives are first
solubilized in glycerin and then mixed with the superabsorber in
proportions ranging from at least 1 to 1,000 parts of actives to at
least 1 to 10,000 parts of the super-absorber depending on its
capacity to hold an aqueous glycerin based active complex.
[0050] In another preferred embodiment of this invention, the
active synergistic anti-oxidants are first micro-encapsulated in
such protective phospholipid vehicles as oral liposomes or by other
state of the art micro-encapsulation techniques, as already noted
and which are well known in this industry for protection of oral
drugs, vitamins, amino acids and peptides.
[0051] The active ingredients are as follows:
[0052] 1. Glutathione, and preferably L-glutathione in an amount
between at least 0.01 mg. to 20 mg., preferably from 0.10 to 10 mg,
most preferably from 1.0 mg to 5.0 mg per cigarette.
[0053] 2. The antioxidant enzyme catalase in amounts of 0.1 parts
per million to 10,000 parts per million and its synergistic
antioxidant partner, superoxide dismutase also in a range from 0.1
ppm to 10,000 ppm.
[0054] Optional Ingredients
[0055] 3. L-selenomethionine or I-selenocysteine at a concentration
to yield at least 0.01 mcgm to 10 mcgm of selenium, preferably 1.0
to 2.5 mcg selenium per cigarette.
[0056] 4. L-cysteine and/or its ester, n-acetyl-I-cysteine in a
range of 0.1 mg to 10.0 mgs., preferably from 0.5 mg to 5.0 mgm and
most preferably from 1.0 mgs to 2.5 mgm, per cigarette.
[0057] 5. Vitamin C as ascorbic acid or as an ascorbyl palmitate or
other ascorbic acid esters alone or microencapsulated such as in
liposomes from 0.1 mg to 60.0 mg, preferably from 0.5 mg to 30.0
mgm, most preferably from 1.0 mgm to 3.0 mgm per cigarette.
[0058] In each instance, the above noted level of ingredients are
based upon a single cigarette filter whether contained within the
filter as being absorbed upon the filter material or as a
rupturable capsule or as a separate stand alone filter for use with
cigars, pipes and unfiltered cigarettes. When used in cigars or as
additives to pipe tobacco, the gross amounts of the above-noted
ingredients can be adjusted in proportion to the amount of tobacco
as compared to the amount of tobacco contained in the typical
cigarette.
[0059] Other optional ingredients can be used in the tobacco or in
the filter which may include those ingredients which are known to
bind, or chemically alter noxious molecules, such as aldehydes
found in tobacco smoke. The putative anti-oxidants of this
invention are used to neutralize the free radicals found in tobacco
as well as those generated by tobacco smoke in the oral cavity, as
the antioxidants are inhaled from the filter in the smoke with each
puff.
[0060] Experimental Data
[0061] The effects of tobacco gas phase smoke were studied in cell
structures. The cell lines studied included WI-38 fetal lung
fibroblasts and both 26 and 83 year old dermal fibroblasts. Cells
were grown in flasks until confluence was reached prior to study
and kept as in an incubator and in culture media as is standard for
cell culture techniques. To test the hypothesis that tobacco gas
phase free radicals cause alterations in all physiology and cell
viability, a smoking device was assmbled so that the smoke of one
cigarette, passing through an accessory cellulosic filter, can be
bubbled through the cell culture media. The lit cigarette smokes
itself by the action of a vacuum pump. The control test is that of
one cigarette. In the test case, the antioxidant complex is placed
in the accessory cellulosic filter, so that this becomes the gas
phase antioxidant treated smoke.
[0062] In the control or test study, the cell culture media is
placed on the fibroblast cell line being studied and the survival
of the cells is monitored for 48 hours using the Alamar Blue Dye
technique.
[0063] In the experiment, the cells were first trypsinized with
0.25% trypsin and then are plated into 96 well plates and incubated
at 37.degree. C. Cells were then treated with the tobacco smoke
which was bubbled in the culture media with suitable dilutions of
this aquenous solution. Suitable positive (untreated) cells and
negative (ethanol treated) cells were used as controls. As noted,
the free radical scavenger antioxidant complexes tested were placed
in liposomes as a coating within the accessory cellulosic filter
affixed to the cigarette.
[0064] The compositions studied were as follows:
[0065] A. 0.2 mg L-glutathione 0.01 mcgm L-selenomethionine
[0066] B. 0.2 mg L-glutathione 0.01 mcgm L-selenomethionine 500 ppm
SOD
[0067] C. 0.2 mg L-glutathione 0.01 mcgm L-selenomethionine 500 ppm
catalase
[0068] D. 0.2 mg L-glutathione 0.01 mcgm L-selenomethionine 500 ppm
super oxide dismutase 500 ppm catalase
[0069] Results:
[0070] The following data was drawn from some of these studies:
1 Untreated Treated WI-38 Fetal cells 60% 84% 26 yr old 52% 80% 83
yr old 10% 78%
[0071] Chart 2: WI-38 Fetal Cell Line--Alamar Blue Dye Test
2 Cell Viability Untreated smoke: 52% Treated smoke: Composition A
83% Composition B 92% Composition C 100% Composition D 100%
[0072] As noted, tobacco gas phase smoke resulted in significant
cell death. This effect is accentuated in the senescent cell lines
compared with the younger cell lines, including the WI-38 fetal
cells. The free radical scavengers reduced gas phase free radical
species and significantly reduced acute cell mortality in all the
cell lines of the various ages that were studied. Addition of the
antioxidant enzymes to the free radical scavengers complex based on
glutathione further reduced or even eliminated cell death from the
treated tobacco gas phase smoke. The addition of the full complex
in the filter of the cigarette shows there is marked protection to
cytotoxicity to cell lives in culture media following gas phase
cigarette smoke exposure.
* * * * *