TOBACCO AND TOBACCO PACKAGING MATERIAL FOR PREVENTING OR REDUCING TOBACCO-ASSOCIATED INJURY IN THE AERODIGESTIVE TRACT OF A SUBJECT

Articles of manufacturing for preventing or reducing tobacco smoke- and smokeless-tobacco associated injury in the aerodigestive tract of a subject are disclosed, which comprise at least one agent capable of reducing or preventing tobacco associated loss of peroxidase activity in said aerodigestive tract, said at least one agent being selected from the group consisting of a cyanide chelator and an iron chelator.

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Description
FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to methods of preventing or reducing pathogenesis of oxidant stress-associated diseases of the aerodigestive tract. More particularly, the present invention relates to methods of employing hydroxocobalamin (vitamin B12a, OH—CO), deferoxamine (DES) and reduced glutathione (GSH) to reduce or prevent tobacco-induced cellular or macromolecular damage in the aerodigestive tract.

Harmful Effects of Tobacco Consumption:

The deleterious effects of tobacco abuse are well known. Tobacco is a worldwide public health hazard accounting for significant morbidity and mortality. Although smoking places an abundant oxidant insult to the oropharynx and respiratory tract, the oxidant burden associated with any tobacco consumption (as described hereinbelow) is deleterious to the entire body of the tobacco consumer. Namely, tobacco consumption leads to development or enhancement of atherosclerosis, cardiovascular disease, chronic obstructive pulmonary disease and various forms of cancer, including carcinomas of the mouth, pharynx, esophagus and lung.

There are two principal ways to consume tobacco: smoking and smoke-less consumption. The latter includes spit tobacco, chewing tobacco, chew, chaw, dip, plug, comes in various forms: snuff, snuss and chewing tobacco. Snuff is a fine-grain tobacco that often comes in teabag-like pouches, which users “pinch” or “dip” between their lower lip and gum. Chewing tobacco comes in shredded, twisted, or “bricked” tobacco leaves that users put between their cheek and gum. Whether ifs snuff, snus or chewing tobacco, the user consumes the tobacco letting it sit in the mouth and suck on the tobacco juices, spitting often to get rid of the saliva that builds up. This sucking and chewing allows nicotine (a narcotic drug), to be absorbed into the bloodstream through the tissues of the mouth. Smokeless tobacco has a detrimental effect on the oral cavity plus systemic effects from buccal absorption of nicotine and other chemicals.

Approximately 50 million Americans smoke and countless others are affected by tobacco smoke (TS) as secondary smokers. Children of smokers breathe this second-hand smoke and have more respiratory problems than children of non-smokers.

Studies have estimated that TS has over 3,000 different constituents, of which many are toxic, carcinogenic and/or generate free radical species. Free radicals are atoms or molecules containing an unpaired electron. Oxygen free radicals include the superoxide free radical (.O2−) and the hydroxyl radical (OH.) which, together with hydrogen peroxide (H2O2) and singlet oxygen (1O2), are jointly called reactive oxygen species (ROS). Due to their high reactivity they may lead to chemical modification and impairment of the components of living cells, such as proteins, lipids, carbohydrates and nucleotides.

Tobacco smoke mediated oxidant injury is similar to that induced by smog, thereby increasing such noxious stimuli to primary and secondary smokers in polluted atmospheric environments.

Most of constituents of TS have been identified in so-called mainstream and side stream TS. The former is that volume of smoke drawn through the mouthpiece of the tobacco product during puffing while side stream smoke is that smoke emitted from the smoldering cigarette in between puffs. Although tar and nicotine are retained in the filter of cigarettes, this applies mainly to mainstream smoke, when comparing filter and non-filter cigarettes. Mainstream smoke emission is also markedly reduced both in low and in ultra low tar yield cigarettes. However, the emissions of toxic and carcinogenic components in side stream smoke are not significantly reduced in filter cigarettes when compared to non-filter counterparts. Thus, side stream smoke is a major contributor to environmental smoke, affecting both the smoker and their non-smoking counterparts, so called secondary smokers.

Evidence shows that cigars as well as cigarettes are highly toxic and addictive. Tobacco smokers have a similar increased risk for oral and laryngeal cancers. Evidence indicates that one cigar generates levels of carcinogenic particles exceeding those generated by three cigarettes. Fumes from cigars are also of greater consequence to secondary smokers. Epidemiologic studies reveal greater frequencies of heart disease, emphysema, and cancers of the mouth and pharynx in cigar smokers when compared to matched non-smokers. Cigar smokers may spend one full hour smoking a single large cigar and commonly hold an unlit cigar in the mouth, allowing further exposure to toxins by local absorption. Thus, consumption of cigars may produce an equal or greater burden of toxic exposure to TS than cigarettes. Recently, sales of cigars have risen, partly due to their gaining popularity with women and the advent of the female friendly “cigar bar”.

Oral diseases associated with tobacco consumption: Tobacco, whether smoked or chewed, 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.

Over 30,000 new cases of cancer of the oral cavity are diagnosed annually, accounting for 2-4 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. Oral squamous cell carcinoma (SCC) is the most common malignancy of the head and neck with a worldwide incidence of over 300,000 new cases annually. The disease is characterized by a high rate of morbidity and mortality (approximately 50%) and in this respect is similar to malignant melanoma (1-4). The major inducer of oral SCC is exposure to tobacco which is considered to be responsible for 50-90% of cases world-wide (5, 6). As such, the incidence of oral SCC in tobacco smokers is 4-7 times higher than in non-smokers (7, 8). Moreover, the higher TS-related risk for oral SCC is manifested by a reduction in the mean age of development of the disease by 15 years as compared to non-smokers (9). Various malignancies are particularly associated with smokeless tobacco consumption. These include oral cancer and cancer of the gastrointestinal tract including esophagus and bladder. Leukoplakia, a tobacco induced white patch on the buccal mucosa, as found in smokers, is a localized irritation due to direct contact of smoked or smokeless tobacco and it is directly related to the frequency and years of tobacco abuse. Although leukoplakia is a benign oral lesion, it has a malignant potential.

In addition, tobacco contributes to other oral symptoms or pathologies of the mouth and teeth. Tobacco may cause halitosis, may numb the taste buds, and interfere with the smell and the taste of food. It may stain teeth and contribute to dental caries. Smokers have more dental tartar (calculus) than non-smokers. Tobacco is associated also with destructive periodontal (gum) disease and tooth loss. Acute necrotizing ulcerative gingivitis (“trench mouth”) is a destructive, painful inflammatory condition occurring mainly in tobacco smokers. Swelling of the nasal and sinus membranes have also been associated, purportedly, in individuals who are “allergic” to TS.

Oral submucous fibrosis occurs mainly in India and is a chronic, progressive premalignant condition. The etiology is chronic chewing of tobacco or areca nut or both. The fibrosis results in restriction of mouth opening and involves the palates, tonsillar fossa, buccal mucosa and underlying muscle. Associated with this condition is also oropharyngeal carcinomas, also with a high frequency in India and associated in 70% of cases with chewing tobacco. Smokeless tobacco and areca nut usage is also common in Pakistan, Bangladesh and Java and in these and Indian immigrants to the United States and United Kingdom.

Tobacco smoke also affects the skin adversely. Dr. Douglas Model of England in 1985 added to the medical lexicon the term “smoker's face” from a study with pictures of 116 cases and suitable non-smoking controls (10). Akin to photodamage, those with smoker's face appear older and have more wrinkles.

Molecular damage resulting from exposure to TS: Tobacco smoke induces oxidative damage to lipids, DNA and proteins, particularly via protein-SH groups as a consequence of containing high levels of both free radicals as well as aldehydes, including acetaldehyde (ethanol), propanol and acrolein, as well as other deleterious molecules.

Oxidant injury: Tobacco smoke is divided into two phases; tar and gas-phase smoke. Tar contains high concentrations of free radicals. Many tar extracts and oxidants are water-soluble and reduce oxygen to superoxide radical which can dismutate to form the potent oxidant H2O2. Oxidants in gas-phase smoke are reactive carbon- and oxygen-centered radicals with extremely short half lives.

Cells subjected to oxidative stress develop severely affected cellular function and suffer 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 TS have also been demonstrated to have an increased intracellular DNA damage, a precursor of mutations and development of malignancies. It has been shown that TS elicits protein carbonylation in plasma and that, in contrast, exposure of human plasma to gas-phase but not to whole TS produces oxidative damage to lipids.

Redox-active metals: Redox-active metal ions, such as iron and copper, in the presence of H2O2 and other low-reactive free radicals found in TS, such as superoxide radicals, participate in the deleterious Haber-Weiss and Fenton reactions, in which the highly reactive hydroxyl free radicals are produced.

Aldehydes: Studies have shown that exposure of plasma to TS results in protein damage in the form of protein carbonylation (11) and in oxidation of plasma lipids and antioxidants (12). The source of the accumulation of protein carbonylation was found to be due to aldehydes present in TS (13, 14). In addition, it was shown that several salivary enzymes such as amylase, lactic dehydrogenase (LDH), and acid phosphatase were considerably affected by TS (14, 15), where both TS-based aldehydes, such as acrolein and crotonaldehyde, as well as oxygen free radicals were implicated as the causative agents affecting the above enzymes (14, 15).

Physiological Antioxidants:

Glutathione: Glutathione, a sulfur-containing tripeptide (L-γ-glutamyl-1-cysteine-glycine) is the most abundant non-protein thiol in mammalian cells and is recognized as the primordial antioxidant. Glutathione, in its reduced form, “GSH”, acts as a substrate for glutathione-S-transferase and glutathione peroxidase, enzymes catalyzing reactions involved in detoxification of xenobiotic compounds and in antioxidation of ROS and other free radicals. This ubiquitous protein plays a vital function in maintaining the integrity of free radical sensitive cellular components. Under states of GSH depletion, including malnutrition and severe oxidative stress, cells may then become injured from excess free radical damage and die.

Oral peroxidase: Oral peroxidation is the pivotal enzymatic activity of the salivary antioxidant system (16-19). Oral peroxidase activity is composed of the combined activity of two peroxidases, salivary peroxidase (SPO) and myeloperoxidase (MPO). Salivary peroxidase, which is secreted by the major salivary glands, mainly the parotid gland (18), contributes 80% of the total oral peroxidase (OPO) activity, while MPO, produced by leukocytes (20), contributes the remaining 20% of OPO activity. Oral peroxidase performs two functions preventing oxidant injury; it reduces the level of H2O2 excreted into the oral cavity from the salivary glands, by bacteria and by leukocytes, and it inhibits the metabolism and proliferation of various bacteria in the oral cavity.

Oral peroxidase is involved in destroying TS-associated H2O2. Tobacco smoke-associated hydrocyanic acid (HCN) is metabolized by the liver to thiocyanate ion (SCN−). This SCN− is specifically sequestered from the plasma by the parotid gland and is secreted by this gland into the oral cavity. Its concentration in the saliva of non-smokers ranges from 0.3-1.5 mM, while the respective range in smokers is approximately 1.4-4.0 mM, depending on the number of cigarettes smoked per day, with a prolonged t1/2 of 9.5 h (21). Following its secretion in saliva, SCN− reacts with, and eliminates H2O2 in the oral cavity in a reaction catalyzed by OPO, as described in FIG. 2a. However, it has been shown that if OPO is damaged or depleted, as occurs upon exposure to TS, the H2O2 in the oral cavity is not eliminated and remains available for further reaction with redox-active metal ions which are secreted via the parotid gland saliva (22, 23).

In the reaction of SCN−+H2O2→OSCN−+H2O, which is catalyzed by OPO, H2O2 oxidizes SCN−, a detoxification product of cyanide secreted mainly by the parotid gland. In this reaction, SCN− acts as the electron-donating component, similarly to GSH in other biological systems (20, 24, 25). Two potent antibacterial oxidizing products evolve from this reaction: hydrogen hypothiocyanite (HOSCN) and its conjugated anion, OSCN−. The antibacterial activity of HOSCN and OSCN− stems from their ability to react with sulfhydryl groups of bacterial enzymes that are vital for glycolysis, such as hexokinase, aldolase and pyruvate kinase (20, 25-28).

The importance of OPO in oral disease prevention has been demonstrated in several studies. For example, studies using animal models or the Ames test have shown that saliva inhibits the mutagenicity of known oral cancer inducers, such as TS, 4NQO and benzopyrene (29, 30). Biochemical studies have also demonstrated that saliva inhibits production of ROS such as superoxide free radical and H2O2 from betel quid tobacco, the most potent inducer of oral cancer (31). These observations are further supported in the observation that patients with oral lichen planus, a premalignant lesion, have reduced salivary antioxidant capacity (32).

Several prior art approaches have been employed in order to reduce or prevent incidence of oral disease resulting from oxidant injury.

For example, cigarette filters are used to trap TS tar but do not the gas-phase compounds.

One approach has employed a filter for TS providing chemo-sorptive properties to reduce aldehyde concentration in TS (33).

Another approach has employed oral megadoses of antioxidants in attempts to reduce generation of H2O2 resulting from the “respiratory burst” reaction associated with phagocytic activity of macrophages and neutrophils. It has been shown that smokers have a higher “respiratory burst” reaction than non-smokers and that this may be associated with the increased incidence of aerodigestive tract disease in the former.

In yet another approach, dipeptide compounds with pharmaceutical properties to increase glutathione levels were employed (34).

A further approach utilized a glycine carboxylic acid alkyl mono-ester of glutathione to increase cellular GSH levels (35).

In yet a further approach, administration of a combination of glutathione and selenium was suggested for preventing oxidant injury resulting from exposure to TS (36).

In another approach, administration of a combination of glutathione, ascorbic acid, selenium and a sulfur-containing amino acid was suggested in order to prevent oral oxidant injury (37).

In yet another approach, administration of a combination including some or all of the following antioxidants; L-glutathione, L-selenomethionine, L-selenocysteine, ascorbyl palmitate, ascorbic acid esters, L-cysteine, N-acetyl-1-cysteine, tocopherol acetate, tocopherol succinate, vitamin A, a zinc salt, methionine and taurine was suggested in order to provide intra-oral protection from oxidant injury (38)

The present inventors have previously described novel smoking filters and oral compositions for reducing tobacco associated damage in the aeurodigestive tract (see U.S. Pat. No. 6,789,546). These compositions include active agents which are capable of reducing or preventing tobacco associated loss of peroxidase activity in the aerodigestive tract.

U.S. Pat. No. 5,922,346 teaches a composition for reducing free radical damage induced by tobacco products and environmental pollutants comprising, as active ingredients, reduced glutathione and a source of selenium selected from the group consisting of elemental selenium, selenomethionine and selenocysteine, the active ingredients being combined with suitable carriers and flavorings for their intra-oral administration as gels, lozenges, tablets and gums in concentrations for reducing free radical damage induced by tobacco products and other environmental pollutants to the oral cavity, pharynx and upper respiratory tract of a user and secondary smokers.

U.S. Pat. No. 5,906,811 teaches a method for reducing free radical damage induced by tobacco products and environmental pollutants comprising administering in a suitable carrier in concentrations for effectively reducing said free radical damage to the oro-pharynx and upper respiratory tract of a user a combination of from 0.01 and 10% (weight) glutathione, from 1.0 to 25% (weight) ascorbic acid, from 0.001 to 10% (weight) of a source of selenium and from 0.001 to 2.0% (weight) of a sulphur containing amino acid.

These aforementioned attempts to reduce tobacco damage are used as an adjuvant treatment following or prior to tobacco consumption, but not concomitantly with tobacco consumption.

U.S. Pat. No. 5,829,449 teach a composition for inclusion within a cigarette, cigar or pipe tobacco for reducing free radical damage to the oro-pharyngeal cavity, respiratory tract and lungs from tobacco smoke, said composition comprising L-glutathione and a source of selenium selected from the group consisting of L-selenomethionine and L-selenocysteine. U.S. Pat. No. 5,829,449 clearly states that the composition is supplied by smoke inhalation and not by direct contact with the aerodigestive tract (i.e., wet tissue).

U.S. Pat. No. 6,138,683 teaches a composition for inclusion within a quantity of smokeless tobacco, selected from the group consisting of chewing tobacco and snuff, for reducing free radical induced damage to the oro-pharyngeal cavity of the user, said composition comprising L-glutathione and a source of selenium in combination with said smokeless tobacco.

SUMMARY OF THE INVENTION

According to the present invention there is provided a smoking product comprising an agent being capable of reducing or preventing tobacco associated death of cells in the aerodigestive tract of a subject, the smoking product being designed and configured so as to enable physico-chemical interaction between said agent and said tobacco smoke when in use by the subject.

According to one aspect of the present invention there is provided, an article of manufacturing comprising tobacco and a tobacco packaging material, at least a portion of said tobacco and/or tobacco packaging material being in contact with an aerodigestive tract of a subject in use thereof, and whereas said at least a portion of said tobacco and/or tobacco packaging material comprises at least one agent capable of reducing or preventing tobacco associated loss of peroxidase activity in said aerodigestive tract, said at least one agent being selected from the group consisting of a cyanide chelator and an iron chelator.

According to another aspect of the present invention there is provided an article of manufacturing comprising a tobacco packaging material at least a portion of which being in contact with an aerodigestive tract of a subject in use thereof, wherein said at least a portion comprises an agent capable of reducing or preventing tobacco associated loss of peroxidase activity in said aerodigestive tract.

According to further features in preferred embodiments of the invention described below, said tobacco packaging material comprises a filter and said at least one agent is impregnated in a paper of said filter being in contact with said aerodigestive tract of said subject in use thereof.

According to still further features in the described preferred embodiments, said tobacco is smokeless tobacco.

According to still further features in the described preferred embodiments said tobacco is smoked tobacco.

According to still further features in the described preferred embodiments said tobacco packaging material is selected from the group consisting of a rolling paper, a filter paper, a suns bag packaging, a cigarette, a pipe and a tin sheet packaging.

According to still further features in the described preferred embodiments the article of manufacturing is selected from the group consisting of a snuff, a cigarette, a snus, a Gutka, a plug, a twist, a scrap and tobacco water.

According to still further features in the described preferred embodiments said agent comprises a cyanide chelator.

According to still further features in the described preferred embodiments said agent is hydroxocobalamin.

According to still further features in the described preferred embodiments said agent is capable of reducing or preventing tobacco smoke-associated death of cells in the digestive tract of a subject.

According to still further features in the described preferred embodiments said cells are lymphocytes and/or epithelial cells.

According to still further features in the described preferred embodiments said agent comprises an iron chelator.

According to still further features in the described preferred embodiments said agent is deferoxamine.

According to still further features in the described preferred embodiments said agent comprises an antioxidant.

According to still further features in the described preferred embodiments said agent is glutathione.

The present invention successfully addresses the shortcomings of the presently known configurations by providing methods of preventing or reducing tobacco smoke-associated injury in the aerodigestive tract.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is schematic diagram depicting the construction of a filter paper impregnated with agents of the present invention according to the present invention.

FIG. 2a is a diagram depicting the pathway of cyanate metabolism resulting from exposure to TS-derived HCN.

FIG. 2b is a data plot depicting reduced OPO activity following in vivo exposure (smoking) to TS in smoker and non-smoker subjects. The OPO activity of 17 smokers and 16 non-smokers was measured prior to and following smoking 1 cigarette.

FIG. 2c is a data plot depicting reduced OPO activity following in vitro exposure of saliva to TS in smoker and non-smoker subjects. The OPO activity of 7 smokers and 11 non-smokers was measured prior to and following exposure to 1 cigarette.

FIG. 3 is a histogram depicting KCSN-mediated resistance to TS exposure-induced decreases in OPO activity in saliva of non-smoker subjects exposed to the TS from 1 cigarette in vitro. Each value represents the average value obtained in experiments on 3 subjects±standard deviation (SD).

FIGS. 4a-b are photographs of Western immunoblotting analysis depicting increased levels of salivary protein carbonylation in representative non-smoker saliva following in vivo exposure of saliva (smoking) to the TS of 1 cigarette (FIG. 4a). Proof that equal quantities of proteins were analyzed per sample is shown via Coomassie Blue staining (FIG. 4h) of the samples. Abbreviations: amylase (Amy), protein rich proteins (PRP's), lysozyme (Lys). Lane 1: prior to smoking, Lane 2: 10 mins after smoking, Lane 3: 30 mins after smoking, Lane 4: 60 mins after smoking.

FIGS. 5a-b are photographs of Western immunoblotting analysis depicting increased levels of salivary protein carbonylation in representative non-smoker saliva following in vitro exposure of saliva to the TS of 1 cigarette (FIG. 5a). Proof that equal quantities of proteins were analyzed per sample is shown via Coomassie Blue staining (FIG. 5b) of the samples. Lane 1: prior to exposure to TS, Lane 2: 10 mins following exposure to TS, Lane 3: 30 mins following exposure to TS, Lane 4: 60 mins following exposure to TS.

FIG. 6 is a data plot depicting TS dose-dependent decrease in OPO activity in saliva exposed in vitro to 3 exposures of TS over a 1 h period. Each data point represents mean±standard error of the means (SEM) of results from experiments on saliva from 4 subjects.

FIG. 7 is a data plot depicting the effect of increasing concentrations of dapsone (4-aminophenylsulfone) on OPO activity in TS-treated saliva. Abbreviations: NO.1, NO.2, NO.3, NO4 and NO.5 correspond to saliva from 5 different subjects and plasma corresponds to commercial MPO.

FIG. 8 is a data plot depicting the effect on OPO activity in saliva treated with TS in the presence or absence of 150 μM dapsone. Saliva samples were normalized with respect to initial OPO activity. OPO exposed to TS in the presence (□) or absence (♦) of dapsone, control saliva exposed to air in the presence (▴) or absence () of dapsone.

FIG. 9 is a data plot depicting the effect of GSH, deferoxamine (DES) and ascorbate on OPO activity in TS-treated saliva. Treatments: TS only (⋄, n=4), TS+1 mM GSH (▪, n=3), TS+1 mM deferoxamine (Δ, n=3), TS+1 mM ascorbate (X, n=3). Data plotted as mean±average OPO activity.

FIG. 10 is a data plot depicting the effect of 100 μM FeCl3 or ascorbate on OPO activity in TS-treated saliva. Data points represent data plotted as mean±SD using saliva samples obtained from 3 subjects.

FIG. 11 is a data plot depicting the effect of purified aldehydes present in TS on OPO activity in saliva from 4-5 subjects. Treatment: exposure to air for 3 h (⋄), 2 mM acetylaldehyde (▪), 20 μM crotonaldehyde A), 80 μM acrolein (X).

FIG. 12 is a histogram depicting the effect of 18 h of dialysis on OPO activity in TS-treated saliva having lost 68% of initial OPO activity levels. 1: control OPO activity in non-TS-treated saliva prior to [1] and following [2] dialysis, OPO activity in TS-treated saliva prior to [3] or following [4] dialysis and time-control following 18 h without dialysis [5].

FIG. 13 is a histogram depicting recovery of OPO activity in saliva treated with KCN following 18 the of dialysis. OPO activity in non-TS-treated saliva prior to [1] or following [2] dialysis, OPO activity in saliva following 2 min KCN treatment prior to [3] or following [4] dialysis and time-control OPO activity in KCN-treated saliva following 18 h without dialysis.

FIG. 14 is a histogram depicting OH—CO mediated prevention of OPO activity loss in saliva resulting from TS-treatment. OPO activity in non-TS-treated saliva in the absence [1] or presence [2] of 1 mM OH—CO (n=4), OPO activity in TS-treated saliva in the absence (n=4) [3] or presence of 0.5 mM OH—CO (n=3) [4], 1 mM OH—CO (n=3) [5] or 2 mM OH—CO (n=3) [6]. Each value represents data calculated as average±SD of results from 3-4 experiments using saliva from 3-4 subjects.

FIG. 15 is a histogram depicting OH—CO mediated prevention of OPO activity loss in KCN-treated saliva. Each value represents data calculated as average±SD of results from 3 experiments using saliva from 3 subjects.

FIG. 16 is a data plot depicting death of lymphocytes incubated at 37° C. in the presence of TS and saliva.

FIG. 17 is a photograph of Western immunoblotting analysis depicting increased levels of protein carbonylation in lymphocytes treated with TS in the presence of saliva. Lane 1: incubation in PBS alone, Lane 2: incubation in the presence of saliva, Lane 3: incubation in PBS+TS, Lane 4: incubation with TS+saliva. Incubations were performed for 80 min at 37° C.

FIG. 18 is a photograph of Western immunoblotting analysis depicting the effects of saliva and uric acid on lymphocyte protein carbonylation. Lane 1: incubation in PBS alone, Lane 2: incubation in the presence of saliva, Lane 3:

incubation in the presence of 10 μM uric acid, Lane 4: incubation in the presence of 100 μM uric acid. Incubations were performed for 20 min at 37° C.

FIG. 19 is a data plot depicting the effects of various antioxidants; 1 mM GSH, 1 mM NAC(N-acetylcysteine) and 1 mM deferoxamine (DES), on survival of lymphocytes incubated in the presence of TS and saliva at 37° C. Asc: ascorbate.

FIG. 20 is a photograph of Western immunoblotting analysis depicting the effects of several antioxidants on protein carbonylation levels in lymphocyte treated with TS and saliva. Lane 1: incubation in the presence of TS and saliva, Lane 2: incubation in the presence of TS, saliva and 1 mM NAC, Lane 3: incubation in the presence of TS, saliva and 1 mM ascorbate, Lane 4: incubation in the presence of TS, saliva and 1 mM GSH, Lane 5: incubation in the presence of TS, saliva and 1 mM deferoxamine (DES). Incubations were performed at 37° C. for 20 min.

FIG. 21 is a photograph of Western immunoblotting analysis depicting the effects of several volatile aldehydes on lymphocyte carbonylation levels. Lane 1: incubation in the presence of PBS alone, Lane 2: incubation with 80 μM acrolein, Lane 3: incubation with 20 μM crotonaldehyde, Lane 4: incubation with 2 μM acetaldehyde, Lane 5: incubation with 80 μM acrolein+1 mM GSH, Lane 6: incubation with 20 μM crotonaldehyde+1 mM GSH, Lane 7: incubation with 2 μM crotonaldehyde+1 mM GSH. Incubations were performed at 37° C. for 20 min.

FIG. 22 is a photograph of Western immunoblotting analysis depicting the effects of saliva and acrolein on the lymphocyte protein carbonylation levels. Lane 1: incubation in PBS alone, Lane 2: incubation in the presence of saliva, Lane 3: incubation with 80 μM acrolein, Lane 4: incubation with 80 μM acrolein+saliva. Incubations were performed at 37° C. for 20 min.

FIG. 23 is a histogram depicting survival of lymphocytes incubated at 37° C. for 20 min in the presence of saliva, various antioxidants and redox-active iron. Column 1: incubation in PBS alone, Column 2: incubation in the presence of saliva, Column 3: incubation in the presence of 1 mM ascorbate, Column 4: incubation in the presence of 1 mM deferoxamine (DES), Column 5: incubation in the presence of 90 μM FeCl3, Column 6: incubation in the presence of saliva+1 mM ascorbate, Column 7: incubation in the presence of saliva+1 mM ascorbate+1 mM deferoxamine (DES), Column 8: incubation in the presence of 90 μM FeCl3+1 mM ascorbate, Column 9: incubation in the presence of 90 μM FeCl3+1 mM ascorbate+1 mM deferoxamine.

FIG. 24 is a histogram depicting survival of lymphocytes incubated at 37° C. for 80 min in the presence of TS, ascorbate and deferoxamine (DES). Column 1: incubation in PBS alone, Column 2: incubation in the presence of TS, Column 3: incubation in the presence of 1 mM ascorbate, Column 4: incubation in the presence of 1 mM deferoxamine, Column 5: incubation in the presence of TS+1 mM ascorbate, Column 6: incubation in the presence of TS+1 mM ascorbate+1 mM deferoxamine.

FIG. 25 is a photograph of Western immunoblotting analysis depicting the effects of GSH and deferoxamine (DES) on protein carbonylation levels in lymphocytes incubated for 20 or 80 minutes at 37° C. in the presence of TS and saliva. Lane 1: incubation in the presence of TS+saliva for 20 minutes, Lane 2: incubation in the presence of TS+saliva for 80 minutes, Lane 3: incubation in the presence of TS+saliva+1 mM GSH for 20 minutes, Lane 4: incubation in the presence of TS+saliva+1 mM GSH for 80 minutes, Lane 5: incubation in the presence of TS saliva 1 mM GSH+1 mM deferoxamine for 20 minutes, Lane 6: incubation in the presence of TS+saliva+1 mM GSH+1 mM deferoxamine for 80 minutes, Lane 7: incubation in the presence of TS+saliva+1 mM GSH+5 mM deferoxamine for 20 minutes, Lane 8: incubation in the presence of TS+saliva+1 mM GSH+5 mM deferoxamine for 80 minutes.

FIG. 26 is a diagram depicting mechanisms of cyanate metabolism following exposure to TS.

 DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of methods, pharmaceutical compositions, oral compositions, filters and tobacco products for preventing or reducing tobacco smoke-associated injury in the aerodigestive tract of a subject. Specifically, the present invention can be used to prevent or reduce loss of OPO activity or CN−—, redox-active metal ion- or aldehyde-induced cell death resulting from TS-associated oxidative stress, all of which being capacities not provided by prior art methods.

The principles and operation of the present invention may be better understood with reference to the drawings and accompanying examples.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or exemplified in the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

Tobacco consumption, such as in the form of smoking, chewing, dipping or snuffing, is associated with pathogenesis of many diseases of the aerodigestive tract.

Thus, various prior art methods of reducing or preventing aerodigestive tract oxidant injury resulting from insults such as TS have been described.

The present inventors have previously described novel smoking filters and oral compositions for reducing tobacco associated damage in the aeurodigestive tract (see U.S. Pat. No. 6,789,546). These compositions include active agents which are capable of reducing or preventing tobacco associated loss of peroxidase activity in the aerodigestive tract.

None of these tobacco compositions and tobacco packaging means, however, are designed for reducing the damage associated with smokeless tobacco when in use by the subject and in particular chewing, snuff and snus.

There is thus a widely recognized need for, and it would be highly advantageous to have, compositions and means for preventing or reducing any tobacco associated oxidant injury in the aerodigestive tract devoid of the above limitation.

While reducing the present invention to practice, the present inventors uncovered that previously described agents (see U.S. Pat. No. 6,789,546) can be implemented in any tobacco and tobacco related products which come in direct contact with the aerodigestive tract (e.g., saliva) and as such can be ameliorate tobacco damage simultaneously with its use (e.g., chewing).

Thus, according to one aspect of the present invention there is provided an article of manufacturing comprising tobacco and a tobacco packaging material, at least a portion of the tobacco and/or tobacco packaging material being in contact with an aerodigestive tract of a subject in use thereof, and whereas the at least a portion of the tobacco and/or tobacco packaging material comprises an agent capable of reducing or preventing tobacco associated loss of peroxidase activity in the aerodigestive tract.

According to another aspect of the present invention there is provided an article of manufacturing comprising a tobacco packaging material at least a portion of which being in contact with an aerodigestive tract of a subject in use thereof, wherein the at least a portion comprises an agent capable of reducing or preventing tobacco associated loss of peroxidase activity in the aerodigestive tract.

As used herein the term “tobacco” refers to any tobacco species (e.g., crude or extract) which is compatible with human use.

On top of tobacco, the present invention also envisages the use of the agents of the present invention (in line with the above described aspects) with other smoked, dipped, chewed, snuff or snused herbs, compatible with human consumption and which causes damage to the aerodigestive tract due to the loss of peroxidase activity.

As used herein the phrase “tobacco packaging material” refers to any auxiliary means which packages the tobacco or facilitates its consumption (carrier). Examples include, but are not limited to, rolling paper, snus bags, filter paper, tin sheets and the like.

Thus, for example, the agent may be impregnated in (attached to, absorbed in, coated with) a filter paper which comes in direct contact with the aerodigestive tract.

FIG. 1 illustrates a cigarrete filter configuration of the tobacco smoke (TS) filter of the present invention which is referred to hereinunder as a cigarette filter 10. Cigarette filter 10 is constructed of a paper lining 12 and a filter core 14 which is composed of glass fiber and is positioned adjacent to a tobacco filling 16. To enable effective delivery the agent of the present invention can be impregnated in filter lining 12. Such filters have been previously described in patent documents (39, 40), the teachings of which are herein incorporated by reference.

Alternatively, the rolling paper may be treated with the agent such that the agent is confined to that region of the paper which comes in contact with the aerodigestive tract (say about 1 cm margines).

As used herein, “aerodigestive tract” refers to saliva-lined tissues such as the lips, mouth, buccal cavity, tongue, oropharynx, throat, larynx, esophagus, upper digestive tract, saliva glands, saliva, as well as the similar mucous-lined tissues of the respiratory tract, such as the respiratory mucosa, alveoli, trachea, and lungs.

The following provides a list of non-limiting examples of agents which can be used in accordance with the teachings of the present invention.

For example, as is illustrated in Examples 1 of the Examples section which follows, antioxidants such as CN− chelators can be used to treat TS-associated loss of OPO activity. Examples of CN− chelators, such as OH—CO and additional antioxidants which can be used by this aspect of the present invention are given hereinbelow.

Preferably, the CN− chelator (e.g., OH—CO) is administered in a manner which enables establishment of a concentration of 0.5-2 mM, preferably 1 mM in body fluids, such as saliva.

CN− chelators can be effectively employed to prevent or reduce TS-associated injury in the aerodigestive tract since they act to sequester CN− which is injurious to OPO. Such capacity of OH—CO, also known as the non-CN−-bound form of cyanocobalamin, hydroxocobalamin or vitamin B12a, to prevent TS-induced loss of OPO activity represents a marked improvement over prior art methods of preventing TS-mediated oxidant injury of the aerodigestive tract since such protection has never been demonstrated or suggested by prior art methods employing other antioxidants such as GSH, ascorbate or deferoxamine, as shown in Example 1 of the Examples section, below.

As is illustrated in Example 2 of the Examples section which follows, antioxidants functional as redox-active metal ion chelators can be used to treat TS-associated death of cells, such as lymphocytes.

Redox-active metal ion chelators such as deferoxamine, and physiological antioxidants, such as GSH are examples of antioxidants suitable for use by this aspect of the present invention, other examples are given hereinbelow.

Redox-active metal ion chelators are used in a manner which enables establishment of about 1 mM concentration in body fluids (e.g., saliva). Preferably, deferoxamine is administered in a manner which enables establishment of a concentration of about 1 mM, more preferably about 5 mM in body fluids. More preferably, a mixture of deferoxamine and GSH is used in a ratio of about 1:1, preferably 5:1, respectively. When used in combination, deferoxamine and GSH body fluid concentrations of about 1 mM each are desirable although a deferoxamine concentration of 5 mM and a GSH concentration of 1 mM are also therapeutically effective. The use of antioxidants, such as GSH and redox-active metal chelators, such as deferoxamine, preferably in combination, represents a significant improvement over prior art methods of preventing or reducing TS-mediated oxidant injury in the aerodigestive tract.

Examples of CN− chelators suitable for use according to the present invention include, for example, epselen, vitamins A, C and E, selenium compounds, OH—CO, flavenoids, quinones (e.g., Q10, Q9), retinoids and carotenoids.

Examples of redox-active metal ion chelators suitable for use according to the present invention include, for example, epselen, desferioxamine, zinc-desferioxamine, polyamine chelating agents, ethylenediamine, diethylenetriamine, triethylenetetramine, triethylenediamine, tetraethylenepentamine, aminoethylethanolamine, amino ethylpiperazine, pentaethylenehexamine, triethylenetetramine-hydrochloride, tetraethylenepentamine-hydrochloride, pentaethylenehexamine-hydrochloride, tetraethylpentamine, captopril, penicilamine, N,N′-bis(3-aminopropyl)-1,3-propanediamine, N,N,Bis (2 aminoethyl) 1,3 propane diamine, 1,7-dioxa-4,10-diazacyclododecane, 1,4,8,11-tetraaza cyclotetradecane-5,7-dione, 1,4,7-triazacyclononane trihydrochloride, 1-oxa-4,7,10-triazacyclododecane, 1,4,8,12-tetraaza cyclopentadecane, and 1,4,7,10-tetraaza cyclododecane.

The iron chelator deferoxamine is also known as DES, desferal and desferioxamine.

Articles of the present invention can further comprise at least one flavorant such as wintergreen oil, oregano oil, bay leaf oil, peppermint oil, spearmint oil, clove oil, sage oil, sassafras oil, lemon oil, orange oil, anise oil, benzaldehyde, bitter almond oil, camphor, cedar leaf oil, marjoram oil, citronella oil, lavendar oil, mustard oil, pine oil, pine needle oil, rosemary oil, thyme oil, and cinnamon leaf oil.

The active ingredients may be introduced to the article of manufacturing as described above (e.g., snuff), such as in the form a dry powder, either as a mixture of antioxidants, or as a complex in protective liposomes, nanospheres or other acceptable delivery vehicles. This powder may be added in the final process of manufacturing and may also contain suitable flavors or fragrances as not infrequently used in this industry.

Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

Example 1 Hydroxocobalamin Provides Protection from Tobacco Smoke-Induced Loss of Aerodigestive Tract Antioxidant Defenses

Many common and highly debilitating oral diseases, such as cancer, periodontitis and gingivitis, result from, or are aggravated by consumption of tobacco products, such as tobacco smoking. For example, oral cancer, a frequently lethal and highly debilitating disease, results from tobacco consumption in 50-90% of cases world-wide. One widely accepted mechanism whereby cancer progression is promoted is via oxidant injury, such as protein damage caused by exposure to free radicals.

The present inventors have therefore analyzed the effects of TS on aerodigestive tract antioxidant defenses and have invented means to prevent such effects, as described below.

Exposure to Tobacco Smoke Leads to a Decrease in Oral Peroxidase and to an Increase in Protein Damage:

Oral peroxidase is the critical salivary enzymatic defense against upper digestive tract oxidant injury resulting in, for example, macromolecular damage associated with progression of diseases such as malignancies of the upper digestive tract, periodontitis and gingivitis. The mechanism of OPO mediated protection from antioxidant injury resulting from exposure to TS HCN is schematized in FIG. 2a.

The present inventors, as described below, have therefore analyzed the effects of TS on OPO activity and on salivary protein carbonylation, a well-known indicator of protein damage induced by exposure to oxidative stress and/or TS (11, 12, 42).

Methods and Materials:

Saliva collection: Whole saliva was collected under non-stimulatory conditions from healthy smokers having smoked at least 20 cigarettes a day for at least 10 years and from non-smoking subjects, as previously described (43).

Generation of tobacco smoke: Tobacco smoke was obtained from popular commercial cigarettes containing 14 mg of tar and 0.9 mg of nicotine (‘Time’ cigarettes, Dubek Ltd., Tel Aviv, Israel).

In vivo exposure of saliva to TS: The OPO activity in 2 ml saliva samples of smoker and non-smoker subjects was measured prior to and following the smoking of 1 cigarette. Subjects were prevented from being exposed to TS for 1 h prior to the experiment.

In vitro exposure of saliva to TS: The in vitro exposure of saliva samples to TS was performed using a cigarette combined with a vacuum system, as previously described (11, 12, 14, 15). Briefly, 4-5 ml saliva samples were placed in 50 ml vaccuum flasks with a sidearm to which cigarettes were connected. A vacuum was applied to the flasks and the smoke from 1 cigarette was drawn into the flask in 4-5 “puffs”. Saliva samples for analysis of OPO activity were drawn immediately prior to exposure to TS and at 0, 30 and 60 min after completion of exposure of samples to the TS (˜10 min). Flasks were incubated in a metabolic shaker at 37° C. Immediately following collection, samples were centrifuged at 800×g for 10 minutes at 4° C. to remove squamous cells and cellular debris. The resulting supernatants were subsequently analyzed for OPO activity and salivary protein carbonylation.

Analysis of OPO activity: OPO activity was measured according to the 2-nitrobenzoic-thiocyanate (NBS-SCN) assay, as previously described (20). Briefly, DTNB is reduced to NBS by addition of β-mercaptoethanol and decreases in NBS concentration by reaction with OSCN−, a product of OPO, are monitored spectrophotometrically by measuring absorbance at 412 nm at pH 5.6 (18). One unit of enzyme activity was defined as the activity required to cleave 1 μmol of NBS/min at 22° C., using a molar extinction coefficient of 12,800 (20).

Western immunoblotting analysis of protein carbonylation: Salivary proteins were separated by SDS-PAGE using a 10% gel and electro-blotted onto nitrocellulose membranes, as previously described (14). The Oxyblot Kit (Intergen Co, Purchase N.Y.) was used with anti-dinitrophenyhydrazine (DNPH) antibodies to label carbonylated proteins. Protein carbonylation levels were then visualized by reacting labelled blots with a secondary HRP-conjugated anti-rabbit antibody followed by ECL detection of secondarily labelled proteins, as previously described (14).

Statistical analysis of results: Ranges, means and SDs and SEMs were computed from the results derived from the smoker and non-smoker experimental subgroups for the in vivo and in vitro studies. Statistical analysis to compare results from subgroups was performed via 2-sample T-test for differences in means using p<0.05 as criteria to establish statistically significant differences.

Results:

Smoking tobacco smoke leads to impaired oral peroxidase activity in vivo: Following at least 1 h without exposure to TS, OPO activity levels in saliva samples from smoker subjects were found to be 82% and 85% that of non-smoker subjects in the subjects employed for the in vivo and in vitro studies, respectively, however statistical analysis indicated that these differences were not statistically significant, therefore it was concluded that baseline OPO activity levels were similar in both smokers and non-smokers (Table 1).

TABLE 1 Basal oral peroxidase activity levels in non-smoker and smoker subjects employed for the in vitro and in vivo studies are similar. In vivo studies (U/ml) In vitro studies (U/ml) Smokers Non-smokers Smokers Non-smokers N 17 16 7 11 Range 180-1,728 193-1,019 397-1,094 236-1,166 Mean 696 573 595 517 SD 417 252 237 299 SEM 101 62.9 90 91

Immediately following the smoking of 1 cigarette, a sharp drop in OPO activity levels was observed in both smoker and non-smoker subjects, with levels dropping further in the non-smokers (42.5% of pre-smoking levels, p<0.01) than in the smokers (58.5% of pre-smoking levels, p<0.01) (FIG. 2b). In the absence of subsequent exposure to TS, OPO activity levels returned to 90-100% of initial pre-smoking levels in both groups at 30 min post-smoking.

In vitro exposure to tobacco smoke results in decreased oral peroxidase activity: A different set of non-smoker and smoker subjects than those employed for the in vivo study were employed for the in vitro study. Similarly to the results obtained in the in vivo experiments described above, OPO activity levels were found to be significantly decreased at Time 0 following exposure to the TS from 1 cigarette in both study groups with loss of OPO activity being more pronounced in non-smoker saliva than in smoker saliva (FIG. 2c).

The literature reports that the saliva of non-smokers contains 0.3-1.5 mM SCN−, while that of heavy smokers contains 1.4-4.0 mM SCN−, it was postulated that the higher quantities of SCN− in heavy smokers provide protection against TS-induced reduction in OPO activity. Thus, in order to ascertain why OPO activity was slightly higher and more resistant to TS in smokers relative to non-smokers, the effect of the exogenous addition of SCN in the form of potassium thiocyanate (KSCN) to non-smoker saliva exposed to TS was analyzed. Addition of 0.5-5.0 mM KSCN to the saliva of 3 non-smoking subjects in the in vitro system and measuring OPO activity before and after smoking 1 cigarette demonstrated that addition of SCN− indeed provides significant protection against loss of OPO activity (FIG. 3).

In vivo exposure of saliva to TS causes significant salivary protein carbonylation: In vivo exposure of saliva to TS, via smoking of 1 cigarette, was found to cause significant increases in levels of salivary protein carbonylation, as assessed by Western immunoblotting analysis (FIG. 4a). The highest levels of protein carbonylation were observed at 10 min post-smoking (FIG. 4a, Lane 2). The major salivary proteins, such as amylase, acidic proline rich proteins and lysozyme were the ones found to be most carbonylated by TS.

In vitro exposure of saliva to TS causes significant salivary protein carbonylation: Similarly to the in vivo studies described above, in vitro exposure of saliva to the TS of 1 cigarette was found to cause significant increases in levels of carbonylation of major salivary protein, as assessed by Western immunoblotting analysis (FIG. 5a). No recovery in the level of protein carbonylation was found in the in vitro studies, as provision of new saliva is not possible.

These results therefore identify loss of OPO activity and concomitant macromolecular damage, such as salivary protein carbonylation, as resulting from exposure to TS.

Hydrocyanic Acid (HCN) Mediates Tobacco Smoke-Associated Loss of Oral Peroxidase Activity:

In order to elucidate the mechanism(s) whereby TS causes loss of OPO activity, the effect of various oxidants and antioxidants on OPO activity in saliva exposed to TS in vitro were analyzed, as described below.

Materials and Methods:

Generation of tobacco smoke: Tobacco smoke was obtained from popular commercial cigarettes containing 14 mg of tar and 0.9 mg of nicotine (‘Time’ cigarettes, Dubek Ltd., Tel Aviv, Israel).

In vitro exposure of saliva to TS: The in vitro exposure of saliva samples to TS was performed as described above with the modification that the same saliva samples were subjected to multiple exposures to the TS of 1 cigarette at 20 min intervals.

Analysis of OPO activity: Analysis of OPO activity in TS-treated saliva samples was performed as described above.

Treatment of saliva with TS in the presence of oxidants, antioxidants, and inhibitors: The various reagents and materials were added to saliva prior to treatment with TS at the specified concentrations. Unless otherwise specified, all chemicals were obtained from Sigma Chemical Corp. (St. Louis, Mo., USA).

Statistical analysis: Compilation and statistical analysis of results was performed as described above for in vitro studies.

Results:

Exposure of saliva to the TS of 3 cigarettes resulted in a TS dose-dependent decrease in OPO activity to 22% of pre-TS exposure levels (FIG. 6).

Dapsone a, has been shown to specifically inhibit SPO but not MPO at acidic pH (44). Thus, in order to determine which of the MPO or SPO activity components of OPO activity are lost as a result of exposure to TS, OPO activity in TS-treated saliva was measured in the presence of increasing concentrations of 50-150 dapsone. Loss of OPO activity due to exposure to TS was found to be 60-85% in the presence of 150 μM of dapsone whereas control MPO activity was unaffected, (FIG. 7) and loss of OPO activity was similar in the presence or absence of 150 μM dapsone in saliva containing ˜40% MPO (FIG. 8) indicating that TS affects SPO and MPO activities to a similar extent.

In the presence or absence of 1 mM of ascorbate or the antioxidants GSH or deferoxamine, loss of OPO activity in TS-treated saliva was found to be similar (FIG. 9) and exposure of saliva with 1 mM ascorbate or the oxidant 100 μM FeCl3 for 2 h did not inhibit OPO activity (FIG. 10).

In the presence of 2 mM 80 μM acrolein, and 20 μM crotonaldehyde, major aldehydes present in TS, OPO activity in TS-treated saliva was unaffected (FIG. 11).

Levels of OPO activity in TS-treated saliva having lost ˜68% of initial OPO activity were restored to 94% of initial levels following subsequent treatment with 18 h of dialysis (FIG. 12). Thus, loss of OPO activity in TS-treated saliva can be treated, presumably, via removal of low molecular weight molecules.

Cyanide is a known inhibitor of heme peroxidase, and the gas-phase TS of various cigarette brands have been found to contain 2-233 μg of KCN (45). Thus, in order to test the hypothesis that dialysis treatment restores OPO activity in TS-treated saliva via removal of KCN, saliva samples were treated with 150 μM KCN and analyzed for OPO activity. This concentration of KCN was observed to cause a loss of ˜65% of OPO activity after only 2 min of incubation, which loss was considerably reversed by dialysis (FIG. 13), thereby indicating that KCN is indeed capable of inhibiting OPO activity.

Hydroxocobalamin prevents tobacco smoke-associated loss of salivary peroxidase: Since CN− ion was found to be involved in TS-associated loss of the OPO activity, the present inventors have attempted to provide a means of preventing such loss of OPO activity via the use of a CN− chelator.

Addition of increasing amounts of OH—CO, a known chelator of CN, was shown to prevent, to a significant extent, the loss of OPO activity saliva 40 min after treatment with TS (FIG. 14). Similarly, addition of 1 mM OH—CO to saliva treated for 2 min with 1 mM KCN was sufficient to completely prevent the KCN-associated loss of 40-60% of OPO activity (FIG. 15). Furthermore, preincubation of saliva with OH—CO could prevent both TS- and KCN-associated loss of OPO activity, indicating that cyanide indeed mediates TS-associated loss of OPO activity.

These results therefore indicate that, according to the present invention, OH—CO can be employed to effectively reduce or prevent the occurrence of diseases, such as malignancies of the aerodigestive tract, periodontitis and gingivitis associated with exposure to TS.

Example 2 The Antioxidants Deferoxamine and Glutathione Prevent Upper Aerodigestive Tract Lymphocyte Death Associated with Exposure to Tobacco Smoke

Many diseases of the aerodigestive tract are associated with consumption of tobacco products or betel nut chewing. For example, oral SCC is the most common malignancy of the head and neck, having a high rate of morbidity and mortality. In as many as 90% of the cases, this cancer is induced by exposure of oral epithelial tells to tobacco products such as TS, or chewed betel nut. This exposure always occurs in the presence of saliva and presumably is induced by free radicals. Thus, the effects of TS on cells in the presence of saliva was examined, as described below.

Materials and Methods:

Mutagenic alterations of oral mucosal cells induced by TS occur in the presence of saliva (22, 23). Thus, in order to explore the interaction between TS and saliva on cells, intact cells were exposed to TS, alone or in the presence of saliva. Lymphocytes were employed since they are highly sensitive to oxidant injury.

Saliva collection: Saliva was collected from healthy subjects, 3 males and 3 females ranging from 21-47 years of age, under non-stimulatory conditions in a quiet room during the morning between 8 am and noon. Saliva collection was performed at least 1 h after eating by spitting into a recipient for 10 minutes, as previously described (43). Following collection, saliva was immediately centrifuged at 800×g for 10 min at 4° C. to remove cells and cell debris and the resulting supernatant was used for biochemical analyses.

Lymphocyte isolation: Blood from 10 consenting, healthy, non-smoking subjects, 5 males and 5 females ranging from 18-55 years of age, was drawn into EDTA-containing vacutainers. Lymphocytes were isolated by Ficoll-Hypaque (Sigma) gradient centrifugation according to the manufacturer's instructions and lymphocytes were suspended at 107 cells/ml PBS (Beit-Ha'emek Industries, Israel), and used immediately in experiments.

Generation of tobacco smoke: Tobacco smoke was obtained from popular commercial cigarettes containing 14 mg of tar and 0.9 mg of nicotine (‘Time’ cigarettes, Dubek Ltd., Tel Aviv, Israel).

Treatment of lymphocytes with TS in the presence of saliva: Exposure of lymphocytes to TS was carried out by attaching a Time cigarette (capable of removing particles>0.1 mm in diameter) in which the filter tip was removed to a Cambridge filter which was combined with a vacuum system to aspirate gas-phase TS inside sealed 250 ml flasks containing lymphocytes suspended in 12-15 ml PBS, as previously described (14, 15). A reproducible vacuum was created in the flask and upon application of vacuum for 5 s, 80-100 ml of TS from the lit cigarette was drawn into the flask. The chemical addititives GSH, NAC, deferoxamine, ascorbate (Asc), uric acid or FeCl3 (Sigma) were added at the specified concentrations. Saliva treatment of lymphocytes was performed by suspending these in PBS supplemented with 30% (v/v) saliva. Following treatment with the TS of 0.5 cigarettes, flasks were incubated at 37° C. for 20 min in a metabolic shaker and subjected to a further 4 treatments with TS, as described above.

Measurement of lymphocyte survival: Lymphocyte viability was assessed by Trypan Blue exclusion assay.

Western immunoblotting analysis of lymphocyte protein carbonylation: Lymphocytes were washed twice in PBS following treatment by centrifugation at 2,000 rpm for 2 minutes to remove saliva and other components of the incubation medium. Lymphocytes were then centrifuged at 14,000 rpm for 1 minute and lysed by sonication for 10 seconds in lysis buffer containing 20 mM Tris pH 7.4, 1 mM EGTA, 1 mM PMSF, 50 μM NaVO4 and 50 mM NaF. Lysates were centrifuged at 14,000 rpm for 1 minute and supernatants were harvested for analysis. Carbonylation analysis was performed as described above.

Statistical analysis: Results for statistical analysis were obtained from the control subgroup (lymphocytes in PBS) and from the various treatment subgroups. Means, SDs and SEMs were computed and results between the subgroups were analyzed and compared via one-way analysis-of-variance (46) using the Bonferroni Multiple-Comparison Test Model (47) to determine significant differences between computed means. The means between each pair of means was analyzed via T-test For Paired Differences and means between each two subgroups were compared via Two Sample T-test For Differences in Means (48).

Results:

During an 80 minute incubation of lymphocytes in PBS alone or in PBS supplemented with 30% (v/v) saliva, neither cell death nor protein carbonylation (FIGS. 16 and 17, respectively) occurred. Treatment of lymphocytes with TS in the absence of saliva resulted in time-dependent increases in cell death and protein carbonylation levels, peaking after 80 minutes with a death rate of 44% (p<0.01). Addition of saliva to lymphocyte suspensions during TS treatment synergistically potentiated the lethal effect of the TS, as demonstrated by an 86.2% (p<0.01) death rate and highly elevated protein carbonylation levels (FIGS. 16 and 17, respectively).

Addition of saliva to lymphocytes incubated for 80 minutes in PBS alone resulted in a mild reduction of protein carbonylation levels (FIG. 17). This was shown to be due to uric acid, the major non-enzymatic antioxidant in saliva (18, 28, 49), since addition of uric acid to the suspension medium instead of saliva led to reduced carbonylation levels (FIG. 18).

Supplementation of suspension medium with 1 mM ascorbate or 1 mM NAC did not alter the death rate of lymphocytes exposed to TS and saliva, whereas, addition of 1 mM GSH partially prevented this lymphocyte loss. At 20 and 80 minutes following TS and saliva treatment, the lymphocyte survival rates dropped to 57.1% and 13.8% respectively, whereas in the presence of GSH these survival rates were 93.4% (p=0.0001) and 24.3% (p=0.0037) respectively (FIG. 19).

Similarly to GSH treatment, addition of 1 mM deferoxamine significantly prevented lymphocyte death at 80 minutes, with a survival rate of 23.7% (p=0.005) (FIG. 19).

The unique protection offered by GSH at 20 minutes, in contrast to the lack of protection by all other antioxidants examined, was also demonstrated by assessing lymphocyte protein carbonylation levels (FIG. 20).

Role of aldehydes: The demonstrated protection from TS+saliva-induced lymphocyte death by GSH led to the hypothesis that this effect was mediated by aldehydes associated with TS. Indeed, addition of various exogenous aldehydes known to be present in TS to lymphocytes suspended in PBS similarly led to cell death. Namely, in the presence of 80 μM acrolein, 20 μM crotonaldehyde or 2 mM acetaldehyde survival rates after 20 minutes were 63.3%, 80.3% and 93.6%, respectively, and at 80 minutes these death rates were 57.3%, 75.3% and 92.7%, respectively. This mimicking of the TS+saliva-induced lethality by exogenous aldehydes was further supported by protection from cell death being mediated by addition of GSH, similarly to GSH-mediated viability restoration in the presence of TS and saliva. Addition of GSH in the presence these aldehydes resulted in lymphocyte survival rates at 20 minutes of 93.3%, 82.3% and 93.3%, respectively and at 80 minutes these survival rates were 87.4%, 81.3% and 93.6%, respectively. As the survival rate of control lymphocytes was 95.7%, it was concluded that acetaldehyde does not play a role while acrolein, and to a slightly lesser degree crotonaldehyde, are the aldehydes causing cell death.

The pattern of injury induced by these aldehydes and the protection provided by GSH was similar in the concomitant levels of protein carbonylation (FIG. 21). However, in contrast to the profound synergistic effect demonstrated by treatment with saliva and TS, treatment with acrolein, the most potent thiol demonstrated, with saliva did not significantly alter the survival rates (57.9% and 66.4% respectively) and did not enhance carbonylation levels (FIG. 22). This ruled out the possibility that TS-saliva-based synergism involves TS-based aldehydes.

Role of transition metal ions: The relatively moderate impact which exogenous aldehydes had on lymphocyte survival and the previously noted lack of related aldehyde-saliva synergy raised the possibility that there might be another mechanism mediating the injurious effects of TS on lymphocyte survival. The fact that deferoxamine had a similar protective effect as GSH pointed to the possible role of redox-active iron ions. In order to test this hypothesis, the role of redox-active iron was analyzed via an assay employing detection of redox-active iron mediated potentiation of ascorbate pro-oxidant activity. The role of redox-active iron ions was also analyzed using deferoxamine, a very potent iron chelator.

Addition of ascorbate to saliva-containing medium without exposure to TS resulted in reduction of the lymphocyte survival rate to 78% (p<0.01; FIG. 23, column 6). The modulatory role of redox-active iron was further demonstrated by addition of ascorbate to PBS containing iron but not saliva, as FeCl3. This yielded similar results to those obtained by addition of ascorbate to saliva. Reduction of the lymphocyte survival rate to 77% was demonstrated (FIG. 23, Column 8), which reduction was totally prevented in the presence of deferoxamine (FIG. 23, Column 9).

Neither ascorbate nor deferoxamine were found to have any modulatory effect on the direct moderate lethal effect which TS had on lymphocytes in the absence of saliva (61% survival rate) (FIG. 24). This proved that redox-active iron ions did not originate from the TS but rather from the saliva, as described.

Protection from TS-saliva mediated lymphocyte death by GSH and deferoxamine: Since the two major underlying mechanisms of lymphocyte loss identified above were based on the action of aldehydes and highly reactive free radicals mediated by redox-active iron, experiments aimed at protecting lymphocytes from both injurious mechanisms simultaneously were performed as follows.

Lymphocytes were exposed to TS and saliva in the presence of 1 mM GSH or both 1 mM GSH and deferoxamine at either low (1 mM) or high (5 mM) concentration. Both survival rates and carbonylation levels were examined after 20 and 80 minutes. Very significant protection against the lethal effect of TS was obtained by addition of 1 mM GSH and 5 mM deferoxamine to the saliva-containing medium prior to TS treatment of the lymphocytes. Whereas the survival rates of the non-protected lymphocytes were 57.2% and 14.4% at 20 and 80 minutes respectively, these rates climbed to 90.8% (p=0.0001) and 61.6%, respectively (Table 2). Furthermore, concomitant with this very significant improvement in survival rate, protein carbonylation levels dropped to nearly nil (FIG. 25, lanes 7,8).

TABLE 2 Effect of GSH alone or in combination with 1 mM deferoxamine on % survival of TS + saliva-treated lymphocytes. 20 minute treatment 80 minute treatment Treatment Mean SEM p Mean SEM p — 57.2 3.7 14.4 2.6 1 mM GSH 90.6 2.1 0.0001 28.8 3.4 0.0001 1 mM GSH + 92.2 1.6 0.0001 57.4 5.3 0.0001 1 mM deferoxamine 1 mM GSH + 90.8 2.7 0.0001 61.6 3.0 0.0001 5 mM deferoxamine

A comprehensive mechanism for the induction of disease by TS is suggested in which a new role is attributed to saliva losing its antioxidant capacity and becoming a potent prooxidant milieu in the presence of TS (FIG. 26). This mechanism is based on the results obtained in the current study as well as on the well-known observation that oral cancer mostly occurs in oral epithelial cells exposed to tobacco products (from TS or chewing betel nut) in the presence of saliva.

These results therefore indicate that GSH and deferoxamine, according to the present invention, can therefore be effectively employed to prevent or reduce diseases of the aerodigestive tract associated with cell death caused by tobacco consumption.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

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Claims

1. A article of manufacturing comprising tobacco and a tobacco packaging material, at least a portion of said tobacco and/or tobacco packaging material being in contact with an aerodigestive tract of a subject in use thereof, and whereas said at least a portion of said tobacco and/or tobacco packaging material comprises at least one agent capable of reducing or preventing tobacco associated loss of peroxidase activity in said aerodigestive tract, said at least one agent being selected from the group consisting of a cyanide chelator and an iron chelator.

2. An article of manufacturing comprising a tobacco packaging material at least a portion of which being in contact with an aerodigestive tract of a subject in use thereof, wherein said at least a portion comprises an agent capable of reducing or preventing tobacco associated loss of peroxidase activity in said aerodigestive tract.

3. The article of manufacturing of claim 1, wherein said tobacco packaging material comprises a filter and said at least one agent is impregnated in a paper of said filter being in contact with said aerodigestive tract of said subject in use thereof.

4. The article of manufacturing of claim 1, wherein said tobacco is smokeless tobacco.

5. The article of manufacturing of claim 1, wherein said tobacco is smoked tobacco.

6. The article of manufacturing of claim 1, wherein said tobacco packaging material is selected from the group consisting of a rolling paper, a filter paper, a suns bag packaging, a cigarette, a pipe and a tin sheet packaging.

7. The article of manufacturing of claim 1, selected from the group consisting of a snuff, a cigarette, a snus, a Gutka, a plug, a twist, a scrap and tobacco water.

8. The article of manufacturing of claim 1, wherein said cyanide chelator is hydroxocobalamin.

9. The article of manufacturing of claim 1, wherein said agent is capable of reducing or preventing tobacco smoke-associated death of cells in the digestive tract of a subject.

10. The article of manufacturing of claim 9, wherein said cells are lymphocytes and/or epithelial cells.

11. The article of manufacturing of claim 1, wherein said iron chelator is deferoxamine.

12. The article of manufacturing of claim 1, wherein said at least one agent further comprises an antioxidant.

13. The article of manufacturing of claim 12, wherein said antioxidant is glutathione.

14. The article of manufacturing of claim 1, wherein said tobacco packaging material comprises a filter and said at least one agent is impregnated in a paper of said filter being in contact with said aerodigestive tract of said subject in use thereof.

15. The article of manufacturing of claim 2, wherein said tobacco is smokeless tobacco.

16. The article of manufacturing of claim 2, wherein said tobacco is smoked tobacco.

17. The article of manufacturing of claim 2 wherein said tobacco packaging material is selected from the group consisting of a rolling paper, a filter paper, a suns bag packaging, a cigarette, a pipe and a tin sheet packaging.

18. The article of manufacturing of claim 2, wherein said ron chelator is deferoxamine.

19. The article of manufacturing of claim 2, wherein said at least one agent further comprises an antioxidant.

20. The article of manufacturing of claim 19, wherein said antioxidant is glutathione.

Patent History
Publication number: 20100108086
Type: Application
Filed: Jan 22, 2008
Publication Date: May 6, 2010
Applicant: Technion Research & Development Foundation Ltd (Haifa)
Inventors: Abraham Z. Reznick (Nofit), Rafael M. Nagler (Timrat)
Application Number: 12/523,995
Classifications
Current U.S. Class: Cigar Or Cigarette (131/360); Tobacco Or Tobacco Substitute Product Or Component Part Thereof (131/347); Plug Tobacco (131/366)
International Classification: A24B 1/00 (20060101); A24F 47/00 (20060101); A24B 13/00 (20060101);