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EP2152102A2 - Zusammensetzungen, artikel und verfahren zur prävention oder reduktion von mit tabak assoziierten schäden - Google Patents

Zusammensetzungen, artikel und verfahren zur prävention oder reduktion von mit tabak assoziierten schäden

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Publication number
EP2152102A2
EP2152102A2 EP08738329A EP08738329A EP2152102A2 EP 2152102 A2 EP2152102 A2 EP 2152102A2 EP 08738329 A EP08738329 A EP 08738329A EP 08738329 A EP08738329 A EP 08738329A EP 2152102 A2 EP2152102 A2 EP 2152102A2
Authority
EP
European Patent Office
Prior art keywords
agent
tobacco
group
article
manufacturing
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP08738329A
Other languages
English (en)
French (fr)
Inventor
Rafael M. Nagler
Moshe Gavish
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Technion Research and Development Foundation Ltd
Original Assignee
Technion Research and Development Foundation Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Technion Research and Development Foundation Ltd filed Critical Technion Research and Development Foundation Ltd
Publication of EP2152102A2 publication Critical patent/EP2152102A2/de
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
    • A24B15/18—Treatment of tobacco products or tobacco substitutes
    • A24B15/28—Treatment of tobacco products or tobacco substitutes by chemical substances
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
    • A24B15/18—Treatment of tobacco products or tobacco substitutes
    • A24B15/28—Treatment of tobacco products or tobacco substitutes by chemical substances
    • A24B15/30—Treatment of tobacco products or tobacco substitutes by chemical substances by organic substances
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24D—CIGARS; CIGARETTES; TOBACCO SMOKE FILTERS; MOUTHPIECES FOR CIGARS OR CIGARETTES; MANUFACTURE OF TOBACCO SMOKE FILTERS OR MOUTHPIECES
    • A24D3/00—Tobacco smoke filters, e.g. filter-tips, filtering inserts; Filters specially adapted for simulated smoking devices; Mouthpieces for cigars or cigarettes
    • A24D3/06—Use of materials for tobacco smoke filters
    • A24D3/14—Use of materials for tobacco smoke filters of organic materials as additive
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group

Definitions

  • the present invention in some embodiments thereof, relates to articles, compositions and methods for reducing or preventing tobacco-induced cellular or macromolecular damage.
  • 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.
  • Tobacco consumption leads to development or enhancement of atherosclerosis, cardiovascular diseases, chronic obstructive pulmonary disease, lung cancer, as well as other forms of cancer and peripheral vascular disease.
  • Cardiovascular disease is the main cause of death due to smoking. Cardiovascular disease can take many forms depending on which blood vessels are involved. Main forms include coronary thrombosis, which may lead to a heart attack; cerebral thrombosis, which may lead to collapse, stroke and paralysis; affected kidney arteries, which result in high blood pressure or kidney failure; and blockage of the vascular supply to the legs, which may lead to gangrene and amputation.
  • Tobacco consumers are more likely to get cancer than non-smokers, particularly carcinomas of the mouth, pharynx, esophagus and lung.
  • Other types of cancers associated with tobacco consumption include bladder cancer, cancer of the oesophagus, cancer of the kidneys, cancer of the pancreas and cervical cancer.
  • COPD chronic obstructive pulmonary disease
  • 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.
  • SCC 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.
  • the major inducer of oral SCC is exposure to tobacco which is considered to be responsible for 50-90 % of cases world- wide [Epstein and Scully, SCD Special Care in Dentistry 1997; 17: 120-8; Holleb et al. Textbook of Clinical Oncology. The American Cancer Society, 1991].
  • 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 has 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.
  • 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.
  • TS has over 3,000 different constituents, of which many are toxic, carcinogenic and/or generate free radical species.
  • Oxygen free radicals are atoms or molecules containing an unpaired electron.
  • Oxygen free radicals include the superoxide free radical ( 02 ) and the hydroxyl radical (OH-) which, together with hydrogen peroxide (H 2 O 2 ) and singlet oxygen ( l O2), 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 therefore 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.
  • mainstream and side stream TS 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.
  • mainstream smoke emission is also markedly reduced both in low and in ultra low tar yield cigarettes.
  • the emissions of toxic and carcinogenic components in side stream smoke are not significantly reduced in filter cigarettes when compared to non-filter counterparts.
  • side stream smoke is a major contributor to environmental smoke, affecting both the smoker and their non-smoking counterparts, so called secondary smokers.
  • 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 H 2 O 2 .
  • Oxidants in gas-phase smoke are reactive carbon- and oxygen-centered radicals with extremely short half lives.
  • 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 metal ions such as iron and copper
  • TS low-reactive free radicals found in TS
  • superoxide radicals participate in the deleterious Haber- Weiss and Fenton reactions, in which the highly reactive hydroxyl free radicals are produced.
  • salivary enzymes such as amylase, lactic dehydrogenase (LDH), and acid phosphatase were considerably affected by TS (Nagler et al., 2000, supra; Nagler et al. J Lab CHn Med 2001; 137:363-9], 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.
  • TS-based aldehydes such as acrolein and crotonaldehyde, as well as oxygen free radicals were implicated as the causative agents affecting the above enzymes.
  • Glutathione a sulfur-containing tripeptide (L-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 peroxidation is the pivotal enzymatic activity of the salivary antioxidant system.
  • Oral peroxidase activity is composed of the combined activity of two peroxidases, salivary peroxidase (SPO) and myeloperoxidase (MPO).
  • SPO salivary peroxidase
  • MPO myeloperoxidase
  • Salivary peroxidase which is secreted by the major salivary glands, mainly the parotid gland, contributes 80 % of the total oral peroxidase (OPO) activity
  • MPO produced by leukocytes, contributes the remaining 20 % of OPO activity.
  • Oral peroxidase performs two functions preventing oxidant injury; it reduces the level of H 2 O 2 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.
  • SCN ⁇ thiocyanate ion
  • 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.
  • cigarette filters are used to trap TS tar but do not affect the gas- phase compounds.
  • dipeptide compounds with pharmaceutical properties to increase glutathione levels were employed (see, for example, U.S. Patent No. United States Patent 4,761,399).
  • a further approach utilized a glycine carboxylic acid alkyl mono-ester of glutathione to increase cellular GSH levels (see, for example, U.S. Patent No. 4,710,489).
  • compositions for reducing tobacco associated damage in the aerodigestive tract (see, U.S. Patent No. 6,789,546, which is incorporated by reference as if fully set forth herein).
  • These compositions include active agents which are capable of reducing or preventing tobacco associated loss of peroxidase activity in the aerodigestive tract.
  • U.S. Patent 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.
  • 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. Patent 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
  • U.S. Patent 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. Patent 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).
  • Patent 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.
  • PCT/IL2008/000101 by the present assignee, describes methods, pharmaceutical compositions, oral compositions, filters and tobacco products for preventing or reducing tobacco smoke-associated injury in the aerodigestive tract of a subject, and which 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.
  • Some of the agents described in this document are CN chelators and iron chelators.
  • D-Penicillamine (see, Figures Ia and Ib), is a known compound often considered as a cysteine analog and is further known, inter alia, as a copper chelator (See, Figure Ib).
  • the following background art describes some of the recently disclosed features of D-penicillamine: Handel et al., Clinical and Experimental Pharmacology and Physiology (2000)
  • penicillamine beneficially affects tobacco-associated cellular and macromolecular damage induced by exposure to tobacco smoke, even in the absence of saliva, and thus can serve as a potent agent for treating tobacco-associated damage. Similar effects are exhibited by copper chelating agents.
  • an article of manufacturing comprising tobacco and a tobacco packaging material, wherein at least a portion of the tobacco and/or tobacco packaging material comprises an agent selected from the group consisting of penicillamine and a structural analog of penicillamine.
  • an article of manufacturing comprising tobacco and an agent being incorporated in at least a portion of the tobacco, the agent being selected from the group consisting of penicillamine and a structural analog of penicillamine.
  • an article of manufacturing comprising a tobacco packaging material and an agent being incorporated in at least a portion of the tobacco packaging material, the agent being selected from the group consisting of penicillamine and a structural analog of penicillamine.
  • an article of manufacturing comprising tobacco and a tobacco packaging material, wherein at least a portion of the tobacco and/or tobacco packaging material comprises an agent, the agent being a copper chelating agent.
  • an article of manufacturing comprising tobacco and an agent being incorporated in at least a portion of the tobacco, the agent being a copper chelating agent.
  • an article of manufacturing comprising a tobacco packaging material and an agent being incorporated in at least a portion of the tobacco packaging material, the agent being a copper chelating agent.
  • At least a portion of the tobacco and/or the tobacco packaging material is in contact with an aerodigestive tract of a subject using the article of manufacturing.
  • the agent is penicillamine.
  • the agent is D-penicillamine.
  • the structural analog of penicillamine has the general formula:
  • X is O or NR 6 ;
  • Y is O;
  • A is CR 7 R 8 or CR 7 R 8 -CR 9 R 10 ;
  • R 1 is selected from the group consisting of hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl and a substituted or unsubstituted aryl, whereas when anyone of the alkyl, aryl and the cycloalkyl is substituted, the substituent is independently selected from the group consisting of alkyl, cycloalkyl, alkoxy, aryl and aryloxy;
  • R 2 is hydrogen or alkyl
  • R 3 and R 4 are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl and aryl, or, alternatively, R 3 and R 4 are linked therebetween so as to form a five- or six-membered nitrogen-containing heteroalicyclic ring, or, alternatively, at least one of R 3 and R 4 forms a five- or six-membered heteroalicyclic ring with R 5 ;
  • R 5 is selected from the group consisting of hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl and a substituted or unsubstituted aryl, whereas when anyone of the alkyl, the cycloalkyl and the aryl is substituted, the substituent is independently selected from the group consisting alkyl, cycloalkyl, alkoxy, aryl, aryloxy, carbonyl, aldehyde and carboxy, or, alternatively, R 5 forms a five- or six-membered heteroalicyclic ring with one of R 3 and R 4 ;
  • R ⁇ is selected from the group consisting of hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl and a substituted or unsubstituted aryl, whereas when anyone of the alkyl, the aryl and the cycloalkyl are substituted, the substituent is independently selected from the group consisting of alkyl and cycloalkyl, or, alternatively, R 6 forms with Ri a nitrogen-containing five-, six- or seven-membered ring; and
  • R 7 , R 8 , R 9 and Ri 0 are each independently selected from the group consisting of hydrogen and alkyl.
  • the penicillamine or the structural analog thereof is capable of suppressing an innate immune activity in a subject using the article of manufacturing.
  • the penicillamine or the structural analog thereof is capable of inhibiting inflammation in a subject using the article of manufacturing.
  • the agent is capable of reducing or preventing tobacco smoke-associated damage in a subject using the article of manufacturing.
  • the copper chelating agent is selected from the group consisting of penicillamine, trientine, ethylendiamine, diethylenetriamine, triethylenetetramine, triethylenediamine, aminoethylethanolamine, aminoethylpiperazine, pentaethylenehexamine, triethylenetetramine, captopril, N 5 N'- bis(3-aminopropyl)-l,3-propanediamine, N,N'-Bis(2-animoethyl)-l,3-propanediamine, l,7-dioxa-4,10-diazacyclododecane, 1,4,8,1 l-tetraazacyclotetradecane-5,7-dione, 1,4,7- triazacyclononane, l-oxa-4,7,10-triazacyclododecane, 1,4,8,12- tetraazacyclopentadecane and 1,4,7,10-te
  • the copper chelating agent is a linear or cyclic polyamine.
  • the at least a portion of the tobacco and/or the tobacco packaging material further comprises at least one additional agent capable of reducing or preventing tobacco smoke-associated damage in a subject using the article of manufacturing.
  • the additional agent is selected from the group consisting of an antioxidant, an iron chelating agent, a cyanide chelating agent and an agent capable of reducing or preventing tobacco associated loss of peroxidase activity in an aerodigestive tract of the subject.
  • the agent is desferal.
  • the tobacco packaging material comprises a filter and the agent is impregnated in a paper of the filter.
  • the tobacco is smokeless tobacco.
  • the tobacco is smoked tobacco.
  • the tobacco packaging material is selected from the group consisting of a rolling paper, a filter paper, a snus bag packaging, a cigarette, a pipe and a tin sheet packaging.
  • 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.
  • a method of treating or preventing a tobacco-associated damage in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent selected from the group consisting of penicillamine and a structural analog thereof.
  • an agent selected from the group consisting of penicillamine and a structural analog thereof in the manufacture of a medicament for treating or preventing a tobacco-associated damage.
  • a pharmaceutical composition comprising an agent selected from the group consisting of penicillamine and a structural analog thereof and a pharmaceutically acceptable carrier, the composition being packaged in a packaging material and identified in print, in or on the packaging material, for use in the treatment of a tobacco- associated damage.
  • a method of treating or preventing a tobacco-associated damage in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent, the agent being a copper chelating agent.
  • an agent being a copper chelating agent in the manufacture of a medicament for treating or preventing a tobacco-associated damage.
  • a pharmaceutical composition comprising an agent and a pharmaceutically acceptable carrier, the agent being a copper chelating agent, the composition being packaged in a packaging material and identified in print, in or on the packaging material, for use in the treatment of a tobacco-associated damage.
  • the tobacco-associated damage is effected in a mucosal tissue.
  • the tobacco-associated damage is effected in a non-mucosal tissue.
  • the agent is D-penicillamine.
  • the structural analog of penicillamine has the general formula described hereinabove.
  • the copper chelating agent is selected from the group consisting of penicillamine, trientine, ethylendiamine, diethylenetriamine, triethylenetetramine, triethylenediamine, aminoethylethanolamine, aminoethylpiperazine, pentaethylenehexamine, triethylenetetramine, captopril, N,N'- bis(3-aminopropyl)-l,3-propanediamine, N,N'-Bis(2-animoethyl)-l,3-propanediamine, l,7-dioxa-4,10-diazacyclododecane, 1,4,8,1 l-tetraazacyclotetradecane-5,7-dione, 1,4,7- triazacyclononane, l-oxa-4,7,10-triazacyclododecane, 1,4,8,12- tetraazacyclopentadecane and 1,4,7,
  • the agent is used in combination with at least one additional agent that is capable of reducing or preventing the tobacco-associated damage.
  • the additional agent is an antioxidant.
  • the antioxidant is desferal.
  • an article of manufacturing comprising a filter and an agent comprised with the filter, the agent being selected from the group consisting of penicillamine and a structural analog of penicillamine, as described herein, and the filter being designed and configured so as to enable release of the agent therefrom when in use by a subject.
  • an article of manufacturing comprising a filter and an agent comprised with the filter, the agent being a copper chelating agent, as described herein, and the filter being designed and configured so as to enable release of the agent therefrom when in use by a subject.
  • an oral composition comprising an agent selected from the group consisting of penicillamine and a structural analog of penicillamine, as described herein, the composition being in the form of a toothpaste, powder, liquid dentifrice, mouthwash, denture cleanser, chewing gum, lozenge, paste, gel or candy.
  • an oral composition comprising a copper chelating agent, as described herein, the composition being in the form of a toothpaste, powder, liquid dentifrice, mouthwash, denture cleanser, chewing gum, lozenge, paste, gel or candy.
  • the oral composition further comprises a flavorant.
  • a medical device comprising an agent selected from the group consisting of penicillamine and a structural analog of penicillamine, as described herein, the medical device being designed and configured to deliver the agent to a bodily site.
  • a medical device comprising a copper chelating agent, as described herein, the medical device being designed and configured to deliver the agent to a bodily site.
  • the medical device is for delivering the agent by topical or transdermal application.
  • all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.
  • methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control.
  • the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
  • FIGs. la-b present the 2-D chemical structure of D-penicillamine ( Figure Ia) and of Penicillamine as a copper chelator, in which one atom of copper is combined with two molecules of PenA ( Figure Ib).
  • FIG. 2 is schematic diagram depicting the construction of a filter paper impregnated with agents according to embodiments of the present invention.
  • FIG. 4 is a bar graph presenting the survival rates of H 1299 cells incubated in the presence of medium+saliva and exposed to CS (rhomb ); of H 1299 cells incubated in the presence of medium alone and exposed to CS (inlaid ); of H 1299 cells incubated in the presence of medium+saliva (stripe ); and of H 1299 cells incubated in the presence of medium (
  • FIG. 5b is a bar graph presenting the survival rate of H 1299 cells incubated under the condition described herein for Figure 5a, at 120 minutes, with SDs.
  • FIGs. 6a and 6b are bar graphs presenting the survival rate of H 1299 cells incubated in the presence of medium+saliva+5 mM PenA and exposed to CS; of H1299 cells incubated in the presence of medium+5 mM PenA and exposed to CS; of H 1299 cells incubated in the presence of medium alone and exposed to CS; of H 1299 cells incubated in the presence of medium+saliva; of H 1299 cells incubated in the presence of medium+saliva and exposed to CS; and of H 1299 cells incubated in the presence of medium, as the percent of viable cells out of the control group ( Figure 6a) and out of the total number of cells.
  • FIG. 8 presents data plots depicting the effects of 5 mM Penicillamine (PenA) on survival of H 1299 lung cancer cells incubated in the presence of medium+saliva (A); of H 1299 cells incubated in the presence of medium alone (B); of Hl 299 cells incubated in the presence of medium+saliva and 5mM PenA (C); of H 1299 cells incubated in the presence of medium with 5 mM PenA (D); of H 1299 cells incubated in the presence of medium+saliva and exposed to CS (E); ofH1299 cells incubated in the presence of medium alone and exposed to CS (F); ofH1299 cells incubated in the presence of medium+saliva and 5 mM PenA and exposed to CS (G); and of H 1299 cells incubated in the presence of medium with 5mM PenA and exposed to CS (H).
  • PenA 5 mM Penicillamine
  • FIG. 13 presents bar graphs showing the synergistic effect of 5 mM Desferal and 5mM PenA on the survival of H 1299 human lung cancer cells treated as described herein.
  • FIG. 14 presents bar graphs showing the CS induced changes in mitochondrial membrane potential without treatment (left bars), in the presence of PenA (middle bars) and in the presence of DES (right bars).
  • H 1299 cells incubated in the presence of medium+saliva and exposed to CS (rhomb). H 1299 cells incubated in the presence of medium alone and exposed to CS (inlaid). Samples were also incubated in the presence of 5mM PenA or 5mM DES prior the exposure of CS. Cells were incubated for 30 minutes with JC-I followed by exposure to CS for 120 minutes.
  • FIG. 15 presents bar graphs showing that CS leads to mitochondrial membrane damage.
  • FIG. 16 presents the Western blot analysis showing the effect of 5 mM PenA on the protein levels of total p53 following CS exposure and saliva supplementation in H 1299 cells.
  • Total protein (20 ⁇ g) were loaded onto gradient SDS gels and transferred to nitrocellulose.
  • FIG. 17 presents the Western blot analysis showing the effect of 2 mM GSH on the protein levels of total p53 following CS exposure and saliva supplementation.
  • Total protein (20 ⁇ g) were loaded onto gradient SDS gels and transferred to nitrocellulose.
  • the present invention is of articles, methods and compositions for preventing or reducing tobacco smoke-associated damage.
  • the present invention in some embodiments thereof, is of methods, pharmaceutical compositions, oral compositions, medical devices, filters and tobacco products, which are useful in preventing or reducing tobacco smoke-associated damage, and which utilize penicillamine, structural analogs thereof and/or other copper chelating agents.
  • penicillamine structural analogs thereof and/or other copper chelating agents.
  • Tobacco consumption such as in the form of smoking, chewing, dipping or snuffing, is associated with pathogenesis of many diseases.
  • the present inventors have previously described novel smoking filters and oral compositions for reducing tobacco associated damage in the aerodigestive tract (see, U.S. Patent 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. Some of the present inventors have previously taught tobacco compositions and tobacco packaging means that prevent or reduce loss of OPO activity or CN " , redox-active metal ion- or aldehyde-induced cell death resulting from TS- associated oxidative stress (see, PCT/IL2008/000101).
  • copper chelating agents such as, for example, penicillamine, as well as structural analogs thereof, are highly effective in ameliorating tobacco-associated damage.
  • treatment with penicillamine substantially increased the survival rate of human lung cancer cells exposed to cigarette smoke, both in the presence and absence of saliva.
  • a synergistic effect was observed when penicillamine was used in combination with desferal.
  • copper chelating agents e.g., penicillamine
  • articles of manufacturing e.g., tobacco products, filters, tobacco packaging materials and the like
  • agent encompasses a copper chelating agent, as described herein, and penicillamine, including structural analogs thereof, as described herein.
  • additional agent is used to describe agents other then a copper chelating agent, penicillamine and structural analogs thereof, as described herein.
  • copper chelating agent which is also referred to interchangeably as “copper chelator” describes a compound that is capable of forming a stable organometallic complex with copper or copper ion, typically by donating electrons from certain electron-rich atoms present in the compound to the electron-poor copper or copper ion.
  • one or more molecules are considered as transition metal chelators if the formation of a cyclic complex of the molecule(s) with an ion of the transition metal results in a "chelate effect".
  • chelate effect refers to the enhanced stability of a complexed system containing the chelate, as compared with the stability of a system that is as similar as possible but contains none or fewer rings.
  • the parameters for evaluating the chelate effect of a chelate typically include the enthalpy and entropy changes ( ⁇ H and ⁇ S), according the following equation:
  • ⁇ is the equilibrium constant of the chelate formation and hence represents the chelate effect.
  • transition metal chelates and copper chelates in particular refer to complexes that include copper ion and one or more copper chelator(s) complexed therewith, which are characterized by a large ⁇ value.
  • copper chelators include polyamine compounds, including linear and cyclic polyamine compounds.
  • copper chelating agents include, but are not limited to penicillamine, trientine, both being FDA-approved drugs, ethylendiamine, diethylenetriamine, triethylenetetramine, triethylenediamine, aminoethylethanolamine, aminoethylpiperazine, pentaethylenehexamine, triethylenetetramine, captopril, N,N'- bis(3-aminopropyl)-l,3-propanediamine, N,N'-Bis(2-animoethyl)-l,3-propanediamine, l,7-dioxa-4,10-diazacyclododecane, 1,4,8,1 l-tetraazacyclotetradecane-SJ-dione, 1,4,7- triazacyclononane, l-oxa-4,7,10-triazacyclododecane, 1,4,8,12- tetraazacyclopentadecane and 1,4,7,10
  • Penicillamine in addition to its copper chelating effect, has also been shown to act as an immunosuppressor, and as an inhibitor of NF-AB and AP-I, thus being recognized as an anti-inflammatory agent.
  • penicillamine was found to be highly effective in decreasing the level of cell death upon exposure to cigarette smoke. Penicillamine was highly active both in cells incubated with and without saliva.
  • Penicillamine was shown to act in synergy with desferal, particularly in cells incubated in the presence of saliva.
  • chelating agents such as iron chelating agents and cyanide agents were shown to reduce cell death upon exposure to tobacco smoke, in the presence of saliva, thus suggesting that their activity is related to salivary enzymes.
  • penicillamine structural analog describes a compound which possesses the main structural features of penicillamine (e.g., an amine group, a thiol group, and a carboxy group, spaced therebetween similarly to penicillamine) and hence may exhibit functional features similar to penicillamine.
  • penicillamine structural analogs are collectively represented by the following general Formula:
  • X is O or NR 6 ; Y is O;
  • A is CR 7 R 8 or CR 7 R 8 -CR 9 Ri 0 ;
  • R 1 is selected from the group consisting of hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl and a substituted or unsubstituted aryl, whereas when anyone of said alkyl, aryl and said cycloalkyl is substituted, the substituent is independently selected from the group consisting of alkyl, cycloalkyl, alkoxy, aryl and aryloxy;
  • R 2 is hydrogen or alkyl
  • R 3 and R 4 are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl and aryl, or, alternatively, R 3 and R 4 are linked therebetween so as to form a five- or six-membered nitrogen-containing heteroalicyclic ring, or, alternatively, at least one of R 3 and R 4 forms a five- or six-membered heteroalicyclic ring with R 5 ;
  • R 5 is selected from the group consisting of hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl and a substituted or unsubstituted aryl, whereas when anyone of said alkyl, said cycloalkyl and said aryl is substituted, the substituent is independently selected from the group consisting alkyl, cycloalkyl, alkoxy, aryl, aryloxy, carbonyl, aldehyde and carboxy, or, alternatively, R 5 forms a five- or six-membered heteroalicyclic ring with one of R 3 and R 4 ;
  • R 6 is selected from the group consisting of hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted cycloalkyl and a substituted or unsubstituted aryl, whereas when anyone of said alkyl, said aryl and said cycloalkyl are substituted, the substituent is independently selected from the group consisting of alkyl and cycloalkyl, or, alternatively, R 6 forms with Ri a nitrogen-containing five-, six- or seven-membered ring; and
  • R 7 , R 8 , R 9 and Ri 0 are each independently selected from the group consisting of hydrogen and alkyl.
  • alkyl describes a saturated aliphatic hydrocarbon including straight chain and branched chain groups.
  • the alkyl group has 1 to 20 carbon atoms. Whenever a numerical range; e.g., "1-20”, is stated herein, it implies that the group, in this case the alkyl group, may contain 1 carbon atom,
  • the alkyl is a medium size alkyl having 1 to 10 carbon atoms. In some embodiments, unless otherwise indicated, the alkyl is a lower alkyl having 1 to 4 carbon atoms and even 1 to 2 carbon atoms.
  • the alkyl group may be substituted or unsubstituted, as indicated hereinabove.
  • cycloalkyl describes an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group where one or more of the rings does not have a completely conjugated pi-electron system.
  • the cycloalkyl group may be substituted or unsubstituted, as indicated hereinabove.
  • aryl describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system.
  • the aryl group may be substituted or unsubstituted, as indicated hereinabove.
  • aldehyde describes a carbonyl group in which R' is hydrogen.
  • alkoxy describes both an -O-alkyl and an -O-cycloalkyl group, as defined herein.
  • aryloxy describes an -O-aryl, as defined herein.
  • C-carboxylate and "O-carboxylate” are referred to herein collectively as “carboxy”.
  • Each of the alkyl, cycloalkyl and aryl groups in the general formula herein may be substituted by one or more substituents, whereby each substituent group can independently be, for example, alkyl, cycloalkyl, alkoxy, aryl and aryloxy, carbonyl, aldehyde and carboxy, depending on the substituted group and its position in the molecule.
  • substituents such as heteroaryl, heteroalicyclic, amine, halide, sulfonate, sulfoxide, phosphonate, hydroxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, isocyanate, sulfonamide, thiocarbonyl, acyl halide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C- amide, N-amide, guanyl, guanidine, silyl, and hydrazine, are also encompassed herein, as long as the functionality of the compound remains similar to that of penicillamine.
  • halide and "halo" describes fluorine, chlorine, bromine or iodine.
  • haloalkyl describes an alkyl group as defined above, further substituted by one or more halide.
  • S-sulfonamide and “N-sulfonamide” are collectively referred to herein as sulfonamide.
  • hydroxyl describes a -OH group.
  • thiohydroxy describes a -SH group.
  • thioalkoxy describes both a -S-alkyl group, and a -S-cycloalkyl group, as defined herein.
  • thioaryloxy describes both a -S-aryl and a -S-heteroaryl group, as defined herein.
  • cyano describes a -C ⁇ N group.
  • nitro describes an -NO 2 group.
  • O-carbamate describes an -OC(O)-NR' R" group, with R' and R" as defined herein.
  • N-amide and C-amide are collectively referred to herein as amide.
  • hydrazine describes a -NR'-NR"R'” group, with R', R", and R"' as defined herein.
  • amine describes a -NR'R" group, with R' and R" as described herein.
  • silica describes a -SiR'R"R'" group, whereby each of R', R" and R'" are as defined herein.
  • heteroaryl describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system.
  • heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine.
  • heteroalicyclic describes a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur.
  • the rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system.
  • Representative examples are piperidine, piperazine, tetrahydrofurane, tetrahydropyrane, morpholino and the like.
  • X is O, such that the compound comprises a carboxy group.
  • Ri is hydrogen, the compound comprises a carboxylic acid group.
  • Ri is other than hydrogen, the compound comprises an ester.
  • X is NR 6 , such that the compound comprises an amide group. In cases where R 1 and R 6 form a nitrogen-containing heteroalicyclic group, the amide is a cyclic amide.
  • ester or amide can be further substituted, as described herein.
  • R 3 and R 4 are both hydrogen, thus forming a primary amine at the indicated position.
  • the compound comprises a secondary amine at the indicated position.
  • a tertiary amine is present.
  • a cyclic amine is present.
  • the amine can comprise an additional group, represented by the indicated substituents.
  • the compound comprises a thiol moiety.
  • the compound comprises a thioalkoxy moiety.
  • the compound comprises a thioaryloxy moiety.
  • Each of the thioalkoxy or thioaryloxy can further comprise an additional group, represented by the indicated substituents on the alkyl, aryl and cycloalkyl groups that form a part of thioalkoxy and thioaryloxy.
  • variable A in the general formula above represents a spacer between the amine moiety and the thiol, thioalkoxy or thioaryloxy moiety.
  • the spacer includes 1 or 2 carbon atoms, being optionally substituted as indicated hereinabove.
  • the configuration of the carbon atom that bears the R 2 group, or of any other chiral carbon atom that may be present in the molecule can be R configuration or S configuration. In one embodiment, the carbon atom that bears the R 2 group has R configuration.
  • the agent described herein e.g., a penicillamine structural analog
  • the agent e.g., a penicillamine structural analog
  • the agent is further preferably selected as being capable of inhibiting inflammation in a subject using the article of manufacturing.
  • the present embodiments further encompass pharmaceutically acceptable salts of the agents described herein.
  • phrases "pharmaceutically acceptable salt” describes a charged species of the parent compound and its counter ion, which is typically used to modify the solubility characteristics of the parent compound and/or to reduce any significant irritation to an organism by the parent compound, while not abrogating the biological activity and properties of the administered compound. Examples, without limitation, include an acid additional salt of an amine group.
  • the present invention further encompasses prodrugs, solvates and hydrates of the agents described herein.
  • prodrug refers to a molecule, which is converted into the active compound (the active parent drug) in vivo.
  • Prodrugs are typically useful for facilitating the administration of the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The prodrug may also have improved solubility as compared with the parent drug in pharmaceutical compositions. Prodrugs are also often used to achieve a sustained release of the active compound in vivo.
  • An example, without limitation, of a prodrug would be a compound, having one or more carboxylic acid moieties, which is administered as an ester (the "prodrug"). Such a prodrug is hydrolysed in vivo, to thereby provide the free compound (the parent drug).
  • the selected ester may affect both the solubility characteristics and the hydrolysis rate of the prodrug.
  • solvate refers to a complex of variable stoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by a solute (the agent described herein) and a solvent, whereby the solvent does not interfere with the biological activity of the solute.
  • Suitable solvents include, for example, ethanol, acetic acid and the like.
  • hydrate refers to a solvate, as defined hereinabove, where the solvent is water.
  • an article of manufacturing which comprises tobacco and a tobacco packaging material, wherein at least a portion of the tobacco and/or the tobacco packaging material comprises an agent as described herein. According to another aspect of embodiments of the present invention there is provided an article of manufacturing which comprises tobacco and an agent as described herein being incorporated in at least a portion of the tobacco.
  • an article of manufacturing which comprises a tobacco packaging material and an agent as described herein being incorporated in at least a portion of the tobacco packaging material.
  • agent refers to any of the copper chelating agents, penicillamine and structural analogs thereof described herein.
  • that portion of tobacco and/or the tobacco packaging material which comprises the agent is in contact with an aerodigestive tract of a subject using the article of manufacturing.
  • tobacco refers to any tobacco species (e.g., crude or extract) which is compatible with human use.
  • the agent can be incorporated in the tobacco (or a portion thereof), by mixing, dipping, spraying, coating, or any other chemical or physical attachment.
  • the present embodiments also envisage the use of the agents described herein (in line with the above described aspects) with other smoked, dipped, chewed, snuff or snused herbs, compatible with human consumption and which cause a damage similar to that damage induced by tobacco, as detailed hereinunder.
  • 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.
  • the agent may be impregnated in (attached to, absorbed in, coated with) a filter paper which comes in direct contact with the aerodigestive tract.
  • the articles of manufacturing described herein can therefore be, for example, tobacco products such as smoking products (e.g., cigarettes, non-filter cigarettes, cigars, and other tobacco products as described hereinabove) or products used in the manufacturing of tobacco products (e.g., cigarette filters, rolling papers and the like).
  • tobacco products such as smoking products (e.g., cigarettes, non-filter cigarettes, cigars, and other tobacco products as described hereinabove) or products used in the manufacturing of tobacco products (e.g., cigarette filters, rolling papers and the like).
  • FIG. 2 illustrates a cigarette filter configuration 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 18.
  • the agent of the present invention can be disposed as an aqueous emulsion within a rupturable capsule 16 positioned at the front of filter core 14.
  • the agent may also be dispersed, impregnated in tobacco filling 18 or provided throughout in droplets or beadlets through the employment of gelatin or other colloidal materials, so that the agent can be easily entrained by the smoke passing through filter core 14.
  • Such filters have been previously described, for example, in U.S. Patent Nos. 3,667,478 and 3,339,558, the teachings of which are herein incorporated by reference as if fully set forth herein.
  • 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).
  • Such tobacco filters can be used as follows: prior to lighting up, pressure is applied to rupturable capsule 16, so that the released agents are dispersed within filter core 14, whereby the agent is accessible to the cigarette smoke passing through.
  • the articles of manufacturing described herein are preferably designed and configured so as to enable physico-chemical interaction between the agent and the tobacco smoke. In some embodiments, the articles of manufacturing are designed and configured so as to enable release of the agent therefrom when in use by a subject.
  • 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.
  • an article of manufacturing being a filter, which comprises an agent as described herein and which is designed and configured so as to enable release of the agent therefrom when in use by a subject.
  • the filter is designed and configured as a tobacco smoke filter (see, for example, Figure 2).
  • Such a filter can be incorporated into "filter-tip cigarettes", cigarette holders, gas-masks, protective face-masks, and air-conditioning unit filters.
  • any of the articles of manufacturing described herein further comprises an additional agent that is capable of reducing or preventing a tobacco-associated damage, as described herein, in a subject using the article of manufacturing.
  • the additional agent can be incorporated (or impregnated) in the tobacco or the tobacco packaging material or in the above-described filter.
  • additional agents are antioxidants.
  • the antioxidant is glutathione (GSH). As demonstrated in the Examples section that follows, GSH was found to be highly active in reducing cell death induced by exposure to tobacco smoke.
  • the antioxidant can further include a cyanide (CN " ) chelator, which can be used to treat tobacco-associated loss of OPO activity.
  • CN cyanide
  • An example of such a chelator is OH-
  • CO also known as the non-cyanide-bound form of cyanocobalamin, hydroxocobalamin or vitamin B 12a.
  • Other examples include, but are not limited to, epselen, vitamins A, C and E, selenium compounds, flavenoids, quinones (e.g., QlO, Q9), retinoids and carotenoids.
  • the CN " chelator e.g., OH-CO
  • the CN " chelator is administered in a manner which enables establishment of a concentration of 0.5-2 niM, preferably 1 mM in body fluids, such as saliva.
  • Cyanide chelators can be effectively employed to prevent or reduce tobacco- associated damage in the aerodigestive tract since they act to sequester cyanide which is injurious to OPO.
  • antioxidants include redox-active metal ion chelators, e.g., redox-active iron chelators (also referred to herein as iron chelating agents). Examples include deferoxamine, and zinc-desferioxamine.
  • the chelating agent deferoxamine is also known as DES, desferal and desferioxamine.
  • Redox-active metal ion chelators are used in a manner which enables establishment of about 1 mM concentration in body fluids (e.g., saliva).
  • body fluids e.g., saliva
  • deferoxamine is administered in a manner which enables establishment of a concentration of about 1 mM, more preferably about 5 mM in body fluids.
  • a mixture of deferoxamine and GSH is used in a ratio of about 1:1, preferably 5:1, respectively.
  • 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 additional active agent is desferal.
  • desferal is used in combination with penicillamine, to thereby exert a synergistic effect.
  • the articles of manufacturing described herein can further comprise at least one flavorant such as, but not limited to, 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.
  • at least one flavorant such as, but not limited to, 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
  • agents and additional agents described herein may be introduced to the article of manufacturing as described above (e.g., snuff), such as in the forma 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.
  • the agents described herein are highly efficient in reducing or preventing damages caused by tobacco (e.g., by cigarette smoke), both cellular damage (e.g., cell death) and macromolecular damage (e.g., protein carbonylation), and exhibit their activity also in the absence of saliva.
  • tobacco e.g., by cigarette smoke
  • cellular damage e.g., cell death
  • macromolecular damage e.g., protein carbonylation
  • administering the agents described herein is effected via, for example oral, rectal, transmucosal, transdermal, topical, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • Subject treatable by the method described herein include tobacco consumers, as well as secondary tobacco consumers (non-smokers that are exposed to tobacco smoke).
  • tobacco-associated damage described cellular or macromolecular damage which is induced or exacerbated by exposure to tobacco consumption.
  • tobacco consumption includes, for example, tobacco smoking (including primary and secondary smoking), chewing, sniffing, and the like, as described hereinabove.
  • the tobacco-associated damage can be a cellular damage, resulting in, for example, cell death, cell malfunction, cell mutation, and the like; or a macromolecular damage, resulting in modification of macromolecules such as lipids, DNA and proteins.
  • tobacco-associated damage typically involves ROS and therefore often involves oxidative damage of cells and cell components.
  • tobacco consumption results in tobacco-associated damage to mucosal tissues, particularly 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.
  • mucosal tissues particularly 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.
  • tobacco-associated damage is typically manifested as various diseases and disorders, including, but not limited to, cardiovascular diseases, chronic obstructive pulmonary disease, lung cancer, as well as other forms of cancer and peripheral vascular disease.
  • Exemplary cardiovascular diseases that are therefore treatable by the agents described herein include, but are not limited to, atherosclerosis; coronary thrombosis, which may lead to a heart attack; cerebral thrombosis, which may lead to collapse, stroke and paralysis; affected kidney arteries, which result in high blood pressure or kidney failure; and blockage of the vascular supply to the legs, which may lead to gangrene and amputation.
  • Exemplary cancers that are treatable by the agents described herein, in addition to lung cancer include, but are not limited to, mouth, pharynx, and esophagus cancer, and oral squamous cell carcinoma.
  • Other types of cancers include bladder cancer, cancer of the kidneys, cancer of the pancreas and cervical cancer.
  • COPD chronic obstructive pulmonary diseases
  • Other damages associated with tobacco consumption which are treatable by the agents described herein include, for example, hypertension, fertility problems, retinoic disorders such as macular degeneration and cataracts, ulcers, periodontal diseases, impotence, Diabetes type 2, Back pain, skin ailments such as premature ageing and wrinkling, osteoporosis, earlier menopause, and damaged and/or weakened immune system, as well as leukoplakia, halitosis, acute necrotizing ulcerative gingivitis ("trench mouth”) and oral submucous fibrosis.
  • the agent can be utilized in combination with an additional agent.
  • the additional agent can be, for example, an antioxidant as described herein, an agent capable of reducing or preventing a tobacco- associated damage and/or an agent suitable for use in the treatment of a disease or disorder as described herein.
  • the additional agent is desferal.
  • the agent can be utilized either per se or being formulated into a pharmaceutical composition which further comprises a pharmaceutically acceptable carrier.
  • pharmaceutical compositions which comprise one or more of the agents described above and a pharmaceutically acceptable carrier.
  • a "pharmaceutical composition” refers to a preparation of one or more of the agents described herein, with other chemical components such as pharmaceutically acceptable and suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • the phrase "pharmaceutically acceptable carrier” describes a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • carriers are: propylene glycol, saline, emulsions and mixtures of organic solvents with water, as well as solid (e.g., powdered) and gaseous carriers.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.
  • excipients examples include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the agents described herein into preparations which, can be used pharmaceutically.
  • the pharmaceutical composition is formulated as a solution, suspension, emulsion or gel.
  • the pharmaceutical composition further includes a formulating agent selected from the group consisting of a suspending agent, a stabilizing agent and a dispersing agent.
  • the agents described herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer with or without organic solvents such as propylene glycol, polyethylene glycol.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer with or without organic solvents such as propylene glycol, polyethylene glycol.
  • penetrants are used in the formulation. Such penetrants are generally known in the art.
  • the agents described herein can be formulated readily by combining the agents with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the agents described herein to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active agent doses.
  • compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the agent(s) may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the agents described herein are conveniently delivered in the form of an aerosol spray presentation (which typically includes powdered, liquified and/or gaseous carriers) from a pressurized pack or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the agents and a suitable powder base such as, but not limited to, lactose or starch.
  • the agents described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the agents described herein in water-soluble form.
  • suspensions of the agents may be prepared as appropriate oily injection suspensions and emulsions (e.g., water-in-oil, oil-in-water or water-in-oil in oil emulsions).
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes.
  • Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
  • the suspension may also contain suitable stabilizers or agents, which increase the solubility of the agents to allow for the preparation of highly concentrated solutions.
  • the agents may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen- free water, before use.
  • a suitable vehicle e.g., sterile, pyrogen- free water
  • agents described herein may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions herein described may also comprise suitable solid of gel phase carriers or excipients.
  • suitable solid of gel phase carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols.
  • compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of an agent as described herein effective to prevent, alleviate or ameliorate symptoms of tobacco-associated damage or prolong the survival of the subject being treated.
  • the therapeutically effective amount or dose can be estimated initially from activity assays in animals.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC 5 o as determined by activity assays (e.g., the concentration of the test agent, which achieves a half-maximal reduction in cell death upon exposure to cigarette smoke).
  • activity assays e.g., the concentration of the test agent, which achieves a half-maximal reduction in cell death upon exposure to cigarette smoke.
  • Toxicity and therapeutic efficacy of the agents described herein can be determined by standard pharmaceutical procedures in experimental animals, e.g., by determining the EC 50 , the IC 5O and the LD 50 (lethal dose causing death in 50 % of the tested animals) for a subject compound.
  • the data obtained from these activity assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
  • dosing can also be a single administration of a slow release composition described hereinabove, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions of the present embodiments may, if desired, be presented in a pack or dispenser device, such as an FDA (the U.S. Food and Drug Administration) approved kit, which may contain one or more unit dosage forms containing the active agent.
  • the pack may, for example, comprise metal or plastic foil, such as, but not limited to a blister pack or a pressurized container (for inhalation).
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S.
  • compositions comprising an agent as described herein, formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is detailed hereinabove.
  • the pharmaceutical composition further comprises an additional agent.
  • the additional agent can be, for example, an antioxidant as described herein, an agent capable of reducing or preventing a tobacco-associated damage and/or an agent suitable for use in the treatment of the indicated disease or disorder as described herein.
  • An exemplary antioxidant is desferal.
  • an oral composition which comprises an agent as described herein.
  • the oral composition can be in the form of a toothpaste, powder, liquid dentifrice, mouthwash, denture cleanser, chewing gum, lozenge, paste, gel or candy and 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.
  • Exemplary chewing gum compositions are described in U.S. Patent No.
  • the chewing gums, gels or pastes of these embodiments may include bicarbonates with thickening agents in a concentration from 0.5 % to 5.0 % by weight.
  • Exemplary thickeners with bicarbonate and zinc salts include, but are not limited to, chicle, xanthan, arabic, karaya or tragacanth gums.
  • Alginates, carrageenans and cellulose derivatives such as sodium carboxymethyl, methyl, or hydroxy ethyl compounds are appropriate for the intended preparations; surfactants and abrasives may also be included. Alcohols will otherwise be avoided for their known risk factor for oral cancers.
  • sweetening agents as saccharin, sodium cyclamate, sorbitol, aspartamane and others may be used in concentrations from 0.005 % to 5.0 % by weight of the total composition.
  • Xylitol has been shown to prevent dental caries and decrease gum disease, in part by reducing the putative oral bacteria, especially Streptococcus mutants.
  • Gels and dentifrices may contain fluoride anticaries compounds. These fluoride compounds, such as salts of sodium, potassium, calcium, magnesium, stannous and others have been known to protect teeth from developing cavities. Fluorides may be present in various amounts in the gels, pastes, gums or lozenges ranging from 0.01 % to 3.0 % by weight, preferably from 0.05 % to 2.0 % by weight, most preferably from 0.1
  • agents described herein can also be incorporated into additional articles. These include, for example, various medical devices for delivering the agent to or applying the agent on a desired bodily site.
  • the phrase "bodily site” includes any organ, tissue, membrane, cavity, blood vessel, tract, biological surface or muscle, which delivering thereto or applying thereon the agents described herein is beneficial.
  • Exemplary medical devices are those configured to deliver the agent by topical application, (e.g., an adhesive strip, a bandage, an adhesive plaster, and a skin patch).
  • the agents can be incorporated in the device structure by any methodology known in the art, depending on the selected nature of the device structure.
  • the agents can be entrapped within a porous matrix, swelled or soaked within a matrix, or being adhered to a matrix.
  • compositions and devices described herein the agent can be utilized in combination with an additional active agent, as described herein.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • 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), and was generated as described hereinbelow.
  • NCI- H 1299 cells Human non-small cell lung cancer NCI- H 1299 cells were used as described by the American Type Culture Collection.
  • Culture medium included DMEM with L-glutamine, supplemented with fetal bovine serum (10 %), penicillin-streptomycin solution (1 %). The cells were grown at 37 °C in 5 % CO 2 .
  • Hydroxocobalamin acetate, D(-)Penicillamine, Defroxamine Mesylate (Desferal-DES), thiol-glutathione (GSH) and MTT (Thiazolyl Blue Tetrazolium Bromide) salt were purchased from Sigma-Aldrich, Israel.
  • Cell Death Kit Cell Death Detection ELISA PLUS" TM KIT was purchased from Roche diagnostics, Germany.
  • Protein levels were measured by the Bradford method (1976) [Bradford M. M. 1976. Anal. Biochem. 72:248-254] using BSA as a standard.
  • Saliva collection Whole saliva was collected from healthy volunteers (20-60 years) under non- stimulatory condition, at least 1 hour after last eating, between 8:00-12:00 AM. The volunteers were asked to generate saliva in their mouths and to spit it into a wide test tube for no more then 20 minute. The obtained whole saliva was centrifuged at 1200 x g for 15 minutes to remove cell debris and palate cells, and the supernatant was used for further applications.
  • the four study groups were exposed to the same conditions and further exposed to CS (as detailed hereinafter), and are denoted CS, CS+saliva, PenA CS and PenA CS+saliva.
  • D-penicillamine was used at a concentration of 1 mM or 5mM.
  • H 1299 cells were similarly treated, using, instead of (or in addition to) PenA, GSH, or DES, at the indicated concentration.
  • the H 1299 cells viability was measured at various time points by Trypan Blue exclusion test, both in exposed and control cells.
  • the medium covering the dish was collected and cells were trypsinized and centrifuged at 1200 x g for 10 minutes. Cell pellets were re-suspended in 1 mL of medium and a sample was collect for cell counting. Cells were stained with the vital dye Trypan Blue at final concentration of 0.25 % and were placed on hemacytometer. Visual counting was preformed by inverted microscope. Protein carbonyl assay: Protein carbonyl concentration was determined by enzyme-linked immunosorbent assay (ELISA), using the Zentech PC Test Kit (Zenith Technology, Dunedin, New Zealand). Briefly, protein cell extractions were allowed to react with a dinitrophenylhydrazine (DNP) solution (200 ⁇ l).
  • DNP dinitrophenylhydrazine
  • the DNP-reacted proteins bound non- specifically to an ELISA plate, and the unconjugated DNP and non-protein entities were washed away.
  • the adsorbed DNP-protein was then probed with an anti-DNP-biotin antibody, followed by a streptavidin-linked HRP probe.
  • the chromatin reagent that contained peroxide was added to catalyze the oxidation of TMB.
  • the reaction was stopped by the addition of a stopping reagent (acid, provided with the kit), and the absorbance was measured for each well at 450 run using a spectrophotometer.
  • a stopping reagent ascid, provided with the kit
  • protein carbonyl standards were also included in the assay.
  • FACS Flow cytometric analysis
  • Cells were scraped from the culture dishes, and centrifuged at 1200 g for 10 minutes. Cell pellets were re-suspended with lysis buffer according to the manufacturer's instructions. The lysate was centrifuged at 200 g for 10 minutes. A fraction of the supernatant was transferred to streptavidin-coated microtiter plate modules.
  • Immunoreagent was added (antihistone-biotin and anti-DNA-peroxidase in incubation buffer), and after incubation with gentle shaking for 2 hours, the modules were rinsed three times with incubation buffer. Then, the signal for apoptosis was measured following incubation for 15 minutes with 2,2-azino-bis-[3-ethylbenzothiazoline]-6- sulfonic acid (ABTS) solution, according to the manufacturer's instructions. The level of staining by the ABTS substrate was determined with an ELISA reader, at the wavelength of 405 nm. Reference absorbance was measured using a 490 nm wavelength. The ABTS solution by itself was used as a negative control, positive control was supply by the manufacturer.
  • ABTS 2,2-azino-bis-[3-ethylbenzothiazoline]-6- sulfonic acid
  • TBARS Lipid peroxidation assay
  • Lipid peroxidation was quantified by determining 2-thiobarbituric acid reactive substance (TBARS) formation according to the method described by Buege and Aust [Methods in Enzymol. 52, 302-310, 1978] with some modification.
  • H1299 cells (2 x 10 4 cells) were homogenized 1 ml of PBS.
  • 1 ml of 2-thiobarbituric acid (TBA) reagent consisting of 0.375 % TBA, 15 % trichloroacetic acid, and 0.25 N HCl was added to 0.5 ml of cell homogenat.
  • the mixture of cell suspension and TBA reagent was heated at 100 °C for 20 minutes, chilled quickly in ice-water to room temperature, and centrifuged at 1,500 g for 10 minutes. The supernatant was measured at 535 tun with a spectrophotometer.
  • [ 3 H]PK 11195 an isoquinoline carboxamide derivative, was used for binding studies.
  • PK 11195 is a specific PBR ligand.
  • Cells were scraped from the culture dishes, washed with phosphate-buffered saline (PBS), and centrifuged at 120Og for 10 minutes. Then the cell pellets were re-suspended in 1 mL of 50 mM phosphate buffer, pH 7.4, and centrifuged at 1200 g for 10 minutes.
  • PBS phosphate-buffered saline
  • Binding assays contained 400 ⁇ L of cell membrane (0.4 mg of protein/mL) in the absence (total binding) or presence (nonspecific binding) of 1 ⁇ M unlabeled PK 11 195, up to a final volume of 500 ⁇ L. After incubation for 80 minutes at room temperature, samples were filtered under vacuum over Whatman GF/B filters and washed three times with phosphate buffer. Filters were placed in vials containing 4 mL of Opti-Fluor (Packard, Groningen, The Netherlands) and counted for radioactivity in a scintillation counter after 12 hours. The maximal number of binding sites (Bmax) and equilibrium dissociation constants were calculated from the saturation curve of [ 3 H] PK 11195 binding, using Scatchard analysis.
  • Results for statistical analysis were obtained from the control subgroup (H 1299 cells in medium) and from the various treatment subgroups (with/without saliva and/or exposure to CS and/or additions of 1 mM or 5 mM exogenous D-penicillamine).
  • Figures 5a and 5b present the effect of 5 mM penicillamine (PenA) on the survival rate of H 1299 cells incubated in medium containing PenA or in medium containing 30 % (v/v) saliva and PenA, and exposed to CS.
  • PenA penicillamine
  • FIGs 5a and 5b illustrate the effect of 5 mM penicillamine (PenA) on the survival rate of H 1299 cells incubated in medium containing PenA or in medium containing 30 % (v/v) saliva and PenA, and exposed to CS.
  • PenA penicillamine
  • H 1299 cells were incubated for 120 minutes with or without the presence of saliva in the culture medium and exposed to CS. Cells were also incubated in the presence of PenA in either low (1 mM) or high (5 mM) concentration prior the exposure of CS. Cell viability was evaluated by Trypan blue exclusion dye assay. As shown in Figures 5a and 5b, the addition of 5 mM D-penicillamine to cells incubated with or without exposure to CS (PenA+CS and PenA M groups, respectively) partially affected the survival of H 1299 cells, such that the cells loss at 120 minutes of exposure to CS in the presence of the PenA was reduced by 25 %. Reduction of cells loss was also observed when saliva was supplemented to the medium prior to the exposure to CS.
  • Figures 6a and 6b present the percent of viable H 1299 cells out of control ( Figure 6a) and out of total number of cells ( Figure 6b) upon incubation in medium alone or in medium supplemented with 30 % (v/v) saliva, with or without prior addition of PenA to the medium, and with or without exposure to CS, for 120 minutes. These data clearly demonstrate the drastic effect of PenA on cell survival, with a 20 % increase of cell survival in cells incubated in saliva-containing medium and a 12 % increase of cells survival in cells incubated in medium alone (See, Figure 6a).
  • Figure 7 presents the effect of 1 mM and 5 mM penicillamine (PenA) on the survival rate of Hl 299 cells incubated in medium and PenA or in medium containing 30 % (v/v) saliva and PenA, and exposed to CS, compared with non-treated cells and further demonstrate a dose-dependent effect of penicillamine.
  • PenA penicillamine
  • the survival rate of cells that contained saliva was 40.4 % following 120 minutes of CS exposure, while the addition of 5 mM PenA significantly raised the survival rate up to 76.4 % (*p ⁇ 0.001).
  • the addition of 5 mM PenA also protected from the lethal effect of CS alone, and significantly raised the survival rate from 61.5 % to 75.5 % (**p ⁇ 0.001).
  • the unique protection phenomenon that prevent cells death not only from the lethal synergism effect induced by CS and saliva, but also from the lethal effect of the CS itself, demonstrates a novel protective PenA mechanism.
  • PI Iodide
  • H 1299 cells were incubated for 120 minutes with or without the presence of saliva in the culture medium and exposed to CS. Cells were also incubated in the presence of 5 mM PenA prior the exposure of CS.
  • Figure 8 further presents the data obtained in these fluorescence studies.
  • cellular uptake of PI was increased after 120 minutes of CS exposure.
  • CS exposure caused a significant decrease of cell viability (Pi-positive cells) both in the presence (23.32 % ⁇ 0.36 %) and in the absence (24.515 ⁇ 4.12 %) of saliva.
  • the presence of saliva in the cultured medium did not lead to additional cellular loss.
  • the addition of PenA to the cultured medium completely protected from cell death after 120 minutes of CS exposure with (2.33 % ⁇ 0.29 %) or without (2.98 % ⁇ 0.06 %) the presence of saliva, in a very significant manner.
  • PenA is known as a copper chelator
  • these results may suggest that when no chelation of the redox active copper was effected prior to the exposure of the cells to CS, the rate of cells killing was doubled. Since PenA did not alter the CS-induced cell death in the absence of saliva, it may be suggested that the redox active copper ions originate in the saliva and not in the CS.
  • PenA reacts as an anti-inflammatory agent and exerts its beneficial activity via modulation of the innate immune activity and hence can be utilized for ameliorating tobacco-associated damage to tissues other than the aerodigestive tract.
  • Figure 9 presents the percents of viable H 1299 cells upon incubation in medium alone or in medium supplemented with 30 % (v/v) saliva, with or without prior addition of 2 mM GSH to the medium, and with or without exposure to CS.
  • Glutathione is a tripeptide (L-c-glutamyl-L-cysteinyl-glycine) containing a thiol group. GSH is an important protective antioxidant against free radicals and other oxidants, and has been implicated in immune modulation and inflammatory responses.
  • Glutathione exists in reduced (GSH) and oxidized (GSSG) states.
  • the thiol group of cysteine is able to donate a reducing equivalent (H + +e ⁇ ) to other unstable molecules, such as reactive oxygen species.
  • glutathione In donating an electron, glutathione itself becomes reactive, but readily reacts with another reactive glutathione to form glutathione disulfide (GSSG).
  • GSH protects cells against CS-born aldehydes, which are known to mediate CS damage.
  • GSH GSH
  • Protein carbonylation is a covalent modification of a protein, induced by reactive oxygen intermediates or by-products of oxidative stress, and is the most general and well-used biomarker for severe oxidative protein damage.
  • H 1299 cells were incubated for 120 minutes with or without the presence of saliva in the culture medium and exposed to CS. Cells were also incubated in the presence of 5 mM DES prior the exposure of CS. Cell viability was evaluated by Trypan blue exclusion dye assay.
  • DES significantly protected the cells from the lethal synergistic effect of saliva and CS. While the cellular death rate with no protection was of 55.1 %, it dropped to 20.5 % following the addition of 5 mM DES (p ⁇ 0.005). No significant change on the lethal effect of CS on the lung cancer cells was observed in the absence of saliva.
  • H 1299 cells were incubated for 120 minutes with or without the presence of saliva in the culture medium and exposed to CS. Cells were also incubated in the presence of 5 mM DES, 5 mM PenA and a mixture of DES and PenA, prior the exposure of CS. Cell viability was evaluated by Trypan blue exclusion dye assay.
  • Mitochondrial membrane permeabilization is an essential step leading to apoptosis. Disruption of ⁇ m irreversibly commits cells to undergo death and is an early marker of apoptosis. Furthermore, cigarette smoke was previously shown to induce mitochondrial depolarization in human monocytes. Therefore, reduced ⁇ m , as measured by diminished incorporation of the fluorescent dye JC-I, was used as an early indicator for CS-induced apoptosis.
  • H 1299 lung cancer calls were exposed for 120 minutes to CS with or without saliva. Samples were also incubated in the presence of 5 mM PenA or 5 mM DES prior the exposure of CS. JC-I incorporation was measured by flow cytometry method.
  • Nonyl-Acridine Orange is a fluorescent dye that can bind specifically to unoxidizable cardiolipin (CL), a mitochondrial phospholipid, independent of the mitochondria energetic state
  • NAO flow cytometry was used to measure the quality of H 1299 lung cells exposed for 120 minutes to CS with or without saliva.
  • the potential metal chelators PenA and DES were evaluated for their protection from CL oxidation following CS exposure.
  • a retinoblastoma protein (pRb) can be activated (hypophosphorylated) through induction of cyclin-dependent kinase inhibitors (CDKIs), such as p21, a transcriptional target of p53, or pl ⁇ in response to DNA damage (see introduction).
  • CDKIs cyclin-dependent kinase inhibitors
  • PenA was found to be a novel antioxidant agent. Addition of PenA to the cultured medium prior the exposure to CS resulted in a significant protection against cellular loss and protein modification, irrespective to the presence of saliva.
  • PenA exerts its protective effect via the following possible mechanisms: As a copper chelator for free copper ions that are presents in saliva.
  • Oxidants in cigarette smoke can activate the mitogen-activated protein kinase (MAPK) signaling cascades in lung epithelial cells in-vitro and in-vivo. These signaling pathways lead to the enhanced ability of Jun and Fos family members (components of the AP-I) to activate transcription of a number of AP-I dependent target genes involved in cell proliferation, death and inflammation.
  • PMK mitogen-activated protein kinase
  • PenA can block the binding of AP-I to the DNA and therefore may prevent apoptosis and inflammatory reaction. This mechanism may explain the decrease in cellular death following CS exposure. This possible mechanism of PenA may have therapeutic impact on chronic lung inflammation in heavy smokers;
  • PenA was also found to prevent ⁇ m depletion on cells that exposed to CS per se but not samples that exposed to CS and saliva. Thus, PenA protects from CS direct damage irrespective of saliva. PenA is also capable of preventing p53 downregulation following CS and saliva exposure. This effect may be attributed to CS salivary mediate damage while addition of PenA partially prevented this effect and thus, stabilized p53 and prevents its degradation. Notably, a synergistic effect is demonstrated on cell viability following incubation of H 1299 with both DES and PenA.

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US20180000705A1 (en) * 2016-06-30 2018-01-04 The Procter & Gamble Company Shampoo Compositions Comprising a Chelant
US20180000706A1 (en) * 2016-06-30 2018-01-04 The Procter & Gamble Company Conditioner Composition Comprising a Chelant
US11786447B2 (en) 2016-06-30 2023-10-17 The Procter & Gamble Company Conditioner composition comprising a chelant
US11246816B2 (en) 2016-06-30 2022-02-15 The Procter And Gamble Company Shampoo compositions comprising a chelant
CN108130357A (zh) * 2017-12-14 2018-06-08 云南中烟工业有限责任公司 一种检测胶基型嚼烟对细胞早期凋亡影响的方法
EP3613835A1 (de) 2018-08-24 2020-02-26 The Procter & Gamble Company Behandlungszusammensetzungen mit einem tensidsystem und einem oligoamin
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