OA20552A - Use of isothiocyanate in smokeless tobacco products. - Google Patents

Abstract

A tobacco product reduces or mitigates some of the negative effects of tobacco consumption, particularly cancer development. The tobacco product contains (a) nicotine, (b) at least one of an isothiocyanate or isothiocyanate precursor, (c) myrosinase and (d) a synthetic isothiocyanate. The product can further include at least one of thymoquinone, resveratrol, synthetic vitamin C, thymoquinone resveratrol and a synthetic vitamin C.

Description

USE OF ISOTHIOCYANATE IN SMOKELESS TOBACCO PRODUCTS

[0001J The présent application claims the benefit of, and priority to, pending Provisional Patent Application Ser. No. 62/844,846 filed on May 8, 2019, titied “Use Of Isothiocyanate In Smokeless Tobacco Products”, which is hereby incorporated fully by reference into the présent application.

Field of the Invention

[0002] The field of the invention is a tobacco product for reducing the négative effects of tobacco consumption.

Background

[0003] The background description includes information that may be useful in understanding the présent invention. It îs not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] In 2017, 14 of every 100 U.S. adults aged 18 years or older (14.0 %) currently smoke cigarettes. This means that an estîmated 34 million adults in the US currently smoke cigarettes. More than 16 million Americans live with a smoking-related disease. Accordîng to the Office on Smoking and Health, cigarette smoking causes more than 480,000 deaths every year in the US and this is nearly one in five deaths in the US.

[0005] Smokers are more lîkely than nonsmokers to develop lung cancer. For example, smoking men and women develop lung cancer by 25 tîntes more than non-smoking men and women.

Accordîng to the American cancer society, tobacco smoke îs made up of thousands of Chemicals, including at least 70 known to cause cancer (carcînogen) such as acetaldehyde, acrylonitrile, formaldéhyde, lead, N-Nitorsopiperidine, and vinyl chloride. It is well-known that smoking can cause cancer almost anywhere in our body including bladder, blood, cervix, colon, esophagus, kidney, ureter, larynx, liver, oropharynx, pancréas, stomach, trachea, bronchus, and lung.

[0006] Many smokers are eager to quit smoking, iiowever, it is very difficult due to nicotine addiction. Nicotine is a drug that occurs naturally in tobacco, and is thought to be as addictive as heroîn or cocaïne. Smokers suffer physical and emotional withdrawal symptoms when they stop smoking. One of the common ways to treat nicotine addiction is the use of Nicotine Replacement Therapy (NRS), such as nicotine patches and nicotine gum. In a 2015 study about people who were trying to quit smoking using NRS, about 20 % of smokers in the control group quit smoking. On the other hand, about 52 % of people who used NRS for at least 2 months, managed to quit smoking (Bo et al., American J Epi, 2015, 513- 520). This resuit indicates that NRS effectively works for smokers who want to quit smoking. On the other hand, almost 50% of smokers in the study failed to quit smoking, even though they used NRS. In addition, there are many smokers who do not even try to quit smoking due to the significant effort and time required for quitting.

[0007] Ali publications herein are incorporated by reference to the same exlent as if each individual publication or patent application were specifïcally and individually indîcated to be incorporated by reference. Where a définition or use of a tenu in an incorporated reference is inconsistent or contrary to lhe définition of that term provided herein, the définition of that term provided herein applies and the définition of that term in the reference does not apply.

[0008] Some smokers use a cigarette substitute to quit smoking. Even though the cigarette substïtute does not conlain nicotine, il might reduce the cigarette craving due to the simple motor eifects of smoking behavior. CA2866912 leaches a cigarette substitute where the substitute contains isothiocyanate. Isothiocyanate works as an anti-cancer agent in cancer cells including lung, breast and oral cancer cells. It helps to prevent or reduce cancer development when smokers conlinuously use the substitute. However, many smokers still return to smoke a real tobacco product because of nicotine craving.

[0009] W02009/05142A2 teaches a smokeless tobacco product containing isothiocyanate to prevent and/or reduce cancer development in smokers. As described above, isothiocyanate works as an anti-cancer agent. Therefore, smoking this tobacco product îs likely less harmful with respect to cancer development as compared to other smokers, but still more harmful that with respect to non-smokers.

[0010] In some embodiments, the numbers expressing quantifies of ingrédients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claîms are approximations that can vary depending upon the desired propertîes sought to be obtained by a particular embodiment. in some embodiments, the numerical parameters should be construed in light of the number of reported significant dîgîts and by applying ordinaiy rounding techniques. Notwîthstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the spécifie examples are reported as precisely as practicable. The numerical values presenled in some embodiments of the invention may contain certain errors necessarily resulting from the standard déviation found in their respective testing measurements.

[0011] As used in the description herein and throughout the daims that follow, the meaning of “a,” “an,” and “the” incîudes plural reference unless the context dearly dictâtes otherwise. Also, as used in the description herein, the meaning of “in” incîudes “in” and “on” unless the context dearly dictâtes otherwise.

[0012] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring indivîdually to each separate value falling withîn the range. Unless otherwise indicated herein, each individu al value is incorpora ted into the spécification as if it were indivîdually recited herein. AU methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise dearly contradicted by context. The use of any and ail examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the spécification should be construed as indicating any non-claîmed element essential to the practice of the invention.

[0013] Groupings of alternative éléments or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed indivîdually or in any combination with other members of the group or other éléments found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or délétion occurs, the spécification is herein deemed to contain the group as modified thus fulfilling the written description of ail Markush groups used in the appended claims.

[0014] Thus, there is stiil a need for a tobacco product having increased effectiveness for reducing the négative effects of tobacco consumption.

Summary ofThe Invention

[0015] The inventive subject matter provides apparatus, Systems and methods for a tobacco product having increased effectiveness for reducing the négative effects of tobacco consumption.

[0016] An ingestible product, for example, a smokeless tobacco, a chewing tobacco, a snuff, a snus, a dissolvable tobacco, a cigarette, a twist, a cigar, and a cigarillo, includes nicotine, at least one of isothiocyanate or an isothiocyanate precursor, and myrosinase.

[0017] The nicotnine can be derived from at least one of powdered tobacco leaf and a tobacco leaf extract. The isothiocyanate precursor can be derived from at least one of water cress, water cress extract, garden cress, and garden cress extract. The isothiocyanate can be synthctic. The myrosinase can be derived from at least one of mustard seed and mustard seed extract. The ingestible product can further inlcude at least one of thymoquinone, a black, cumin seed extract, resveratrol and a synthetic vitamin C.

[0018] In some embodiments, the ingestible product includes at least one of powdered tobacco leaf and a tobacco leaf extract as a soruce of the nicotine, and at least one of water cress, water cress extract, garden cress, and garden cress extract as a source of the isothiocsyanate precursor; and mustard seed extract as a source of the myrosinase. The ingestible product can further include synthetic isothiocyanate, thymoquinone, resveratrol, synthetic vitamin C, thymoquinone and a synthetic vitamin C.

[0019] In some embodiments, the nicotine and the isothiocyanate precursor in both or in each can be encapsulated and included in the ingestible product. The encapsolation can be formed by using at least one of micelles and nano-particles. In some embodiments, at least some of the isothiocyanate precursor can be in direct contact with at least one of powdered tobacco leaf and a tobacco leaf extract. The ingestible product can contain a filter.

[0020] 20. A method of preventing or treating cancer in a population, but making available to at least some members of the population an ingestible product comprising nicotine, at least one of isolhiocyanate and an isothiocyanate precursor, and myrosinase.

[0021] The inventive subject matter further includes a method of preventing or treating cancer in a population, but making available to at least some members of the population an ingestible product including nicotine, at least one of isothiocyanate and an isothiocyanate precursor, and myrosinase.

[0022] As used herein, and unless the context dictâtes otherwise, the term coupled to is intended to include both direct coupling (in which two éléments that are coupled to each other contact each other) and indirect coupling (in which at least one additional element is located between the two éléments). Therefore, the terms coupled to and coupled with are used synonymously.

[0023] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictâtes otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictâtes otherwise.

[0024] Ail methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and ali examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended mereiy to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the spécification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0025] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanyïng drawing figures in which like numerals represent like components.

Brief Description of The Drawings

[0026] Fig. 1 shows brightfield (left) and fluorescent (right) images of A549 in the absence/prcsence ofDMSO.

[0027] Fig. 2 shows brightfield and fluorescent images of A549 treated with AITC.

[0028] Fig. 3 shows brightfield and fluorescent images of A549 treated PE1TC.

[0029] Fig. 4 shows brightfield and fluorescent images of A549 with treated Thymoquinone.

[0030] Fig. 5 shows brightfield and fluorescent images of A549 with treated Resveratrol .

[0031] Fig. 6 shows brightfield and fluorescent images of A549 treated with AITC and PEITC.

[0032] Fig. 7 shows brightfield and fluorescent images of A549 treated with AITC, PEITC, and

Thymoquinone.

[0033] Fig. 8 shows brightfield and fluorescent images of A549 treated with Thymoquinone and Resveratrol.

[0034] Fig. 9 shows brightfield and fluorescent images of A549 treated with AITC, PEITC, Thymoquinone, and Resveratrol .

Detailed description

[0035] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive éléments, the inventive subject matter is considered to include ail possible combinations of the disclosed éléments. Th us if one embodiment comprises éléments A, B, and C, and a second embodiment comprises éléments B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, cven if not explicîtly disclosed.

[0036] In some embodiments, the numbers expressing quantities of ingrédients, properties such as concentration, reaction conditions, and so forth, used to describe and claîm certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached daims are approximations that can vary depending upon the desired properties sought to be obtained by a partîcular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported sîgnificant digîts and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the spécifie exampies are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard déviation found in their respective testing measurements.

[0037] The recitation of ranges of values herein is merely intended to serve as a shorlhand method of referring individually to each separate value falling within the range. Unless otherwise îndicated herein, each individual value is incorporated into the spécification as if it were individually recited herein.

[0038] Unless the context dictâtes the contrary, ail ranges set forth herein should be interpreted as being inclusive of their endpoints, and open-ended ranges should be interpreted to include only commercially practical values. Similarly, ail lists of values should be considered as inclusive of întermediate values unless the context indicates the contrary.

[0039] The tobacco products contemplaled herein are referred to as being îngestible because cither (a) they are retained in the mouth such that juices emanating from the product are swallowed, or (b) they are smoked or vaped, such that inhaled components are mixed with saliva and then swallowed. Such îngestible product includes at least one of the following: smokeless tobacco, tobacco leaf extract, chewing tobacco, snuff, snus, dissolvable tobacco, cigarette, twist, a cîgar, and cigarillo.

[0040] Preferred embodiments contain (a) nicotine, (b) at least one of isothiocyanate or isothiocyanate precursor, and (b) myrosinase. The nicotine can be derived from any suitable source of nicotine, for example, powdered tobacco leaf and/or tobacco leaf extract.

[0041] Isothiocyanate (ITC), R-N=C=S awell known anti-cancer molécule, increasing p53 expression, depleting mutant p53 expression, inducing mitotic cell cycle arrest and apoptosis in cancer cells including breast, lung, and cancer cell lines. As used herein, the terre ITC includes isothiocyanate analogs and a precursor ofITC, provided such molécules increase p53 expression, deplete mutant p53 expression, induce mitotic cell cycle arresl and apoplosis in cancer cells. ITC analogs include Ally 1 ITC (AITC), Phenethyl ITC (PEITC), and Benzyl ITC (BITC) which are derived from cruciferous vegetabies (37). In some embodiments, the vegetables and/or vegetable extracts can be added to the ingestible product.

[0042] A precursor ofITC, ethyl glucosinolate (PEGLS), derived from watercress and/or watercress extract, can be hydroiyzed enzymatically into PEITC (4). Myrosinase, an endogenously or exogenously obtained enzyme, can be used to hydrolyze glucosinoiates. Other precursors ofITC include glucotropaeolin for BITC, gluconasturtiin for PEITC, and Allyl Glucosinolate (Sinigrin) for AITC, and can ali be enzymatically converted to ITC. Other sources of the precursor ITC include garden cress and garden cress extracts thaï are rich in Glucotropaeolin.

[0043] In some embodiments, watercress, its extracl, garden cress and/or its extract can be added to provide the ITC precursor, and an endogenous and/or exogenous myrosinase can be added to facilitate the hydrolysis to ITC. Additionaliy or alternatively an ingestible product can include a purified ITC.

[0044] Myrosinase can be derived from at least one of mustard seed and mustard seed extract. In some embodiments, the mixture of the watercress extract (source ofPEGLC) and mustard seed extract (source of myrosinase) can be added to the ingestible product possibly generating PEITC. In another embodiment, the reactant (ex. PEITC) can be directly added to the ingestible product. In some embodiments, ITC can also be synthesized, and added directly to the ingestible product.

[0045] In some embodiments, the product further includes at least one of thymoquinone and a vîtamin C. Thymoquinone can be extracted from Nigella sativa (black cumin seed) or other plant. Thymoquinone exhibits anticancer activity, showing inhibition of metastatic capacity (25), maintaining antioxidant activity, including préservation of anti-oxidant enzyme capacitîes such as glutathione peroxidase, catalase, and glutathione-S-transierase (7,26), and upregulation of p53 expression (28) in cancer cells including lung and breast cancer cells. Extracted or purified thymoquinone and/or the extract of the plant can be added to the ingestible product.

[0046] Resveratrol, anatural slilbene and a non-flavonoid polyphenol, is a phytoestrogen that possesses antî-oxidant, anti-înflammatory, cardioprotective, and anti-cancer properties (Ko et al., the rôle of Resveratrol in cancer therapy, Int J. Mo. Sci., 2017). It has been reported that resveratrol can reverse multidrug résistance in cancer cells, and, when used in combination with clinically used drugs, it can sensitîze cancer cells to standard chemotherapeutic agents. Several novel analogs of resveratrol hâve been developed with improved anti-cancer activity, bioavailability, and pharmacokinetic profile.

[0047] Vitamin C also exhibits anticancer activity, showing upregulation of P53 expression (35). The vitamin C can be extracted from citrus fruits and can be added to the ingestible product.

[0048] The synergistic anti-cancer activity is expected by any combination of ITC, thymoquinone, resveratrol and vitamin C presented in the ingestible product and the results of the synergistic effect are shown in the figures below.

[0049] In some embodiments, a smokable product contains no nicotine, tobacco leaf or tobacco leaf extract, but does contain at least one of ITC, thymoquinone, resveratrol and vitamin C. And instead of tobacco leaf or a tobacco leaf extract, the smokable product includes a nicotine-free plant leaf, or extract, such as one or more nicotine-free herbs. Such product is nicotine-free, with anti-cancer activity, and as such it is contemplated current smokers as well as potential smokers could enjoy smoking the product.

[0050] In some embodiments, at least one of the nicotine, ITC, myrosinase, thymoquinone, resveratrol and vitamin C îs encapsulated, and such encapsulation provides ingrédients physîcally or chemically separated, entrapped, or suspended in the ingestible product. The encapsulation provides at least two advantages. First, it prevents molecular dégradation and/or deformation from any acid produœd from the tobacco leaf or ex tract. Second, a user can fully enjoy the taste of the ingestible product even though the product contains other materials in addition to the tobacco leaf or extract.

[0051] The encapsulation may include binders and/or surfactants. Examples of the binders are dicalicium phosphate, magnésium stéarate, croscarmellose sodium, titanium dioxîde, silica, parabens, lactose, maldodextrin, monodosium glutamate, talc, miscrocrystalline cellulose, gelatin, gelian gum, tartrazine, allura red, îndigotine, indigo line, Polyvinylpyrrolidone, and sodium slearyl fumarate, stearic acid.

[0052] Surfactants are ciassified as anionic or nonionic. Contemplated surfactants for the subject matter herein are non-toxlc, inciuding palmitic acid, stearic acid and oleic acid (Cl 6 -C24) fatty acids, long chain (C 16 - C 24) esters of fatty acids, polyethylene glycol, polyethylene glycol alkyl ethers, polyethylene glycolate mono- or di-fatty acid esters of polyethylene glycol, fatty acid mono, poiyoxyethylene mono- and di-stearate, and polyoxyethylene ether, poiyoxyethylene sorbitan monolaurate, monopalmitatc, monostearate or mono-esters, poiyoxyethylene alkyl phénols and alkyl phenyl ethers, polysorbate, stearylamine, triethano lamine oleate, vegetable oîls, poiyoxyethylene very caillou, sorbitan fatty acid esters, and cholestérol.

[0053] The encapsulation can be micelles, mîcroparticles, and nanoparticles. The micelles are formed using for examples, phospholipid, giycolipids, and lonized fatty acids.

[0054] Examples of suitable materials for mîcroparticles and nanoparticles are silica, alumîna, titania, carbon black, aluminum silicate, calcium silicate, magnésium silicate, potassium silicate, sodium silicate, sodium aluminosilicate, sodium calcium aluminosilicate containing, trîcalcium silicate, silica airgel, talc, iron oxide, other métal oxides combinations thereof

[0055] A method of preventing or treating cancer in a population such as smokers provides an ingestible product containing nicotine, at least one of ITC and an ITC precursor, and myrosinase. The nicotine can be derived from at least one of powdered tobacco leaf and a tobacco leaf extract. The ITC can be derived from at least one of watercress, walercress extract, garden cress, and garden cress extract. The myrosinase can be derived from mustard seed extract. In some embodiments, the ingestible product further contains at least one of molécules inciuding thymoquinone, resveratrol and vitamin C. The product can be packaged in any suitable form, inciuding at least one of a smokeless tobacco, a chewing tobacco, a snuff, a snus, and a dissolvable tobacco, a cigarette, a twist, a cîgar, and a cigarillo.

[0056] In contemplated experiments, the effecl of the ingestible product could be examined in breast cancer cell (MCF7), Hela cells, oral cancer cells, A549 (human lung cancer cells). Cells are plated equal number and incubated with ITC, thymoquinone, and vitamin C îndividually and the combination thereof. The apoptosis progression could be measured using Dow cytometry, and immunostaining against proteins that are responsible for apoptosis, such as TUNEL and caspase-3. Expression levels of wildtype and mutant p53 could be determined by qRT-PCR and immunostaining against wildtype and mutant p53. The cell cycle arrest at G2/M could be observed applying immunostaining against G2-M checkpoînt regulators such as cyclin B 1 and cdc2.

[0057J The celis could be divided into severai groups including 1. No treatment, 2. ITC only, 3 thymoquinone only, 4 vitamin C only,5 resveratrol only 6 ITC + thymoquinone, 7 thymoquinone + vitamin C, 8 ITC + resveratrol 9 vitamin C + ITC, 10 ITC + thymoquinone + vitamin C + resveratrol, 11 thymoquinone + resveratrol, 12 vitamin C + resveratrol. Severai different incubation limes and different concentrations of the molécules are examined to observe the effect of ITC, thymoquinone, vitamin C and resveratrol and the combination thereof.

EXAMPLES

Each of Allvl isothiocvanate (AITC). Phenvl isothiocvanate (PEITC). Thymoquinone. or Resveratrol effectively induces cell death in vitro.

[0058] The experiments were performed with the following procedure.

[0059] A549 cells were plated into 48 well culture plates at a density of 3.0x104 cells/cm². Cells were dissociated with 0.05% trypsin EDTA following a 5-minute incubation at 37°C, neutralized in a 1:1 ratio of standard medium and counted using a hemocytometer prior to plating. Ail wells were settled overnight prior to addition of one of the following treatment compounds: AITC, PEITC, Thymoquinone, or Resveratrol. Ail trealments were performed within 24 hours following initial plating.

[0060] Treatment of the A549 cells occurred at the following time points and concentrations: AITC - 25 μΜ, 50 μΜ and 100 μΜ for 24, 48 and 96 hours. PEITC - 25 μΜ, 50 μΜ, 100 μΜ and 200 μΜ for 24, 48 and 96 hours. TQ - 25 μΜ, 50 μΜ, 10 CpM and 200 μΜ for 24, 48 and 96 hours. Resveratrol - 25p M, 50 μΜ and 100 μΜ for 48 and 96 hours. DMSO was used to dissolve each compound, and cells treated with DMSO without any of tested compounds served as a control. Treated cells received daily treatments using the same concentrations untîl the lasl time point of the experiment.

[0061] Apoptosîs was detected using the CellEvent Caspase 3/7 green détection kît/assay from Thermofisher Scientific. Cells were incubated with diluted assay reagent for 30 minutes at 37°C prior to analysis, and imaged by a brightfield and a fluorescent microscopes.

[0062] CellEvent Caspase-3/7 Green Détection Reagent is intrinsically non-fluorescent, as the four amino acid peptide (DEVD peptide) inhibits the ability of the dye to bind to DNA.

However, following activation of caspase-3/7 in apoptotîc cells, the DEVD peptide is cleaved, enabling the dye to bind to DNA and produce a bright, fluorogenic response. The fluorescent émission of the dye -530 can be observed under a fluorescent microscope.

[0063] Fig. 1 shows a brightfield image on the left and a fluorescent image on the right, correspondîng to the same A549 lung cancer cells treated with DMSO. Fig. 2- 5 show a brightfield image and a fluorescent image with various time points for varions concentrations of the various treatment compounds, AITC, PEITC, Thymoquinone, and Resveratrol.

[0064] Figure 2 shows that A1TC induces cancer cell death. At concentrations 50 μΜ and higher, more cells are exponentially undergone apoptosis at 48 hours than at lower concentrations. At lower concentrations, cell death was observed to occur at a more graduai rate.

[0065] Figure 3 show thaï PEITC induces cell death. At concentrations 100 μΜ and higher, more cells are exponentially undergone apoptosis earlier than at lower concentrations. At lower concentrations, cell death was observed to occur at a more graduai rate.

[0066] Figure 4 shows that Thymoquinone induces cell death. Al concentrations 100 μΜ and higher, more cells are exponentially undergone apoptosis earlier than at lower concentrations. At lower concentrations, cell death was observed to occur at a more graduai rate.

[0067] Figure 5 shows that Resveratrol induces cell death. At concentrations 50 μ.Μ and higher, more cells are exponentially undergone apoptosis earlier than at lower concentrations. On the other hand, the apoptotîc effect was reduced at 25 μΜ relative to higher concentrations of Resveratrol. ït is noteworthy that the apoptotîc effects of resveratrol are only observed at 48 and hours, in fact, the effects of the compound are delayed relative to other compounds. After 4 days of treatment, it was observed that each A549 non-small cell lung cancer cell culture that was administered with the either allyl îsothiocyanate, phenyl isothiocyanate, thymoquinone, or resveratrol had reached an apoptosis rate of up to 95%.

[0068J Figure 6 shows that the combination of ailyl isothiocyanate and phenyl isothiocyanate induces cancer cell death. At concentrations higher than 25 pM phenyl isodiiocyanate , exponentially more cells are undergone apoptosis at 48 hours than at lower concentrations. At lower concentrations of phenyl isothiocyanate, cell death occurs at a more graduai rate.

[0069] Figure 7 shows that the addition of thymoquinone to the previous combination of allyl isothiocyanate and phenyl isothiocyanate induces cancer cell death. At concentrations of 25 μΜ for ail compounds, exponentially more cells are undergone apoptosis at 48 hours than in the cultures without thymoquinone.

[0070] Figure 8 shows that the combination of thymoquinone and resveratrol induces cancer cell death. The rate of cell death observed in this combination is similar to the compound combination observed in Figure 7which also included thymoquinone. Both appear to exponentially induce apoptosis once they hâve passed 48 hours.

[0071] Figure 9 shows that the combination of phenyl isothiocyanate, allyl isothiocyanate, thymoquinone, and resveratrol induces cancer cell death. At concentrations where each compound is higher than 10 μΜ, exponentially more cells are undergone apoptosis at 48 hours than at lower concentrations. At lower concentrations (< 10 μΜ) , the rate of apoptosis appears to be less pronounced.

[0072] The combination of compounds (Fig 6-9) shows that apoptosis is observed at the lowest concentration of 10 μΜ in a combination including ail 4 compounds. A higher degree of apoptosis was observed in the Chemical combinations with concentrations higher than 10 μΜ from a perîod of 24 to 72 hours (upto 80 % apoptosis). Cell apoptosis treated with the combination of compounds is observed upto 72 hours, but not upto 96 hours, as seen in Figs 1 to 5, due lo the brevity of time. One of reasons is that apoptosis rate of 90 % or higher, similar to Figs 1-5, could hâve been reached by 96 hours.

[0073] It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the spécification and the daims, ail terms should be ₅ înterpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be înterpreted as referring to éléments, components, or steps în a non-exclusive manner, indicating that the referenced éléments, components, or steps may be présent, or utilîzed, or combined with other éléments, components, or steps that are not expressly referenced. Where the spécification claims refers to at least one of something selected from the group consisting of A, B, C .... and N, the text should be înterpreted as requiring only one élément from the group, not A plus N, or B plus N, etc.

Claims (29)

1. An ingestible product comprising nicotine, and at least one of (a) isothiocyanate or an isothiocyanate precursor, (b) myrosinase, and (c) resveratrol.

2. The ingestible product of claim 1, formulated as one of the following: a smokeless tobacco, a chewing tobacco, a snuff, a snus, a dissoivable tobacco, a cigarette, a twist, a cigar, and a cigarillo.

3. The ingestible product of any of claims 1-2, further comprising powdered tobacco leaf as a source of the nicotine.

4. The ingestible product of any of claims 1-3, further comprising a tobacco ieaf extract as a source of the nicotine.

5. The ingestible product of any of claims 1-4, further comprising at least one of water cress, water cress extract, garden cress, and garden cress extract as a source of the isothiocyanate precursor.

6. The ingestible product of any of claims 1-5, wherein the isothiocyanate is synthetic (Phenethyl Isothiocyanate or Allyl Isothiocyanate).

7. The ingestible product of any of claims 1-6, further comprising at least one of muslard seed and mustard seed extract as a source of the myrosinase.

8. The ingestible product of any of daims 1-7, further comprising thymoquinone.

9. The ingestible product of any of claims 1-8, further comprising a black cumin seed exlract.

10. The ingestible product of any of claims 1-9, further comprising a synthetic vitamîn C.

11. The ingestible product of any of claims 1-10, further comprising the resveratrol.

12. The îngestible product of any of claims 1-11, comprising the nicotine, and each of (a) the isothiocyanate or an isothiocyanate precursor, (b) the myrosinase, and (c) the resveratrol.

13. The ingestible product of any of claims 1-12, comprising:

at least one of powdered tobacco leaf and a tobacco leaf extracl as a source of the nicotine;

at least one of water cress, water cress extract, garden cress, and garden cress lü extra et as a source of the isothiocyanate precursor; and mustard seed extract as a source of the myrosinase.

14. The ingestible product of claim 13, further comprising a synthetic isothiocyanate.

15

15. The ingestible product of claim 13, further comprising thymoquinone.

16. The ingestible product of claim 13, further comprising synthetic vitamin C.

17. The ingestible product of claim 13, further comprising resveratrol.

18. The ingestible product of claim 13, further comprising thymoquinone, resveratrol and a synthetic vitamin C.

19. The ingestible product of any of claims 1-18, wherein at least one of the nicotine and 25 the isothiocyanate precursor and synthetic isothiocyanate is encapsulated.

20. The ingestible product of any of claims 1-19, wherein the isothiocyanate precursor and synthetic isothiocyanate are encapsulated in at least one of micelles and nanoparti cl es.

21. The ingestible product of any of claims 1-20, wherein at least some of the isothiocyanate and isothiocyanate precursor and synthetic isothiocyanate is in direct contact with at least one of powdered tobacco leaf and a tobacco leaf extract.

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22. The ingestible product of claim 1-21, wherein the ingestible product includes a filter.

23. An ingestible product comprising nicotine, at least one of isothiocyanate and an isothiocyanate precursor, myrosinase and a synthetic isothiocyanate for use in a method of preventing or treating cancer in a population.

24. The ingestible product for use of claim 23, wherein the ingestible product comprises: at least one of powdered tobacco leaf and a tobacco leaf extract as a source of the nicotine;

at least one of water cress, water cress extract, garden cress, and garden cress extract as a source of the isothiocyanate;

a synthetic isothiocyanate and mustard seed extract as a source of the myrosinase.

25. The ingestible product for use of any of daims 23-24, wherein the ingestible product further comprises thymoquinone.

26. The ingestible product for use of any of daims 23-25, wherein the ingestible product further comprises resveratrol.

27. The ingestible product for use of any of daims 23-26, wherein the ingestible product further comprises vitamin C.

28. The ingestible product for use of any of daims 23-27, further comprising packaging the ingestible product as at least one of a smokeless tobacco, a chewing tobacco, a snuff, a snus, and a dissolvable tobacco.

29. The ingestible product for use of any of daims 23-28, further comprising packaging the ingestible product as at least one of a cigarette, a twist, a cigar, and a cigarillo.