DESC:Field of the Invention
The present invention relates to a system to deliver cannabidiol (CBD), through combustible formats containing tobacco / nicotine to offer multiple benefits to users. The present invention further relates to a composition comprising said CBD or delivery through smoke.

Background of the invention and prior art
Various methods or systems for delivering CBD as active agents have been reported in the art. It has been reported that, ~70 cannabinoids produced by Cannabis sativa L. (marijuana), ?9-tetrahydrocannabinol (THC) is the principal psychoactive constituent of at least 70 cannabinoids found in cannabis plant owing to its high potency and abundance in plant preparations (Velasco G et al., 2012, Nature Reviews Cancer 12:436).
THC is known to exert a wide variety of biological effects by mimicking endogenous substances — the so-called endocannabinoids. Specifically, cannabinoids are well known to exert palliative effects in cancer patients. At the same time, the usage of ?9-THC is often limited by their unwanted psychoactive side effects.
Further, a few studies have shown that, under certain conditions, cannabinoid treatment can stimulate cancer cell proliferation in vitro and can interfere with the tumor-suppressor role of the immune system. Likewise, there have been conflicting reports regarding the role (tumor suppressor or oncogenic) of the endocannabinoid system in cancer (Velasco G et al., 2012, Nature Reviews Cancer 12:436). Further there are reports of the therapeutic potentials of cannabinoids. Most of the prior arts illustrate the potential of cannabinoids as an anti-cancer agent and its delivery through products like gum, lozenges, skin creams etc.
US2002/111377 relates to method providing a cannabinoid composition and delivering the cannabinoid transdermally to the subject. The said composition includes at least one cannabinoid selected from group consisting of ?9-THC, cannabinol, cannabidiol, nabilon, levonantradol, (-)-HU-210, (+)-HU-210, 11-hydroxy-?9-THC, ?8-THC-11-oic acid, CP 55940 and R(+)-WIN 55, 212-2. According to ‘377 the composition is delivered in the form of cream, salve, ointment as topical formulation. In another aspect of ‘377 relates to delivering the composition transdermally into a bandage, pad or other type of patch which can be applied to subject’s skin. The method used to relieve the symptoms of variety of diseases including cancer and AIDS, lack of appetite, chronic pain etc.
US2012/034293 provides a method for relieving symptoms associated with illness or associated with the treatment of illness in a mammalian subject comprising the steps of selecting at least one cannabinoid from the group consisting of cannabinol, cannabidiol, nabilon, levonantradol, (-)-HU-210, (+)-HU-210, 11-hydroxy-?9-THC, ?8-THC-11-oic acid, CP 55940 and R(+)-WIN 55, 212-2. The composition includes at least one permeation enhancer from the group consisting of propylene glycol monolaurate, diethylene glycol monoethyl ether, an oleoyl macrogoglyceride, a caprylocaproyl macrogolglyceride and an oeyl alcohol. The ‘293 also provides a method of delivering the selected cannabinoid and permeation enhancer transdermally to treat the illness. The disease as disclosed in ‘293 are cancer and AIDS, lack of appetite, chronic pain, etc which are treated by the method.
WO2011/026144 relates to microneedle mediated drug delivery systems comprising a pharmaceutical composition comprising a pharmaceutically active agents e. g. cannabidiol and prodrugs of cannabidiol. Further, ‘144 provide a method of using microneedle transdermal or topical drug delivery system comprising the pharmaceutical composition to treat pancreatitis and pancreatic cancer.
US2012/0095088, US 2012/0046351 relate to a product and a process wherein cannabinoids and/or other substances associated with medicinal cannabis are contained in food stuffs such that the medicinal cannabis is distributed uniformly in the foodstuff. Foodstuffs consistent with this invention include baked goods, hard candies, ice cream, bases, ice cream, and yogurt.
WO 2010/053580 relates to means and methods for reducing the risk of developing and/or aggravating smoke-related disease thereof. More specifically, the invention relates to means and methods for producing a smokeable product with reduced inflammatory capacity and uses thereof for reducing the risk of developing and/or aggravating smoke-related disease. The objective of ‘580 is achieved by delivering a medicinal active ingredient of marijuana, preferably taken from the group consisting of ?9-tetrahydrocannabinol (THC), cannabidiol and b-caryophyllene, to a subject with decreased risk of cannabis smoking-related illness. The method comprises obtaining a smokeable marijuana product, wherein the marijuana comprised therein has been irradiated so as to reduce the nucleic acid content thereof, and then smoking it so as to deliver a medicinal useful ingredient of marijuana to the subject. The smokeable product according to ‘580 includes cigarettes and cigars of all types and sizes, loose tobacco for rolling cigarettes by the consumer, pipe tobacco, and smokeable products comprising marijuana and/or herbaceous tobacco additives or replacements. This process requires complicated and cumbersome process of reduction of the nucleic acid content. If non irradiated marijuana is smoked besides releasing cannabidiol also concomitantly releases undesirable psychotropic substances such as THC thereby masking the health benefits of cannabidiol.
US6630507 relates to cannabinoids having antioxidant properties. This makes cannabinoids useful in the treatment and prophylaxis of wide variety of oxidation associated diseases, such as ischemic, age-related, inflammatory and autoimmune diseases. The cannabinoids are found to have particular application as neuroprotectants, for example in limiting neurological damage following ischemic insults, such as stroke and trauma, or in the treatment of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and HIV dementia. Nonpsychoactive cannabinoids, such as cannabidiol, are particularly advantageous to use because they avoid toxicity that is encountered with psychoactive cannabinoids at high doses useful in the method of the present invention. A particular disclosed class of cannabinoids useful as neuroprotective antioxidants is formula (I) wherein the R group is independently selected from the group consisting of H, CH3, and COCH3.
US 8293786 relates to pharmaceutically active agents (e.g., prodrugs of cannabidiol) suitable for local and systemic delivery to a mammal, including systemic transdermal delivery and topical delivery. One embodiment of ‘786 discloses compositions are bucally administrable may include formulations in which the cannabidiol prodrug is administered in lozenges, sprays, gels, pastes, dissolvable tablets or dissolvable strips. The disease conditions are selected from nausea, pain, chemotherapy induced vomiting, dermatitis, Rheumatoid arthritis, anti-inflammatory, anti-cancer, diabetic neuropathy and so on.

Further there are reports (http://truth11.com/2012/12/12/cannabidiol-offer-boost-towards-a-healthy-cancer-free-body/) on both cannabinoids (CBD), the non-psychoactive component of the marijuana plant, and tetrahydrocannabinol (THC), the psychoactive component that can “have a protective effect against the development of certain types of tumors” and potentially inhibit the growth of all types of cancer such as brain and breast cancer. (source: National Cancer Institute). Research is also supporting the idea that both THC and CBDs may have anti-tumor properties and retard cancer cell growth.
Nature Reviews Cancer 3, 745-755 (October 2003) discloses Cannabinoids — the active components of Cannabis sativa and their derivatives — exert palliative effects in cancer patients by preventing nausea, vomiting and pain and by stimulating appetite. In addition, these compounds have been shown to inhibit the growth of tumor cells in culture and animal models by modulating key cell-signaling pathways. Cannabinoids are usually well tolerated, and do not produce the generalized toxic effects of conventional chemotherapies.
Mol Cancer Ther November 2007 6; 2921 relates to cannabidiol (CBD), a cannabinoid with a low-toxicity profile, that down-regulates Id-1 expression in aggressive human breast cancer cells. The CBD concentrations effective at inhibiting Id-1 expression correlated with those used to inhibit the proliferative and invasive phenotype of breast cancer cells. CBD was able to inhibit Id-1 expression at the mRNA and protein level in a concentration-dependent fashion. These effects seemed to occur as the result of an inhibition of the Id-1 gene at the promoter level. Importantly, CBD did not inhibit invasiveness in cells that ectopically expressed Id-1. In conclusion, CBD represents the first nontoxic exogenous agent that can significantly decrease Id-1 expression in metastatic breast cancer cells leading to the down-regulation of tumor aggressiveness.
Guzmán M et al., 2001 J Mol Med (Berl). 78(11):613- teaches that the most exciting and promising areas of current cannabinoid research which is the ability of these compounds to control the cell survival/death decision. Thus cannabinoids may induce proliferation, growth arrest, or apoptosis in a number of cells, including neurons, lymphocytes, and various transformed neural and non-neural cells. Regarding the immune system, low doses of cannabinoids may enhance cell proliferation, whereas high doses of cannabinoids usually induce growth arrest or apoptosis.
Further, both in vitro and in vivo studies have shown that cannabidiol inhibit transformed cells growth by causing cell death, blocking cell growth, and blocking the development of blood vessels in a concentration-dependent manner while having little effect on non-transformed cells and may even protect them from cell death. Many studies suggest that CBD may preferentially kill proliferative cells in disease conditions while protecting normal cells (Shrivastava A, et al., . Mol Cancer Ther 10 (7): 1161-72, 2011; Vaccani A, et al., Br J Pharmacol 144 (8): 1032-6, 2005; Torres S, et al., Mol Cancer Ther 10 (1): 90-103, 2011).
There are reports that the cannabinoids as actives are selective in inducing cell death of breast tumor cell lines and has no effect on normal cell lines thereby underscoring its incorporation into the delivery system (Mol. Cancer Ther, July 2011:10:1161). Similar results have also been observed with myelogenic leukemia cells and normal monocytes respectively (Mol. Pharmaco. Sepetmber 2006: 70(3):897). The active CBD is an approved drug for the treatment of Multiple sclerosis where it constitutes 50% of total composition and is marketed under the brand name Sativex. FDA has recently permitted the investigation of active for the treatment of epilepsy in children which further justifies its safety for potential applications.
Although CBD is reportedly effective against various tumors, its molecular mechanism of action is not fully characterized. CBD is reported to be cytotoxic to gliomas and inhibits tumor cell migration in vitro (Vaccani A et al.,. Br J Pharmacol 2005;144:1032–6; Massi P et al., J Pharmacol Exp Ther 2004;308:838–45; Massi P et al., Cell Mol Life Sci 2006;63:2057–66). In addition, CBD induces apoptosis in human leukemia cell lines by activating classical caspase pathways, and enhancing NOX4 and p22phox function (McKallip RJ et al., Mol Pharmacol 2006;70:897–908).

A recent study reports that CBD inhibits human breast carcinoma (Ligresti A et al., J Pharmacol Exp Ther 2006;318:1375–87). Further as mentioned earlier CBD down regulates Id1, a regulator of metastasis in breast cancer cell lines (McAllister SD et al., Mol Cancer Ther 2007;6:2921–7). Furthermore, CBD, in conjunction with THC, induces programmed cell death (PCD) in glioma cells (Marcu JP et al., Mol Cancer Ther 2010;9:180–9).

Prior art findings have shown the active has multiple health benefits. The present invention provides a smoking article which has cannabidiol to be delivered through smoke to the appropriate cells of the subject.

Object of the invention
It is an objective of the present invention to provide a system/product to deliver active agent, through combustible tobacco format.
It is further objective of the present invention to incorporate the active molecules in different tobacco formats to deliver biological effects at the cellular and molecular level.
It is further an objective of the present invention that the delivered actives have selective action against the cells with altered physiological status and have no effect on the normal cells.
It is an objective of the present invention to provide a composition comprising the active to deliver the health benefits to the subject.

Summary of the Invention
The present invention relates to a smokable tobacco product for pulmonary delivery of Cannabidiol (CBD) comprising: CBD; tobacco; and organoleptic ingredients and excipients, in order to deliver CBD through smoke to pulmonary alveolar cells in the range of 1µM CBD/60µg of CSC to 10µM CBD/60µg of CSC (cigarette smoke concentrate), such smoke being formed by pyrolytic burning of said product.
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Brief Description of the Accompanying Drawings
Figure 1 is a schematic illustration of the conventional cigarette.
Figure 2 represents the conventional cigarette comprising the tobacco flakes with the CBD through by injection method.
Figure 3 shows the CSC when challenged with A549 cells to demonstrate reduced viability upon incubation with CBD.
Figure 4 demonstrates that the CBD does not alter the cell viability of CSC challenged normal CHO cells as determined by WST-1 Assay.
Figure 5 illustrates CBD reduces migration of CSC challenged A549 cells.
Figure 6 represents the determination of CBD availability in CSC obtained from CBD incorporated cigarettes using HPLC method.
Figure 7 represents the determination of CBD availability (A) and bioactivity (B) in CSC obtained from incorporated cigarettes by HPLC and A549 based cell viability assay
Figure 8 represents the determination of CBD in CSC obtained from CBD incorporated E cigarettes by A549 based cell viability assay

Description of the Invention
The present invention relates to delivery of bioactive(s), such as CBD, through tobacco containing articles, such as cigarettes. The delivery of CBD using tobacco smoke provides an ideal system. Tobacco is neutral and the smoke is found to be ideal delivery system for transfer of CBD to the alveolar cells.
"Product" according to the present invention includes all forms of combustible products, viz., cigarettes, cigars, etc.
In accordance with the present invention a delivery system/product comprises active agents at pre-defined doses such that it delivers the actives via the smoke route.
"Delivery System" as used herein contemplates, to attenuate or mitigate or reduce or lower or prevent or inhibit or avert abnormal cellular proliferation and protect or defend or guard or shield or shelter the biological system comprises atleast one active agent(s), organoleptic agent(s) which is optional and other acceptable incipient(s) in combinations thereof through smoke, to deliver the said actives into the biological system.
Cigarette smoke condensate (CSC) refers to the sticky particles comprised of thousands of chemicals created by burning tobacco. It is the particulate component obtained from mainstream or side stream tobacco smoke is often used in scientific research to test its biological effects. It does not form part of the composition. It is meant for testing or analysis purposes only.
The delivery system/ product according to the present invention comprises
(i) a composition comprising an active agent, tobacco, organoleptic agents, excipients and combinations thereof;
(ii) cigarette paper;
(iii) filtration zone comprising filter and tipping;
The active agents are selected from cannabidiol and/or its mixtures, isomers, stereomers, salts, polymorphs, complexes and combinations thereof. The cannabidiol is synthetic and is procured from SIGMA ALDRICH.
The active agent in the composition can range from 0.1 to 5 wt%. It has been found that the active agent CBD can be incorporated from 0.1 to 5 wt% in all standard product specific compositions.
The inventors have found that, CBD concentration is to be maintained at 0.1-5 wt% in the composition of the delivery system/product in order to provide the effective amount of CBD i.e., 1-3µM CBD/60µg of CSC through smoke route during product consumption. Thus according to the present invention 0.1-5 wt% CBD in said composition is suitable to deliver the active in effective concentration at the target site.
The present inventors have found in in vitro experiments that the effective concentration of CBD is 1-10µM CBD/60µg of CSC in order to provide enhanced cellular benefits in terms of anti proliferation and anti malignancy effects without affecting the normal cells. Further it has also been demonstrated that effective concentration 1-3µM prevents abnormal cellular proliferation.
According to the present invention, tobacco is present in the composition in an amount of 700-750 mg which constitutes 70% of the composition.
The organoleptic agents and/or acceptable excipients are selected from Flavors / flavorants, taste enhancers, natural extract such as clove, elaichi etc in the range of 0.0001 to 10 wt%, preferably 0.001 to 5 wt% and most preferably, 0.01 to 2 wt%.
Further, the active CBD is to be included into the cigarette rod to deliver the cannabinoids directly into lungs through pulmonary route.
The invention is now illustrated by way of non limiting example(s).
Example 1
Process for incorporating CBD into various forms of combustible product containing tobacco/nicotine
The present invention contemplates utilizing the bioactive such as CBD onto the cigarette flakes through various processes such as spraying, dipping, radiation, osmosis, friction, mixing, blending, infusion, coating, waxing, applying the substance in liquid form to the tobacco and/or nicotine, filter or cigarette paper, by immersing the tobacco, filter, or cigarette paper in a solution of the substance, impregnating by pressure, chemically impregnating, using an adhesive, saturation, brushing, gaseous infusion, immersion or the like.
Example 1a
Figure 2 illustrates the cigarette containing tobacco flakes which have been injected with CBD. It was prepared by dissolving CBD in ethanol which is then sprayed, coated, mixed directly on cut tobacco. The CBD sprayed tobacco was then used for manufacture of cigarettes.

Example 2
Preparation of cigarette smoke condensate extract
The cigarette smoke condensate (CSC) as a representative of cigarette whole smoke was prepared from reference cigarette 3R4F. CSC was prepared as per recommendation; Cigarettes were smoked under ISO conditions using a Cerulean Machine. CSC was collected in a cambridge filter pad and extracted in Dimethyl Sulphoxide (DMSO) as per standard recommended protocol published by CORESA in vitro task force. The filter pad which contains adsorbed CSC was shaken in solution containing DMSO. The volume of DMSO used for extraction was determined by the weight of the filter pad so as to obtain CSC at a concentration of 25 mg\ml. Extracted CSC was frozen in aliquots immediately to avoid repeated freeze thawing cycles.
CSC challenged A549 cells demonstrate reduced viability upon incubation with CBD (Figure 3)
A549 cells (10,000) per well were seeded in a 96 well plate in DMEM containing 10% FCS and penicillin (100 µg/ml), streptomycin (100 µg/ml) and incubated at 37°C in a humidified chamber under 5% carbon dioxide for 24 hours. Later, cells were exposed to DMEM containing cigarette smoke condensate (CSC) at a concentration of 60 µg/ml alone or were incubated concomitantly with CBD (1µM, 3µM and 10µM) for 24 hours. The cellular viability was assayed by WST dye uptake which was measured at 450 nm using spectrophotometry. The results are expressed as % of viable cells as compared to untreated cells (NC). As shown in the figure 3, CBD caused a dose dependent decrease in cell viability in A549 cells at 24 hours post treatment as determined by WST assay. In the current experiment, Total Particulate Matter (TPM) was generated from 3R4F reference cigarettes smoked under ISO conditions. The TPM was resuspended in DMSO to obtain CSC. Solvent control – DMSO. The data was analyzed for statistical significance using Student's t-test
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CBD does not alter cell viability of CSC challenged normal CHO cells as determined by WST-1 Assay (Figure 4)
CHO cells were plated at a density of 10,000 cells per well in a 96 well plate in DMEM medium containing 10% FCS and penicillin (60 µg/ml), streptomycin (100 µg/ml) in humidified chamber under 5% carbon dioxide. 24 hours post plating the cells were left untreated (NC), treated with solvent, CSC alone (60 µg/ml) or CSC concomitantly with 3µM CBD for 24 hours. The cellular viability was assayed by WST dye uptake which was measured at 450 nm using spectrophotometry. As shown in the figure 4, CBD at 3µM dosage did not alter viability of CSC challenged normal CHO cells. The results are expressed as % of viable cells as compared to untreated cells (NC). In the current experiment, Total Particulate Matter (TPM) was generated from reference cigarettes smoked under ISO conditions. The TPM was re-suspended in DMSO to obtain CSC. Solvent control – DMSO. The data was analyzed for statistical significance using Student's t-test.

Determination of cell migration upon treatment of CSC challenged A549 cells with CBD and CBD reduces migration of CSC challenged A549 cells (Figure 5)
A549 cells were plated in a 6 well plate at a density of 1 x 106. 24 hours later when the cells had achieved 100% confluency, a p20 pipette tip was used to create a uniform scratch in the middle of the cell monolayer. The tip of the scratched area was marked to identify it under the microscope. Subsequently, the cells were either left untreated or were treated with CSC (60 µg/ml) in the presence or absence of CBD at concentration of 1µM or 3µM respectively. Using phase contrast microscopy (4X), the migration of the cells into the scratch area was observed in wells left untreated or treated with CSC at 24 and 48 hours respectively. As shown in the figure 5, CSC challenged A549 cells upon concomitant treatment with CBD at 3 µM dosage demonstrated reduced migration. Thus the experiment clarifies that CBD at 3 µM dose reduces cellular migration thereby suggesting that it may limit distant migration of aberrant cells.

Determination of CBD availability in CSC obtained from incorporated cigarettes by HPLC method (Figure 6)
30 µl of either ethanol (solvent control) or ethanol containing CBD (5mg) was directly injected into the control cigarettes respectively. Subsequently the cigarettes were smoked to obtain CSC. Later the CSC obtained from cigarettes were investigated for the presence of CBD based on WHO recommended (http://www.unodc.org/documents/scientific/ST-NAR-40-Ebook.pdf) using HPLC (Figure 6).

Determination of CBD bioactivity in CSC obtained from incorporated cigarettes by cellular viability assay (Figure 7)
30 µl of either ethanol (solvent control) or ethanol containing CBD (1 and 5mg) was directly injected into the 3R4F rod and filter respectively. Subsequently the cigarettes were smoked to obtain CSC.
As shown in the figure 7, CSC obtained from CBD incorporated cigarettes were tested for CBD activity using cell viability assay. CSC obtained from CBD incorporated cigarettes demonstrated reduced cell viability when compared to control cells thereby confirming the bio-activity of CBD incorporated into cigarettes. The decrease in viability of cells treated with CSC from CBD incorporated cigarettes was statistically significant (Students T test). Thus CBD incorporated into cigarette is released in an unmodified form upon smoking of the cigarette thereby confirming its application.
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Determination of CBD bioactivity in e-juice obtained from CBD incorporated E-cigarettes by cellular viability assay (Figure 8)
30 µl of either ethanol (solvent control) or ethanol containing CBD (1 mg or 5mg) was directly mixed with e-juice containing 80% propylene glycol, 20% glycerol and 2.5% nicotine respectively. Subsequently the E-cigarettes were smoked to obtain TPM as described previously (Figure 3).
As shown in the figure 8, TPM obtained from CBD incorporated E-cigarettes were tested for CBD activity using cell viability assay. TPM obtained from CBD (1mg or 5 mg) incorporated E-cigarettes did not alter cell viability when compared to control treated cells thereby confirming the absence of CBD activity (Students T test). Whereas when incorporated into cigarettes, CBD altered cell viability as seen as shown in figure 7. Thus CBD incorporated into E-cigarette is inactive and E-Cigarette is not suitable for use as a delivery vehicle for transfer of CBD.

[5mg of CBD incorporated into Cigarettes is equal to transfer of 3µM of CBD in vivo
Amount of CBD obtained from Filter pad for one cigarette injected with 5 mg of CBD = 187 µg
1 cigarette produces approximately 12 mg of Total particulate material (TPM)
Therefore 12 mg of TPM contains 187 µg of CBD
For cell culture experiments 60 µg of TPM per ml. 60 µg of TPM contains 0.935 µg of CBD
Or 1 ml of TPM containing solution has 0.935 µg of CBD for activity which is 2.977 µM
Molecular weight of CBD 314
Conc. of TPM used for experiments: 60 µg/ml]
,CLAIMS:1) A smokable tobacco product for pulmonary delivery of Cannabidiol (CBD) comprising:
a. CBD;
b. tobacco; and
b. organoleptic ingredients and excipients,
in order to deliver CBD through smoke to pulmonary alveolar cells in the range of 1µM CBD/60µg of CSC to 10µM CBD/60µg of CSC (cigarette smoke concentrate), such smoke being formed by pyrolytic burning of said product.
2) The product as claimed in claim 1 wherein said CBD is in the range of 0.1-5% by weight of the smokable tobacco product.
3) The product as claimed in claim 1 wherein said CBD is delivered through smoke to pulmonary alveolar cells preferably in the range of 1µM CBD/60µg of CSC to 3µM CBD/60µg of CSC (cigarette smoke concentrate)
4) The product as claimed in claim 1, wherein said CBD is incorporated on tobacco.
5) The product as claimed in claim 1 wherein CBD is incorporated in paper and wrapped around tobacco to form a cigarette.
6) The product as claimed in claim 1 wherein said excipients are flavoring agents selected from, clove, elaichi, citrus flavors.
7) The product as claimed in any preceding claim, such that CBD delivered is adapted to act on cancerous alveolar cells.