EA040213B1 - TOBACCO LEAF EXTRACT AND ITS USE FOR THE TREATMENT OF TOBACCO DEPENDENCE - Google Patents

Description

Technical field

The present invention relates to tobacco leaf extract, as well as to a pharmaceutical composition containing this tobacco leaf extract, and to its use in the treatment of tobacco dependence.

Prior Art

In general, according to the definition of the World Health Organization, addiction or addiction is a syndrome in the development of which the use of a substance becomes much more important than other activities that were previously considered much more important. In its extreme form, the state of addiction is characterized by an irresistible craving for a substance that causes the addicted individual to instinctively seek out that substance.

Tobacco addiction leads to public health problems, causing a large number of smoking-related diseases such as cancer (lung, larynx, mouth, lips, etc.), cardiovascular disease, chronic bronchitis, etc. In addition to contributing to the development of the diseases themselves, smoking has a number of side effects (decreased fertility, changes in the epidermis, changes in the nasopharyngeal mucosa, changes in the cerebral arteries, damage to the esophagus and stomach, deficiency of vitamins B and C, etc.).

There are three types of tobacco dependence that are observed simultaneously: physical dependence, which occurs mainly due to the presence of nicotine in tobacco and which is expressed in the form of a strong desire (strong urge to smoke, irritability, nervousness, agitation, anxiety, sleep disturbances, etc. .); physiological dependence, which is associated with the psychotropic effect of nicotine, expressed in delivering pleasure and relaxation, in stimulating intellectual activity, reducing anxiety, counteracting depression and reducing appetite; dependence on the environment or behavioral dependence, which is determined by social pressure and the insistence of friends. On average, physical dependence disappears within a few weeks. The withdrawal syndrome reaches its maximum intensity approximately three to four days after smoking cessation and can last for several weeks. The decrease in physiological dependence is slower and may last for several months.

Tobacco addiction is enhanced by positive and negative reinforcement (conditioned reflex). Nicotine triggers the release of dopamine, a neurotransmitter in the pleasure (reward) circuit. The smoker smokes both to reproduce the sensation of pleasure caused by the release of dopamine (this is a positive reinforcer of the effect of the cigarette) and to eliminate the withdrawal syndrome when the level of dopamine becomes too low (this is the negative reinforcement of the effect of the cigarette).

There are three main pharmacological treatments for tobacco dependence. The first is aimed at complete smoking cessation starting from a predetermined date, and in the case of nicotine substitutes, pharmacological treatment is started after this predetermined cessation date, and in the case of bupropion or varenicline, treatment is started a week before this date (to achieve sufficient drug concentration in plasma at the time of the predetermined smoking cessation date). The second way is to reduce the consumption of tobacco before the start of smoking cessation, and in this case, the reduction phase corresponds to the pre-treatment during which pharmacological treatment is prescribed. The third way is to prevent relapse after successful smoking cessation. To these three methods can be added a gradual reduction without attempts to stop.

The effectiveness of each of these methods is enhanced by the preparation phase of the smoker, who must convince himself of the need to stop smoking, during which his motivation is enhanced. Thus, guidelines for the use of drugs for the pharmacological treatment of tobacco dependence should state that smoking cessation therapy is more likely to be successful if patients are motivated to stop smoking.

Currently, there are four main types of tobacco dependence treatment (which do not include behavioral therapy, acupuncture and hypnosis), namely: nicotine substitutes, bupropion, varenicline, and homeopathy. The principle of operation of each of these four methods is based on the fact that nicotine is a molecule that determines the mechanism of tobacco dependence.

Nicotine substitutes can be administered in various ways: transdermally (through the skin) as patches, orally as chewing gum, lozenges or sublingual tablets, or through the lungs as inhaled preparations. The administration of nicotine substitutes generally reduces the negative reinforcement received from the cigarette by preventing the smoker from quitting withdrawal as much as possible, by having the nicotine contained in the substitutes compensate for the nicotine contained in the cigarettes. The effectiveness of nicotine substitutes depends on the method used, and usually the percentage of quitting smoking is from 15 to 20%. Nicotine substitutes are used in attempts to quit smoking immediately, as well as in pre-treatment before a quit attempt - they help smokers reduce their cigarette consumption by gradually

- 1 040213 smoking cessation (reduction of consumption until complete cessation). They may also be used for the sole purpose of reducing tobacco use. Their effectiveness in preventing relapse has not yet been confirmed.

Bupropion, commercially available from GlaxoSmithKline Laboratories (under the trade name Zyban®), acts on certain brain neurotransmitters such as catecholamines, norepinephrine, and dopamine. Bupropion is a selective catecholamine reuptake inhibitor, which gives it antidepressant properties. Its efficacy is equivalent to that of nicotine patches (approximately 20% quit rate). It is prescribed less frequently than nicotine substitutes because it has a lower benefit/risk ratio with comparable efficacy. Indeed, its use can lead to suicide attempts.

Varenicline, commercially available from Pfizer Laboratories (under the trade name Chantix® or Champix®), is a nicotinic acetylcholine receptor partial agonist. Varenicline binds to these receptors through a dual action: as a partial α4β2 receptor agonist, it creates a response similar to that of nicotine, but at a lower intensity (and thus reduces craving upon withdrawal); and at the beginning of treatment, when the smoker is on therapy but occasionally smokes, he suppresses neurochemical stimulation in the presence of nicotine (a partial α4β2 receptor antagonist). Thus, varenicline reduces the negative (withdrawal effect) and positive (the urge to smoke) reinforcer from smoking cigarettes. In practice, varenicline reduces the pleasure of smoking, which would be a measure of future quit success. It is more effective than nicotine substitutes (with a quit rate of approximately 28%). Varenicline is used in attempts to quit smoking abruptly, as well as in pre-treatment for several weeks before a quit attempt, because it helps smokers reduce cigarette consumption, which in part helps to gradually quit smoking (gradually reducing consumption to zero). In the first study (NCT00789074), 35% of smokers were able to reduce their cigarette consumption by at least 50% after 3 weeks of pre-treatment with varenicline. In the second study (NCT00835900), the percentage reduction in the number of cigarettes smoked at 4 weeks from the start of varenicline pre-treatment was 42%. In the third study (NCT01370356), 47% of smokers were able to reduce their cigarette consumption by at least 50% at 4 weeks from the start of pre-treatment with varenicline. Its effectiveness in preventing relapse has not yet been confirmed. However, the use of varenicline often leads to side effects (nausea) and may cause heart problems or suicide attempts. These side effects mean that, despite being more effective than nicotine substitutes, varenicline is often prescribed only as a secondary treatment after failure of nicotine substitutes.

The fourth way to quit smoking is homeopathic therapy, which is based on the use of extremely small doses of a substance that causes symptoms that one would like to control. In order to quit smoking, tobacco plant extract is often used. It may be noted that Irish patent application IE 960511 discloses the use of homeopathic dilutions of tobacco extract for the manufacture of a medical product intended to restore neuronal function and alleviate nicotine withdrawal. Like other non-traditional methods of quitting smoking, this technique is ineffective in the treatment of heavy smokers.

Despite intensive research in this area, there is still a need to discover new treatments or improve existing treatments for tobacco dependence.

In patent application PCT/FR2007/000786, the Applicant has found that injecting an aqueous solution of tobacco leaf extract into a smoker can treat that smoker's tobacco addiction. This discovery is completely unexpected since the Applicant was able to determine that this aqueous tobacco leaf extract contains small amounts of nicotine. Thus, the mechanism of action in the treatment of tobacco dependence with such an aqueous tobacco leaf extract does not involve the delivery of nicotine to the smoker.

However, the fact that said aqueous tobacco leaf extract contains small amounts of nicotine does not provide information on the composition of the extract and how it was obtained. Indeed, the nicotine content of aqueous tobacco leaf extracts can vary significantly depending on a variety of factors.

The nicotine content of tobacco leaves in the main types of grown tobacco can range from 0.5 to 8% (Davis et al., Production, Chemistry, and Technology), ISBN-13: 978-0632047918, Chapter 8, page 275).

Burley tobacco contains approximately twice as much nicotine as Maryland tobacco and twice as much nicotine as Oriental tobacco (Leffingwell, Chemical constituents of tobacco leaf and differences among tobacco types).

- 2 040213 tobacco), Leffingwell Reports, Volume 1 (No. 2), February 2001).

The application of nitrogen fertilizers affects the nicotine content of tobacco leaves, and the nicotine content in the upper leaves exceeds that in the middle leaves, which in turn exceeds that in the lower leaves (Xiao-Tang et al., Yield and nicotine content of flue -cured tobacco as affected by soil nitrogen mineralization, Pedosphere 18(2): 227-235, 2008, ISSN 1002-0160/CN 321315/P).

Crop rotation affects tobacco leaf nicotine content (Butorac et al., The Effect of Tobacco Monoculture and Crop Rotations on Tobacco Leaf Composition), Agriculturae Conspectus Scientificus, vol. 69 (2004) No. 4 (95-101)).

The chemical composition and nicotine content of tobacco leaves is affected by ultraviolet radiation and light intensity (Andersen et al., Chemical composition of tobacco leaves altered by nearultraviolet and intensity of visible light). Plant Physiol (1973) 51, 723-726).

The methods of obtaining the extract also affect the nicotine content, in particular the duration and conditions of maceration (soaking), methods of sterilization and physical and chemical separation.

Tobacco extract, such as the extract discussed in patent application PCT/FR2007/000786, includes low molecular weight molecules, ie. with a molecular weight less than 10 kDa. Such small molecules include tobacco-specific N-nitrosamines (TSNA for short) and N-nitrosamino acids derived from non-volatile alkaloids, volatile aldehydes, polynuclear aromatic hydrocarbons (such as benzo[a]pyrene), lactones, urethanes. or metals such as, for example, cadmium (see: Wagner et al., Plant Physiol. 1986, 82, 274-279 and IARC Monographs on the Evaluation of Carcinogenic Risks to Human) , v. 49). These compounds can be carcinogenic, mutagenic, or toxic and can cause cancer, as well as damage to the kidneys and bones. Thus, these compounds are detrimental to the health of a patient who has been administered a composition containing such compounds.

Such a tobacco extract includes many proteins with different molecular weights. For example, 759 tobacco proteins are described in the Swiss-Prot database. The RuBisCO protein alone can make up 30 to 50% of the soluble proteins. A complete RuBisCO protein typically contains eight subunits, forming a protein complex of approximately 540 kDa. The toxicity of these proteins when administered subcutaneously as a solution is unknown, and there is no indication that all or part of them can have any therapeutic effect.

The object of the present invention is to provide an effective treatment for tobacco dependence. Preferably, the therapy employed is non-toxic, well tolerated by patients, and involves limited risks, minimizing the likelihood of administering molecules whose toxicity is unknown and which are essentially non-therapeutic.

Despite the fact that tobacco extracts contain thousands of compounds, the Applicant has surprisingly found that proteins contained in tobacco leaf extract elicit a specific IgG immune response that promotes smoking cessation. Thus, the tobacco leaf extract according to the invention, having a high protein content and a specific protein composition, elicits a specific IgG immune response that promotes smoking cessation.

This finding is all the more surprising since aqueous tobacco leaf extracts contain thousands of compounds, of which proteins constitute only one class of compounds. Previously it was not known that tobacco proteins have an effect on tobacco dependence and thus there was no reason to use them in order to quit smoking.

In particular, the Applicant has shown that the tobacco leaf extracts according to the invention have a high protein content and a defined protein composition. This specific protein composition can be obtained by using dried (cured) tobacco leaves. Drying (withering) promotes the degradation of proteins, for example through hydrolytic mechanisms, and increases the free amino acid content of tobacco leaves (Hamilton & Lowe, 1978; Long & Weybrew, 1981; Burton et al., 1983).

The Applicant has surprisingly found that the proteins contained in the tobacco leaf extracts according to the invention induce the production of specific IgG antibodies which promote smoking cessation. The Applicant has shown that the administration of the tobacco leaf extract according to the invention induces a specific IgG immune response that promotes effective recovery from tobacco dependence, even without the addition of an adjuvant drug that can enhance the immune response to the extract. The Applicant has shown that specific IgG immune response, tobacco dependence treatment and smoking cessation are particularly effective when using dried tobacco leaf extracts.

This discovery is all the more surprising since the prior art does not have

- 3 040213 indications that IgG antibodies specific to tobacco leaf extract proteins are likely to help smokers reduce their smoking intensity or quit smoking. To date, neither the activation of a specific IgG immune response after the administration of tobacco extracts nor the role of tobacco extracts in smoking cessation has been described. To date, no relationship has been described between the immune system (which is known to produce specific IgG antibodies) and the nervous system (which is known to be involved in the perception of the act of smoking and related behavior) that can contribute to smoking cessation.

Preferably, the treatment of tobacco dependence according to the present invention requires the administration of the pharmaceutical composition according to the invention only a few times, preferably only one administration is required, as a result of which the reinforcing effect of cigarettes is no longer felt by the patient or is felt significantly weaker, and in particular the urge to smoke is reduced (positive reinforcement). ) and the attractiveness of cigarettes.

The essence of the invention

The first aspect of the present invention relates to a tobacco leaf extract containing at least 5 wt.% proteins based on the total weight of the dry extract, where the molecular weight of the proteins exceeds 10 kDa, and the proteins essentially do not contain molecules whose molecular weight is less than 10 kDa. Preferably, the proteins are selected from the group consisting of the following families of proteins: lignin-forming anionic peroxidase, glucan-endo-1,3-beta-glucosidase, endochitinase, pathogenesis-related protein, osmotin and proteinase inhibitor, and mixtures thereof.

The invention further relates to a pharmaceutical composition comprising tobacco leaf extract and its use in the treatment of tobacco dependence.

The invention further relates to a pharmaceutical composition comprising tobacco leaf extract and its use in quitting smoking.

The invention further relates to a method for treating tobacco dependence, comprising administering a tobacco leaf extract to a subject suffering from tobacco dependence or tobacco addiction.

The invention further relates to a method for treating tobacco dependence, comprising administering a pharmaceutical composition comprising tobacco leaf extract to a subject suffering from tobacco dependence.

In the context of the present invention, the treatment of tobacco dependence or addiction includes all forms of tobacco use, namely: smoked tobacco, in the form of manufactured or rolled cigarettes, cigarillos or cigars, pipe tobacco, as well as smokeless tobacco, such as snuff, chewing tobacco or snus.

The invention also relates to a method for producing a tobacco leaf extract containing at least 5 wt.% proteins based on the total weight of the dry extract, where the molecular weight of the proteins exceeds 10 kDa, and the proteins essentially do not contain molecules whose molecular weight is less than 10 kDa, where the method includes the following steps:

a) drying (withering) of tobacco leaves;

b) grinding dried tobacco leaves to obtain ground dried tobacco leaves;

c) extracting the ground dried tobacco leaves with mechanical agitation with a solvent, for example an aqueous solvent, preferably an aqueous buffer solution, the pH of which is from 6.0 to 8.5;

d) separating solid residues from the ground dried tobacco leaf extract solution by filtration or centrifugation, resulting in a solid, residue-free ground dried tobacco leaf extract solution;

e) filtration at a constant volume of the solution obtained in step d in the presence of a solvent, preferably an aqueous solvent, taken in an amount of 2 to 12 volumes, preferably 3 to 10 volumes, preferably 4 to 8 volumes, preferably 6 volumes, where the volume is equal to the volume of the extract, using a membrane with a cut-off value of 10 kDa;

f) optionally lyophilizing the protein solution obtained in step e.

The invention also relates to a kit comprising a pharmaceutical composition comprising a tobacco leaf extract containing at least 5 wt.% proteins based on the total weight of the dry extract, where the molecular weight of the proteins exceeds 10 kDa, and the proteins essentially do not contain molecules whose molecular weight is less than 10 kDa.

Brief description of graphic materials

In FIG. 1 shows a picture of the electrophoretic separation of the tobacco leaf extract according to example 1: fractionation on a 4-12% NuPage gel in MES buffer (English 2-(N-morpholino)ethanesulfonic acid, Russian the presence of a reducing agent.

In FIG. 2 shows IgG induction following injection of tobacco leaf extract into mice according to Example 1.

In FIG. 3 shows immunophenotyping to characterize activated

- 4 040213 immune cells.

In FIG. 4 shows the activation of natural killer cells by tobacco leaf extract according to example 1.

In FIG. 5 shows the effect of the tobacco leaf extract according to Example 1 on T and B lymphocytes.

In FIG. 6 shows the excitation of inflammatory cytokines and IFNy by tobacco leaf extract according to example 1.

In FIG. 7 shows the excitation of TH2 cytokines, chemokines and hematopoietic growth factor by tobacco leaf extract according to example 1.

In FIG. 8 and 9 show the evolution of tobacco consumption in patients treated with tobacco leaf extract according to Example 1.

Information confirming the possibility of carrying out the invention

tobacco leaf extract

The present invention relates to a tobacco leaf extract containing, based on the total weight of the dry extract, at least 5 wt.%, preferably at least 10 wt.%, preferably at least 15 wt.%, preferably about 20 wt.% proteins whose molecular weight exceeds 10 kDa, which are essentially free of molecules whose molecular weight is less than 10 kDa.

The dry extract is preferably prepared prior to lyophilization of the tobacco leaf extract according to the invention, then placed under a vacuum bell, preferably in the presence of P ₂ O ₅ , until a constant weight of the extract is reached.

Preferably, the content of molecules whose molecular weight is less than 10 kDa is less than 5 wt.% of the total weight of the extract, more preferably less than 2.5 wt.% and more preferably less than 1 wt.%.

According to the present invention, approximately means plus or minus 1% and is due to measurement inaccuracy.

The content of proteins with a molecular weight greater than 10 kDa, usually determined in tobacco leaf extracts, is approximately 1%. Thus, the tobacco leaf extract according to the present invention has a much higher content of proteins whose molecular weight exceeds 10 kDa than already known extracts.

Preferably, the proteins present in the extract of the present invention are selected from the group consisting of the following protein families: lignin-forming anionic peroxidase, glucan-endo-1,3-beta-glucosidase, endochitinase, pathogenesis-related protein, osmotin and proteinase inhibitor, and also their mixtures.

The names of the proteins present in the extract according to the present invention correspond to their names in the Swiss-Prot database, which is a biological database that lists protein sequences.

According to one embodiment, the tobacco leaf extract of the invention comprises at least one protein belonging to the glucan-endo-1,3-beta-glucosidase family and preferably selected from the acid isoform PR-Q' of beta-1,3-endoglucanase (PR36401 according to database UniProt), from the main vacuolar GLB isoform of beta-1,3-endoglucanase (P27666 according to the UniProt database) and mixtures thereof.

According to one embodiment, the tobacco leaf extract of the invention comprises at least one protein belonging to the endochitinase family and preferably selected from acid endochitinase P (P17513 according to the UniProt database), acid endochitinase Q (P17514 according to the UniProt database), endochitinase B (P24091 according to the UniProt database) and their mixtures.

According to one embodiment, the tobacco leaf extract of the invention comprises at least osmotin (P14170 according to the UniProt database).

According to one embodiment, the tobacco leaf extract of the invention comprises at least one lignin-forming anionic peroxidase (P11965 according to the UniProt database).

According to one embodiment, the tobacco leaf extract of the invention comprises at least one pathogenesis-associated protein, preferably selected from pathogenesis-associated protein R (P13046 according to the UniProt database), pathogenesis-associated protein PR4A (PR29062 according to the UniProt database), pathogenesis-associated protein with the pathogenesis of the PR-4B protein (PR29063 according to the UniProt database) and their mixtures.

According to one embodiment, the tobacco leaf extract of the present invention comprises at least one protein belonging to the proteinase inhibitor family and preferably selected from proteinase inhibitor I-B (Q03199 according to the UniProt database), proteinase inhibitor I-A (Q03198 according to the UniProt database) and mixtures thereof. .

According to one embodiment, the tobacco leaf extract also includes polysacha

- 5 040213 reeds, the molecular weight of which exceeds 10 kDa, which are preferably soluble in water.

Preferably, the tobacco leaf extract according to the present invention comprises, based on the total weight of the dry extract, at least 5 wt.%, preferably at least 10 wt.%, preferably at least 15 wt.%, preferably about 20 wt.% proteins selected from the group consisting of the following families of proteins: lignin-forming anionic peroxidase, glucan-endo-1,3-beta-glucosidase, endochitinase, pathogenesis-related protein, osmotin and proteinase inhibitor, as well as mixtures thereof.

According to one embodiment, the tobacco leaf extract of the present invention is substantially free of high molecular weight proteins.

According to the present invention, the term high molecular weight protein refers to a protein whose molecular weight exceeds 500 kDa, preferably whose molecular weight exceeds 400 kDa, more preferably whose molecular weight exceeds 300 kDa, more preferably whose molecular weight exceeds 200 kDa, even more preferably whose molecular weight exceeds 150 kDa, and even more preferably the molecular weight of which exceeds 100 kDa.

According to one embodiment, the tobacco leaf extract of the present invention is substantially free of proteins whose molecular weight exceeds 50 kDa.

According to the present invention, the phrase essentially does not mean that the content of molecules is less than 15 wt.% of the total mass of proteins in the extract, preferably the content of molecules is less than 10 wt.% of the total mass of proteins in the extract, more preferably less than 7.5 wt. % of the total weight of proteins in the extract, more preferably less than 5 wt.% of the total weight of proteins in the extract, preferably less than 2.5 wt.% of the total weight of proteins in the extract, more preferably less than 1 wt.% of the total weight of proteins in the extract and even more preferably less than 0.5 wt.% of the total weight of the proteins in the extract.

Preferably, the content of high molecular weight proteins is less than 15 wt.% of the total weight of proteins in the extract, preferably less than 10 wt.% of the total weight of proteins in the extract, more preferably less than 7.5 wt.% of the total weight of proteins in the extract, more preferably less than 5 wt.% of the total weight of the proteins in the extract, more preferably less than 2.5 wt.% of the total weight of the proteins in the extract, more preferably less than 1 wt.% of the total weight of the proteins in the extract, and even more preferably less than 0.5 wt. % of the total mass of proteins in the extract.

According to one embodiment, the tobacco leaf extract of the present invention is substantially free of RuBisCO proteins.

In one exemplary embodiment, leaf tobacco is of the Nicotiana Tabacum species or the Nicotiana Rustica species. Tobacco leaves can be obtained from brown or light tobacco, and the tobacco can be selected from Virginia tobacco, Burley tobacco, Oriental tobacco, Latakia tobacco, Louisiana black tobacco, Maryland tobacco, Kentucky tobacco, California tobacco, Tex-Mex tobacco and mixtures thereof.

Of course, the extract does not necessarily consist of pure tobacco leaf extract, and proteins extracted from marijuana can be added to treat tobacco and marijuana addiction.

In one specific embodiment, the tobacco leaf extract is obtained from a 1/1/1 blend of brown tobacco, Virginia tobacco and Burley tobacco, taken in a ratio of 1/1/1.

According to one specific embodiment, the tobacco leaf extract is obtained from Burley tobacco.

According to one embodiment, tobacco leaf extract is obtained from dried (cured) tobacco leaves.

According to one specific embodiment, the tobacco leaf extract is obtained from air-dried tobacco leaves (air-dried tobacco leaves).

According to one specific embodiment, the tobacco leaf extract is obtained from air-dried tobacco leaves dried for a time suitable for local climatic conditions, preferably for a minimum time of one month.

According to the present invention, air drying (withering) or natural drying means drying carried out in the presence of normal outdoor air or indoor air. Air drying can be carried out in an open or closed structure, with or without a canopy. Air drying can, for example, be carried out in a natural drying shed. In this case, freshly picked or pre-cured tobacco leaves are left to dry under the influence of ambient air. Tobacco leaves can, for example, be hung in ventilated, unheated sheds. Tobacco leaves can, for example, be left to dry naturally until they turn brown. At this stage, there is practically no sugar left in the leaf. Preferably, the drying is carried out for a time suitable for local climatic conditions, preferably for a minimum time of one month. For example, when harvesting in central France, drying can be carried out between September and December.

- 6 040213 December. During the drying period, the tobacco leaves can be turned or aerated one or more times to ensure uniform drying and avoid condensation and leaf rot or destruction. The air drying process can be used, for example, to dry Burley tobacco.

According to one specific embodiment, tobacco leaf extract is obtained from tobacco leaves that have been dried in a natural drying shed.

According to another specific embodiment, tobacco leaf extract is obtained from tobacco leaves that have been dried in a heated drying shed. In this case, drying can be carried out in a shed or structure heated to a suitable temperature.

According to another specific embodiment, the tobacco leaf extract is obtained from flue-cured tobacco leaves. In this case, drying can be carried out in a structure or shed heated to a suitable temperature. Heat can be supplied to the structure or shed by piping connected to an external heat source. As a result of this controlled heating, yellow-orange leaves are obtained. The leaves thus obtained have a high sugar content. In this way, for example, Virginia tobacco can be dried.

According to another specific embodiment, the tobacco leaf extract is obtained from sun-dried tobacco leaves. In this case, the tobacco leaves may be laid out on stands and exposed to the sun for a period of 12 to 30 days. Under the influence of direct sunlight and heat, the leaves turn yellow or orange and retain a high sugar content. Oriental type tobaccos are usually dried in this way.

According to another specific embodiment, the tobacco leaf extract is obtained from fire-cured tobacco leaves. In this case, small pieces of burning wood may be located under the tobacco leaves, and as a result of this drying, the leaves acquire a smoky flavor.

The tobacco leaf extract of the present invention is preferably an aqueous extract.

The tobacco leaf extract of the present invention is preferably a brown aqueous extract.

Preferably, the tobacco leaf extract may be prepared by a process comprising extracting the ground and dried tobacco leaves with a solvent, e.g., an aqueous solvent, then separating solid residues from the ground dried tobacco leaf extract solution, then diafiltration at a constant volume of the ground dried tobacco leaf extract solution containing no solid residues, where diafiltration is carried out using a membrane with a cut-off value of 10 kDa in the presence of an aqueous solvent taken in an amount of from 2 to 12 volumes, preferably from 3 to 10 volumes, preferably from 4 to 8 volumes, preferably 6 volumes, where the volume is equal to the volume of the extract.

Of course, other types of solvents may be chosen to carry out the extraction of the tobacco leaf extract. The extraction performed in step c can be carried out with an organic or inorganic solvent or with a mixture of organic and/or inorganic solvents.

To ensure high extraction efficiency and yield and to obtain a high quality extract having the desired purity and composition, the person skilled in the art can easily select the appropriate solvent(s) based on the following criteria:

the polarity of the solvent or mixture of solvents (polar or non-polar);

the physical state of the solvent or mixture of solvents (eg liquid, solid, supercritical or gaseous);

the chemical nature of the solvent or mixture of solvents (eg organic or inorganic);

the charge of the solvent or mixture of solvents (eg, ionic or non-ionic);

the origin of the solvent or mixture of solvents;

miscibility of the solvent or mixture of solvents;

solubility of a solvent or mixture of solvents.

Based on the desired extraction yield and the purity and composition of the extract, a person skilled in the art will be able to determine the procedure for selecting a solvent(s) that meets these criteria.

Preferably, the tobacco leaf extract of the present invention is prepared by the tobacco leaf preparation method discussed below.

pharmaceutical composition

The second aspect of the present invention relates to a pharmaceutical composition comprising, as an active ingredient, the tobacco leaf extract discussed above.

The pharmaceutical composition according to the present invention further comprises at least one pharmaceutically acceptable excipient, such as pharmaceutically acceptable diluents, such as water.

- 7 040213

Preferably, the pharmaceutical composition comprises proteins present in the tobacco leaf extract according to the invention, the content of which is from 1 to 1000 µg/ml, preferably from 10 to 500 µg/ml, preferably from 50 to 300 µg/ml, preferably from 60 to 200 µg /ml, preferably from 80 to 150 μg/ml.

The pharmaceutical composition according to the invention is preferably an aqueous composition.

According to one specific embodiment, the pharmaceutical composition may further comprise a negative reinforcement enhancing active, such as, for example, a nicotine substitute, varenicline or bupropion.

According to one embodiment, the pharmaceutical composition further comprises an adjuvant. Examples of adjuvants include swelling agents such as, for example, sugar such as lactose, sucrose, trehalose, sorbitol, glucose, raffinose, mannitol, preferably lactose, sucrose, trehalose, glucose or mannitol, an amino acid such as arginine, glycine or histidine, preferably glycine, or polymers such as dextran or polyethylene glycol, or mixtures of these substances. According to this embodiment, the pharmaceutical composition comprises from 50 to 99 wt.% of excipients, preferably from 80 to 97 wt.% of the total weight of the pharmaceutical composition.

According to one specific embodiment, the pharmaceutical composition may further include proteins extracted from marijuana.

According to one embodiment, the pharmaceutical composition according to the present invention is in a form suitable for subcutaneous administration.

According to another embodiment, the pharmaceutical composition is in a form suitable for administration via an adhesive (adhesive or stick-on) transdermal therapeutic system. Preferably, the pharmaceutical composition is in the form of a patch.

According to another embodiment, the pharmaceutical composition is in a form suitable for administration by spraying or evaporation. Preferably, the pharmaceutical composition is in the form of a nebulizer, steam inhaler or aerosol.

The pharmaceutical composition according to the invention is preferably obtained by the method for preparing a pharmaceutical composition comprising tobacco leaf extract, which is discussed below.

Application of pharmaceutical composition

The third aspect of the present invention relates to the use of a pharmaceutical composition for the treatment of tobacco dependence.

According to the present invention, the treatment of tobacco dependence or the treatment of addiction to tobacco more specifically means reducing the addictive reward from a cigarette. The administration of the pharmaceutical composition according to the invention significantly reduces the positive reinforcement from the cigarette and in particular the urge to smoke.

The present invention also relates to a composition comprising the tobacco leaf extract disclosed above for use in the treatment of tobacco dependence.

In other words, the present invention also relates to a method for treating tobacco dependence or addiction, comprising administering a composition comprising the tobacco leaf extract disclosed above.

Preferably, the administration of the composition of the invention allows the patient to quit smoking, reduce tobacco consumption or prevent relapse after quitting by attenuating the positive reinforcement from tobacco and significantly reduce the urge to smoke, thereby facilitating quitting. Preferably, administration of the composition will allow any type of smoker, i. including heavy smokers, quit smoking and reduce tobacco use or prevent relapse after quitting.

Preferably, the administration of a composition according to the invention, further comprising proteins extracted from marijuana, allows the simultaneous treatment of dependence or addiction to tobacco and marijuana.

According to one embodiment, the composition of the present invention is administered by subcutaneous injection. According to this embodiment, the pharmaceutical composition is in a form suitable for subcutaneous administration.

According to one embodiment, the pharmaceutical composition is in a dosage form of 0.03 to 10 ml, preferably 0.1 to 5 ml, preferably 0.5 to 2 ml. Thus, the pharmaceutical composition is preferably administered by subcutaneous injection in an amount of 0.03 to 10 ml, preferably 0.1 to 5 ml, preferably 0.5 to 2 ml.

According to one embodiment, the composition of the present invention is administered via an adhesive transdermal therapeutic system containing the tobacco leaf extract disclosed above. According to this embodiment, the pharmaceutical composition is in a form suitable for administration by adhesive transdermal therapy.

- 8 040213 system. Preferably, the pharmaceutical composition is in the form of a patch.

The patch may be in the form of a reservoir-type patch containing one or more compartments, or a matrix-type patch. The practical implementation of the patches can be determined by one of ordinary skill in the art based on their general knowledge in the art in order to achieve a controlled and continuous systemic administration of tobacco leaf extract over the entire period of wearing the patch, e.g., for a period of time ranging from about 2 to 24 hours.

A reservoir-type patch includes one or more separate reservoirs containing the active ingredient(s) (including tobacco leaf extract) in solution or suspended in a polymer matrix, which is in contact with the skin through a semi-permeable polymer membrane, which allows you to control the release rate of the active substance (s) .

The matrix type patch comprises a polymeric mass within which the active substance(s) are dissolved or dispersed in suitable proportions. Such active substances are released by diffusing through the polymer chains of the matrix.

In one specific embodiment of this type of patch, the adhesive covers the entire release surface of the matrix and is an integral part of it. Thus, this patch is an active type adhesive patch, well known to those skilled in the art, which has a simplified manufacturing process and allows thin, sufficiently flexible patches to be comfortably placed on the patient's skin.

In order to ensure good penetration of said active substances under the skin or into the bloodstream at a suitable dosage, at a suitable diffusion rate and for a suitable period of time, the person skilled in the art can set the following parameters:

the ratio of surface areas and volumes of each of the compartments of the patch;

optional addition of one or more hydrophilic additives;

optional addition of one or more activators or diffusion inhibitors;

optional addition of one or more solubilizers;

optional addition of one or more stabilizers;

optional addition of one or more absorption promoters; and more generally, the addition of all types of additives well known to those skilled in the art that successfully control the flow and stability of the tobacco leaf extract.

A person skilled in the art should be able to select a technique to establish the parameters listed above depending on the desired solubility and stability.

Various parameters of the method of making the patch can be easily adapted by a person skilled in the art to obtain the desired dosage.

As is known in the art, such a patch includes a release liner that is designed to protect the adhesive side applied to the skin after the patch has been made and during its storage period. As known in the art, the person skilled in the art can, for example, use polyester films, one side of which can be treated with anti-tack silicone polymers.

According to another embodiment, the composition of the present invention is administered using a spray or vaporization system containing the tobacco leaf extract disclosed above. According to this embodiment, the pharmaceutical composition is in a form suitable for administration by spraying or evaporation. Preferably, the pharmaceutical composition is in the form of a nebulizer, steam inhaler or aerosol container.

The nebulizer, steam inhaler, or aerosol container may be made in the form of a nebulizer, vaporizer, or aerosol container having a reservoir including one or more compartments. The practical implementation of nebulizers, steam inhalers, or aerosol containers can be determined by one of ordinary skill in the art, based on their general knowledge in the art, in order to achieve controlled and sustained systemic administration of tobacco leaf extract.

The nebulizer, steam inhaler, or aerosol container has one or more separate reservoirs containing the active ingredient(s) (including tobacco leaf extract) in solution or suspension. The active substance (s) is administered by spraying or evaporation on the treated area of the body. Spraying or evaporation can be carried out, for example, on the skin. The active substance(s) may be administered by spraying or evaporation onto the mucous membranes.

In order to ensure good penetration of said active substances under the skin or into the bloodstream at a suitable dosage, at a suitable diffusion rate and for a suitable period of time, the person skilled in the art can easily set the following parameters:

the ratio of surface areas and volumes of each spray compartment or reservoir, pa

- 9 040213 inhaler or aerosol container;

optional addition of one or more hydrophilic additives;

optional addition of one or more activators or diffusion inhibitors;

optional addition of one or more solubilizers;

optional addition of one or more stabilizers;

optional addition of one or more absorption promoters; and more generally, the addition of all types of additives well known to those skilled in the art that successfully control the flow and stability of the tobacco leaf extract.

A person skilled in the art should be able to select a technique to establish the parameters listed above depending on the desired solubility and stability.

Various parameters of the method of making a nebulizer, steam inhaler or aerosol container can be easily adapted by a person skilled in the art to obtain the desired dosage.

According to the first embodiment, the pharmaceutical composition is in a form intended for single administration only. According to this embodiment, the pharmaceutical composition is administered only once, thus reducing or suppressing the positive reinforcement from tobacco and significantly reducing the urge to smoke.

According to another embodiment, the pharmaceutical composition is in a form intended for repeated administration, preferably two or three times. According to this embodiment, the pharmaceutical composition is preferably administered on days 0 and 10, or on days 0, 10 and 30, thereby reducing or suppressing the positive reinforcement from tobacco and significantly reducing the urge to smoke. In this exemplary embodiment, the dosage administered the second time, and optionally the third time, is identical to the dosage administered the first time.

In one specific embodiment, the pharmaceutical composition of the present invention may be administered with a composition comprising a negative reinforcement enhancing active such as a nicotine substitute, varenicline, bupropion, or mixtures thereof. According to this embodiment, several specific administration options may be considered:

(i) administering a pharmaceutical composition according to the invention prior to an attempt to quit smoking to reduce cigarette consumption and then administering a negative reinforcer active to limit the effect of negative reinforcement and in particular withdrawal symptoms;

(ii) co-administration of a pharmaceutical composition of the invention and a negative reinforcer active during an attempt to quit smoking in order to quit smoking immediately;

(iii) administering the negative reinforcer active during the quit attempt, for example within 12 weeks of the start of the quit attempt, and then administering the pharmaceutical composition of the invention, for example within 2 weeks of the end of the administration of the active on smoking. negative reinforcement to prevent relapse.

Preparation of tobacco leaf extract

The fourth aspect of the present invention relates to the preparation of a tobacco leaf extract according to the present invention.

Preferably, the method for producing tobacco leaf extract according to the present invention includes the following steps:

a) drying (withering) of tobacco leaves,

b) grinding dried tobacco leaves to obtain ground dried tobacco leaves,

c) extracting the ground dried tobacco leaves with mechanical agitation with a solvent, for example an aqueous solvent, preferably an aqueous buffer solution, the pH of which is between 6.0 and 8.5,

d) separating solid residues from the ground dried tobacco leaf extract solution by filtration or centrifugation, resulting in a solid, residue-free ground dried tobacco leaf extract solution,

e) filtration at a constant volume of the solution obtained in step d in the presence of a solvent, preferably an aqueous solvent, taken in an amount of 2 to 12 volumes, preferably 3 to 10 volumes, preferably 4 to 8 volumes, preferably 6 volumes, where the volume is equal to the volume of the extract, using a membrane with a limiting transmission value of 10 kDa,

f) optionally lyophilizing the protein solution obtained in step e.

According to one specific embodiment, the drying of step a carried out in the open air is called natural drying. In this case, drying is carried out in the presence of conventional air.

- 10 040213 spirit, outdoors or indoors. Drying can take place in an open or closed structure, with or without a canopy. Air drying can, for example, be carried out in a natural drying shed. In this case, freshly picked or pre-cured tobacco leaves are left to dry under the influence of ambient air. Tobacco leaves can, for example, be hung in ventilated, unheated sheds. Tobacco leaves can, for example, be left to dry naturally until they turn brown. At this stage, there is practically no sugar left in the leaf. Preferably, the drying is carried out for a time suitable for local climatic conditions, preferably for a minimum time of one month. For example, when harvesting in central France, drying may take place between September and December. The tobacco leaves can be turned over one or more times during the drying period to ensure uniform drying and avoid condensation and leaf rot or destruction. The air drying process can be used, for example, to dry Burley tobacco. According to one specific embodiment, the drying of step a is carried out in a natural drying shed.

According to another specific embodiment, the drying of step a is carried out in a heated drying shed. In this case, drying can be carried out in a shed or structure heated to a suitable temperature.

According to another specific embodiment, the drying of step a is carried out by flue drying. In this case, drying can be carried out in a structure or shed heated to a suitable temperature. Heat can be supplied to the structure or shed by piping connected to an external heat source. As a result of this controlled heating, yellow-orange leaves are obtained. The leaves thus obtained have a high sugar content. In this way, for example, Virginia tobacco can be dried.

According to another specific embodiment, the drying of step a is performed by sun drying. In this case, the tobacco leaves may be laid out on stands and exposed to the sun for a period of 12 to 30 days. Under the influence of direct sunlight and heat, the leaves turn yellow or orange and retain a high content of sugars. Oriental type tobaccos are usually dried in this way.

According to another specific embodiment, the drying of step a is performed by fire drying. In this case, small pieces of burning wood may be located under the tobacco leaves, and as a result of this drying, the leaves acquire a smoky flavor.

Preferably, the drying of step a promotes the degradation of high molecular weight tobacco proteins, such as, for example, RuBisCO, by hydrolytic mechanisms.

Steps c-e correspond to extraction, filtration, and diafiltration steps similar to those discussed in US Pat. 0040]).

Preferably, the extraction of step c is performed at a temperature of 4 to 20°C, preferably 4 to 10°C.

Preferably, the extraction of step c is carried out for 12 to 36 hours, preferably 22 to 26 hours, preferably 24 hours.

According to one specific embodiment, the solvent used in step c is an aqueous solvent. Preferably, the aqueous solvent used in step c is an aqueous ammonium bicarbonate buffer, preferably at a concentration of 2 to 6 g/l, preferably 4 g/l.

Of course, other types of solvents may be used to carry out the extraction of step c. The extraction of step c can be carried out with an organic or inorganic solvent or with a mixture of organic and/or inorganic solvents.

In order to ensure high efficiency and high extraction yield and to obtain a high quality extract having the desired purity and composition, a suitable solvent(s) can be easily selected by one skilled in the art based on the following criteria:

the polarity of the solvent or mixture of solvents (polar or non-polar);

the physical state of the solvent or mixture of solvents (eg liquid, solid, supercritical or gaseous);

the chemical nature of the solvent or mixture of solvents (eg organic or inorganic);

the charge of the solvent or mixture of solvents (eg, ionic or non-ionic);

the origin of the solvent or mixture of solvents;

miscibility of the solvent or mixture of solvents;

solubility of a solvent or mixture of solvents.

Based on the desired extraction yield and the purity and composition of the extract, one skilled in the art will be able to determine the appropriate solvent(s) to select.

- 11 040213 meeting the named criteria.

Preferably, the extraction of step c is performed by suspending the ground and dried tobacco leaves in a buffer solution; preferably the concentration of ground and dried tobacco leaves in the buffer solution is 30 to 70 g/l, preferably 40 to 60 g/l and more preferably 50 g/l. The suspended solids are then removed by filtration, eg Buchner funnel filtration (step d), to obtain ground and dried tobacco leaves free of solids.

Preferably, the aqueous solvent used in step d is water for injection (abbreviated water d/i).

Preferably, more than 99% of molecules with a molecular weight of less than 10 kDa are recovered in step e, and in particular free amino acids, proteins and peptides with a molecular weight of less than 10 kDa, as well as protein residues with a molecular weight of less than 10 kDa resulting from the destruction of proteins during step a.

Preferably, before carrying out step f, the protein solution obtained in step e is sent to the sterilizing filtration step e'.

The tobacco leaf extract according to the invention, obtained at the completion of the above process, may be stored as obtained at the end of sterilizing filtration step d', or it may be lyophilized as a result of step f.

Preparation of a pharmaceutical composition comprising tobacco leaf extract

The fifth aspect of the present invention relates to the preparation of a pharmaceutical composition comprising a tobacco leaf extract according to the invention.

The pharmaceutical composition can be obtained by a method that includes the following steps:

a) obtaining a tobacco leaf extract as described above;

b) adjusting the protein concentration to achieve a protein concentration of 100 to 200 μg/ml;

c) adding a pharmaceutically acceptable excipient(s); And

d) optional addition of adjuvant(s) to the resulting extract, preferably the addition of mannitol.

The tobacco leaf extract used in step b can either be the extract obtained immediately after the diafiltration step on the 10 kDa membrane, or the extract obtained after the sterilizing diafiltration step, or the tobacco leaf extract which has been lyophilized and then reduced, for example, in saline or water.

Preferably, the concentration in step b can be adjusted:

either by dilution with water to lower the protein concentration, or by diafiltration to increase the protein concentration.

A particularly preferred pharmaceutically acceptable excipient in step c is water.

The adjuvants that can be added in step d can in particular be swelling agents such as, for example, sugars such as lactose, sucrose, trehalose, sorbitol, glucose, raffinose, mannitol, preferably lactose, sucrose , trehalose, glucose or mannitol; an amino acid such as arginine, glycine or histidine, preferably glycine; or polymers such as dextran or polyethylene glycol; or mixtures of the listed compounds.

According to one specific embodiment, step d includes at least the addition of mannitol.

Kit containing a pharmaceutical composition comprising tobacco leaf extract

A sixth aspect of the present invention relates to a kit comprising dosages of tobacco leaf extract or pharmaceutical composition according to the invention.

According to a first embodiment, the kit comprises one or more doses of tobacco leaf extract, preferably in lyophilized form, as well as one or more doses of saline or water for the preparation of the pharmaceutical composition according to the invention immediately prior to administration.

According to another embodiment, the kit includes one or more dosages of a pharmaceutical composition ready for administration to a patient.

The kit may optionally include one or more syringes for administering the pharmaceutical composition by subcutaneous injection.

The kit may optionally include one or more patches for transdermal administration of the pharmaceutical composition.

The following are examples to illustrate the present invention.

- 12 040213

Description of exemplary embodiments of the invention

Example 1 Preparation of a tobacco leaf extract according to the invention.

Burley tobacco leaves were used to obtain the extract.

Grind 100 g of Burley tobacco leaves, dried by natural drying for about three months (in central France, between September and December). The ground leaves are suspended for 24 hours at 4 to 10° C. in 1892 g of water d/i to which 8 g of ammonium bicarbonate has been added. The suspended solids are then removed by filtration with a Buchner funnel.

The extract is then clarified filtered on a 0.2 µm filter. A brown extract is obtained. The extract is then weighed to determine the volume of d/i water that is used in the diafiltration step at constant volume; in this case, the mass of the liquid extract was 1680 g.

Then, the extraction of proteins of the obtained extract of the ground and dried tobacco leaf, which does not contain solid residues, is performed: 10080 g of water for injection is added to the extract of the ground and dried tobacco leaf, which does not contain solid residues. Then, 11760 g of the solution thus obtained was subjected to diafiltration at a constant 6-fold volume with a membrane with a cut-off value of 10 kDa (MerckMillipore) until the weight of the ultraconcentrate was reduced to 1680 g.

The ultraconcentrate obtained after diafiltration is a mixture of proteins in which proteins with a molecular weight of less than 10 kDa are found at a concentration 99% lower than their initial concentration. The color of the obtained ultraconcentrate is between B2 and B3 according to the measuring scale established by EUROPEAN PHARMACOPOEIA 5.0, 2.2.2, Degree of coloration of liquids (Degree of coloration of liquids).

The resulting diafiltrate can be lyophilized after the addition of mannitol, for example in a lyophilizer SMH 150, resulting in a lyophilized tobacco leaf extract.

Example 2. Determination of the protein composition of the tobacco leaf extract obtained in example 1.

The proteins of the tobacco leaf extract obtained in Example 1 are first separated on a polyacrylamide gel. To do this, the analyzed extract is added to NuPage® Bis-Tris gel (4-12%) and electrophoresis is performed at 200 V for 35 minutes, after which it is brought into contact with 20 ml of Instant Blue dye, which is kept for 1 hour and then washed with water overnight. Cut out selected bands of polyacrylamide gel (6 bands) and then decolorize with 50 mM NH4CO3/CH3CN (50/50) buffer. Disulfide bridge reduction is then performed in a 10 mM dithiothreite/50 mM NH4CO3 solution for 40 min at 55°C. The reduced cysteine residues are then alkylated in a 100 mM iodoacetamide/50 mM NH4CO3 solution for 30 minutes at room temperature in the dark.

The protein solution thus obtained is subjected to cleavage by the action of an enzyme, obtaining protein or peptide fragments: cleavage is carried out by the action of trypsin (Trypsin V5111, Promega), taken in an amount suitable for staining polyacrylamide gel bands in 50 mM NH4CO3 solution overnight at 37° C.

The peptides from the mixture of peptides resulting from the enzymatic treatment were separated by nanoliquid chromatography (U3000 Nano LC System, ThermoFischer Scientific) with pre-concentration on a PepMar C18 pre-treatment microcolumn (5 µm; 100 A; 300 µm x 5 mm; ThermoFischer Scientific) followed by elution on a PepMar C18 nanocolumn (3 µm; 100 A; 75 µm x 250 mm; ThermoFisher Scientific) in a linear gradient mode; Buffer A 0.1% HCOOH in H ₂ O/CH ₃ CN (95/5), Buffer B 0.1% HCOOH in H2O/CH3CN (20/80), Gradient 0 to 60% B over 60 min, flow rate 300 µl/min).

The peptides are then analyzed by mass spectrometry on an LTQ Velos analyzer (Dual Pressure Linear Ion Trap; ThermoFisher Scientific) equipped with a nanospray source (ThermoFisher Scientific) and connected to a U3000 NanoLC device. Data acquisition was performed using Excalibur 2.1 software (ThermoFisher Scientific) in positive mode with MS (mass spectral) scan cycle from m/z 400 to 1600 in Resolution Enhanced mode followed by MSMS (tandem mass spectrometry) scan. , TMS) in the Normal Resolution mode (normal resolution) on 20 MS ions with the highest signal intensity (charge 2 or more) in the CID mode, in a helium atmosphere, with a collision energy of 35 eV. Previously fragmented MS ions were dynamically excluded for 30 seconds with a mass tolerance of 50 mmu (abbr. millimass unit = one thousandth of an atomic mass unit).

To identify the masses of peptides and their fragments, these masses were compared with data available in databases. To do this, MS and MSMS data were processed using the ProteomeDiscoverer 1.4 software in accordance with the MASCOT (Version 2.4) search algorithm, and queries were made to the UniprotKB/Swiss-Prot database (released in April 2015), shortened to TOBAS types.

- 13 040213

Protein identification was confirmed using a p confidence level of less than 0.05. Only proteins were retained that were identified as containing at least 2 peptides with the highest level of confidence.

Quantitative indicators of each of the proteins identified in this way allowed only a relative classification of proteins among themselves, that is, such a classification was correlated with the concentrations of these proteins. The absolute values of such indicators depend on the conditions of the experiment, the type of test sample and the quantities taken.

The following is a list of 12 proteins identified and classified in this way, listed in descending order of their scores (from most present to least present):

P36401; E13H_TOVAS; Glucan-endo-1,3-beta-glucosidase, acid isoform PR-Q' (EC 3.2.1.39) ((1->3)-beta-glucan-endohydrolase) ((1->3)-beta-glucanase) (Beta-1,3-endoglucanase) (PR-35); they say weight 36995 Yes

P17513; CHIP_TOBAC; Acid endochitinase P (EC 3.2.1.14) (Pathogenesis-related protein P) (PR-P); they say weight 27469 Yes

P17514; CHIQ_TOBAC; Acid endochitinase Q (EC 3.2.1.14) (Pathogenesis-related protein Q) (PR-Q); they say weight 27633 Yes

P27666; E13F_TOBAC; Glucan-endo-1,3-beta-glucosidase, major vacuolar isoform of GLB (EC 3.2.1.39) ((1->3)-beta-glucan-endohydrolase) ((1->3)-beta-glucanase) (Beta -1,3-endoglucanase, basic) (Glucanase GLB); they say weight 40443 Yes

P14170; OSMO_TOBAC; Osmotin; they say weight 26681 Yes

P24091; CHI2_TOBAC; Endochitinase B (CHN-B) (EC 3.2.1.14); they say weight 34721 Yes

P11965; PERX_TOBAC; Lignin-forming anionic peroxidase (EC 1.11.1.7) (TORA); they say weight 34674 Yes

P13046; PRR1_TOBAC; The pathogenesis-associated protein R basic form (thaumatin-like protein E22); they say weight 24667 Yes

P29062; PR4A_TOBAC; Pathogen-related protein PR-4A; they say weight 16221 Yes

Q03199; IPIB_TOBAC; Proteinase I-B Inhibitor (PI-IB) (Microbial serine proteinase inhibitor, major isoform); they say weight 11916 Yes

Q03198; IPIA_TOBAC; Proteinase inhibitor I-A (PI-IA) (Microbial serine proteinase inhibitor, minor isoform); they say weight 11880 Yes

P29063; PR4B_TOBAC; Pathogen-related protein PR-4B; they say weight 16235 Yes

Thus, the tobacco leaf extract method allows certain proteins to be concentrated and other proteins to be removed. Thus, in the tobacco leaf extract obtained in Example 1, out of the 759 Nicotiana tabacum proteins listed in Swiss-Prot, only 6 families and 12 proteins are mainly present. These 12 proteins, which are mainly present in the tobacco leaf extract obtained in Example 1, have a molecular weight ranging from 10 to 50 kDa.

Example 3 Determination of protein content.

The protein content of the tobacco leaf extract obtained in Example 1 (before and after the diafiltration step) was determined by the Bradford method.

The extract obtained in example 1 was dissolved in saline.

Three protein standard solutions were prepared: to obtain standardized solutions at an exact concentration of 100 µg/ml, 50 µl of a 2 mg/ml bovine serum albumin solution (Sigma Aldrich, product number P0834) was diluted 20-fold with 950 µl of saline.

Each test tube contained 80 µl of protein sample and 920 µl of Bradford's reagent (Sigma Aldrich, item number B6916). After adding the Bradford reagent to each tube, the tubes were gently shaken to create a funnel and then incubated for 5 min at room temperature. The samples were transferred into 1.5 ml cuvettes and the absorbance of the samples was measured at 595 nm for 1 h.

Protein concentration was determined by comparison with prepared standard protein solutions.

The measured amounts of protein were:

before diafiltration: 295.45 +/- 9.91 µg/ml after diafiltration: 160.61 +/- 1.31 µg/ml.

The electrophoretic separation pattern of the tobacco leaf extract according to Example 1 was determined after the diafiltration step. NuPage gel was coated with proteins at 2, 5, 10, and 15 μg.

NuPage 4-12% gel was prepared for 35 minutes, then placed in 20 ml of Coomassie Blue for 1 hour and washed with water overnight. The gel was analyzed with a ChemiDoc™ XRS Biorad system using Image Lab™ software.

Three main bands were found corresponding to approximately 15 and 30 kDa (FIG. 1). Two other bands were also traced, corresponding to approximately 40 kDa.

- 14 040213

The results show that in this tobacco leaf extract, the amount of detected proteins whose molecular weight exceeds 100 kDa is small compared to the amount of proteins whose molecular weight is from 10 to 50 kDa.

Example 4 Effect of Diafiltration Volume on the Removal of Molecules Below 10 kD from a Tobacco Leaf Extract of the Invention.

During the diafiltration step, the removal of molecules with a molecular weight of less than 10 kDa from the tobacco leaf extract obtained in example 1 was monitored, depending on the amount of diafiltration used. Monitoring was performed by gas chromatographic analysis with an analytical indicator (nicotine) having a molecular weight of less than 10 kDa.

The results are presented in the following table.____________________________

Samples with appropriate diafiltration volume Analytical indicator peak area Percentage of Analytical Indicator Remaining Before diafiltration 54913 100 % Diafiltration with x3 volume 2630 4.79% Diafiltration with x4 volume 809 1.47% Diafiltration with x5 volume 247 0.045% Diafiltration with hb volume But* But

* n/a - undetectable

The removal efficiency of molecules whose molecular weight is less than 10 kDa increases with increasing diafiltration volume, and such molecules are completely removed if the diafiltration volume is 6 times the original volume.

Example 5. Biological activity of the extract of example 1 in relation to human mononuclear cells in vitro and in experiments on mice in vivo.

Materials and methods

Applied Products

PMA (phorbol myristacetate)/Ionomycin (Sigma-Aldrich, item number P8139/I0634)

ABS (bovine serum albumin) (Sigma Aldrich, item number A7020)

PBS (phosphate buffered saline) (PAA Laboratories, item number H15-002)

Tween 20 (Sigma Aldrich, item number P1379)

Goat anti-mouse IgG (g-chain specific) - alkaline phosphatase (Southern Biotech, item number 1030-04)

Biotin Anti-Mouse IgE Conjugate (Biolegend, item number BLE406904) Streptavidin Alkaline Phosphatase (Southern Biotech, item number 7100-04)

Cell Acquisition and Stimulation Mode

Peripheral blood mononuclear cells from different healthy donors were separated by density gradient centrifugation (separation medium: LMS 1077, PAA Laboratories). The obtained mononuclear cells were stimulated with the extract of example 1 at various dilutions for 24 or 48 hours. After stimulation for 24 or 48 hours, the supernatant was removed and stored at -80°C for subsequent cytokine profile analysis using the Luminex method. After stimulation for 24 hours, the cells were also subjected to phenotypic analysis by flow cytometry.

Cytometric phenotypic study

To detect various membrane markers, cells were first stained with a viability marker (eFluor 450 stain (65-0863-14 eBioscience) and then with antibodies directly bound to fluorochromes. T and B lymphocyte populations were identified using CD3 and CD19 markers. NK cells (from the English natural killer cells, natural killer cells) were detected either using the NKp46 marker, or using a combination of CD56 and CD16 markers, or using the expression of CD56 in the absence of CD3. .gate) to analyze various activation markers (CD69, CD25, HLA-DR).

The list of antibodies used in the phenotypic cytometric study is presented in the following table.

- 15 040213

Clone Product number and supplier Anti-CO45-Chrome Orange J.33 A96416; Beckman Coulter Ahtm-CD56 ARS B159 555518; Becton Dickinson Ahtm-CD25PC7 M-A251 557741; Becton Dickinson Ahth-CD25 PC5 B1.49.9 IM2646; Beckman Coulter Ahth-CD69 FITC FN50 557049; Becton Dickinson Ahtm-CD69 PE L78 341652; Becton Dickinson Ahtm-CD3ECD UCHT1 A07748; Beckman Coulter Anti-KrR46 ARS 9E2/NKp46 558051; Becton Dickinson AHTH-CD19 FITC HIB19 11-0199-42; eBioscience Anti-HLA-DR PE G46-6 347401; Becton Dickinson

An isotype control was used in each experiment and a compensation matrix was created for this multivariate phenotypic study.

Stained samples were analyzed using a Navios cell count (Beckman Coulter 10-colour) with a minimum data set of 100,000 events per tube. The collected data was then analyzed using the Kaluza software.

Cytokine Secretion Profile Analysis by Luminex and ELISA Methods

Based on the principles of ELISA (short for enzyme-linked immunosorbent assay) and cytometry, this technique allows multiple analytes to be examined simultaneously.

Unique color-coded microbeads bind to capture antibodies specific to each analyte. After incubation with the analyzed samples, detection of antibodies associated with fluorochrome is performed, which allows analysis using an optical system that includes two lasers (Bio-plex 200 Bio-rad systems). The first laser is used to identify the microbead, and the second laser is used to quantify the detected antibodies associated with the fluorochrome. The Bio-Plex Pro Human Cytokine Grp1 panel 17 Network Kit (Bio-rad) can test 17 analytes in samples taken from various stimulation experiments performed on cells. The main concentrations of each cytokine can be obtained from cells stimulated in a culture medium that does not contain the tobacco leaf extract of Example 1.

The IL-8 ELISA assay is based on the traditional ELISA sandwich assay technique. Commercially available kits provided by Eurobio-Diaclone (Besancon) were used.

Immunization of mice with tobacco leaf extract of example 1

Seven week old female C57BL/6 and Balb/c mice were purchased from the Charles River Company (L'Arbresles, France). Mice were immunized on days 0 and 21 with ip (abdominal) injections of 200 μl of the tobacco leaf extract of Example 1, previously lyophilized and reconstituted in 5 ml of saline. Blood was collected from all mice before and after immunization. After centrifugation, the blood serum was frozen at -20°C.

ELISA IgG and IgE

For the ELISA experiment, one vial of the product was reconstituted in 2.5 ml carbonate bicarbonate buffer (0.1 M, pH 9). Nunc Maxisorb 96W plates were incubated with 100 µl of tobacco leaf extract lyophilized as described in Example 1, then reconstituted and incubated for 18 hours. The plates were washed with PBS-0.05% Tween buffer and then the plates were saturated with 200 µl of PBS-1% buffer ABS for 1 h at room temperature. After washing the plates with PBS-0.05% Tween buffer, 100 μl of mouse serum diluted to 1/20 in PBS-1% ABS was incubated for 2 hours at room temperature.

IqG Elisa

After washing with Tween 20 PBS buffer, 100 µl of a goat anti-mouse IgG secondary antibody coupled to alkaline phosphatase (Southern Biotechnology, item number 1030-04) and diluted with PBS-1% ABS was incubated for 1 hour at temperature. After washes with PBS-0.05% Tween, 100 μl of para-nitrophenyl phosphate substrate (denoted pNPP, Sigma) alkaline phosphatase was added and left for 15 min at room temperature in the dark. The reaction was then stopped by adding 50 μl of 3 M NaOH and optical densities (OD) were read at 405 nm.

IgE Elisa

After washing with PBS with Tween 20 buffer, 100 µl of rat anti-mouse IgE secondary antibody bound to biotin (Biolegend, item number BLE406904) and diluted in PBS-1% ABS was incubated for 1 h at room temperature. After washes with PBS-0.05% Tween, 100 μl

- 16 040213 streptavidin associated with alkaline phosphatase (Southern Biotechnology, product number 7100-04) and incubated for 1 hour at room temperature (112000th dilution in PBS-1% ABS according to the supplier's recommendations). After additional washes with PBS-0.05% Tween, 100 μl of pNPP alkaline phosphatase substrate was added and left for 15 min at room temperature in the dark. The reaction was then stopped by adding 50 μl of 3 M NaOH and optical densities (OD) were read at 405 nm.

results

Tobacco Leaf Extract of Example 1 Induces a Specific IgG Humoral Response in Mice

After two immunizations of mice with tobacco leaf extract, lyophilized as described in example 1 and reconstituted in 5 ml of saline, IgG excitation caused by the components of the product was observed. Thus, in mice of 4/5 breeds C57BL/6 and 4/5 Balb/c, an increase in the level of IgG caused by tobacco leaf extract was observed on the 28th day, i.e. 7 days after the second vaccination (Fig. 2). The only mouse in each group that did not experience a reaction had baseline levels of IgG antibodies. Antibody titers in Balb/c mice were lower than in C57BL/6 mice (FIG. 2).

However, no humoral IgE response was detected after 2 vaccinations of mice with tobacco leaf extract. Optical densities (OD) were less than 0.05 in all analyzes performed on different sera.

Tobacco leaf extract preferentially activates natural killer cells in peripheral blood.

After extraction of mononuclear cells from peripheral blood by density gradient processing (Ficoll), different cell populations (T and B lymphocytes, NK cells, etc.) can be characterized (FIG. 3). Thus, NK cells (natural killer cells) were identified in the CD3 negative population by the CD56 and CD16 markers or by the NKp46 marker. After incubation of mononuclear cells with the tobacco leaf extract obtained in example 1, an increase in the expression of activation markers CD69, CD25 and HLA-DR was observed on NK cells, identified by the CDI6+CD56+ (CD3-) markers (Fig. 4A) and by the NKp46 marker ( Fig. 4B). When using a 2-fold diluted tobacco leaf extract and detecting CD69 expression, more pronounced results were obtained. Thus, more than 60% of NK cells express the CD69 marker within 24 h after contact with the tobacco leaf extract (Fig. 4A-B). At rest, less than 5% of NK blood cells express CD69.

The effect of tobacco leaf extract on the activation of peripheral blood lymphocytes is much weaker. Thus, at rest, 2% of T lymphocytes express CD69, and this expression increases to 6% after 24 h of culturing with tobacco leaf extract (Fig. 5A). CD25 expression on T lymphocytes activated with tobacco leaf extract does not increase (FIG. 5A). Similarly, tobacco leaf extract did not significantly affect peripheral blood B lymphocytes because less than 3% of B lymphocytes expressed CD69 after 24 h of culture with this extract (Fig. 5B).

Profile of cytokines generated by the tobacco leaf extract obtained in Example 1 on human blood mononuclear cells

Peripheral blood mononuclear cells were contacted with a tobacco leaf extract, lyophilized as described in example 1, and reconstituted in 5 ml of PBS at various dilutions (1/2 or 1/20) for 24 h, after which cytokines, chemokines were studied using the Luminex method. and growth factors present in the supernatant.

In FIG. 6 shows intensive generation of inflammatory cytokines (ΓΕ-1β, IL-6, TNFa, IL-17) under the influence of tobacco leaf extract. For example, baseline levels of these cytokines in the absence of mononuclear cell contact with tobacco leaf extract are less than 10 picograms/ml (FIG. 6A).

It was shown that after incubation with a two-fold diluted tobacco leaf extract, the concentrations of cytokines ΓΕ-1β, IL-6 and TNFa exceeded 1000 picograms/ml (Fig. 6A). This overt excitation of inflammatory cytokines was also observed when the tobacco leaf extract was diluted to 1/20 (FIG. 6A). The results obtained on the test material taken from different donors are quite uniform. Among the TH1 cytokines (IL-2, IFNy, IL-12) conferring cell-mediated immunity, only IFNy was observed to be significantly stimulated by tobacco leaf extract diluted to 1/2 and 1/20 (Fig. 6B). Tobacco leaf extract induces the generation of very low concentrations of TH2 cytokines (IL-4, IL-5, IL-13) of the order of a few picograms/ml (FIG. 7A). In contrast, tobacco leaf extract stimulates the production of IL-10 by mononuclear cells, and its concentration in the supernatant of cells contacted with two-fold diluted tobacco leaf extract is 750 picograms/ml (Fig. 7A). IL-10 was originally thought to be a TH2 cytokine, but it can also be produced by regulatory T lymphocytes, referred to as Tr1.

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The inventors have also shown that tobacco leaf extract can also induce the production of hematopoietic growth factors such as G-CSF, which promotes the proliferation and recruitment of neutrophils and chemokines, such as MCP-1, which is known to have a chemotactic effect on macrophages ( Fig. 7B). However, tobacco leaf extract was shown to have no significant effect on other growth factors such as IL-7 or GM-CSF (FIG. 7B). The cytokine/chemokine profile remained the same when performing studies in the supernatant collected after 48 hours of cultivation with tobacco leaf extract.

Conclusion

The tobacco leaf extract obtained in Example 1 can elicit a specific humoral IgG response directed against proteins present in the product in two mouse species with different background genotypes.

IgE humoral response was not observed after two administrations of the substance to the same mice.

Treatment of peripheral blood mononuclear cells with the tobacco leaf extract obtained in Example 1 in vitro causes preferential activation of NK cells. This activation is reflected in increased expression of CD69, CD25 and HLA-DR molecules.

The tobacco leaf extract obtained in Example 1 induces the production of inflammatory cytokines such as IL-1e, IL6, IL-17, IL-8 and TNFa in vitro. These cytokines are present in high concentrations in the culture supernatant collected 24 hours after stimulation.

The tobacco leaf extract obtained in Example 1 also induces the production of IFNy, IL-10, G-CSF and MCP-1. However, this extract does not significantly affect the production of TH2 cytokines (IL-4, IL-5, IL-13).

Example 6. Toxicological study of the tobacco leaf extract obtained in example 1.

Materials and methods

All studies described below were performed using the tobacco leaf extract obtained in Example 1.

In all cases, the introduction is made subcutaneously. Toxicological studies were performed on rodents (Han Wistar rats). Toxicological studies were carried out in accordance with the Organization for Economic Co-operation and Development (OECD) Good Laboratory Practice Principles (Mutual Acceptance of Data, abbreviated MAD) in Evaluation Data, November 26, 1997 (C(97) 186 Final version) and in accordance with the Good Laboratory Practices (GLP) published by the French Ministry of Employment and Solidarity (No 2000/5bis, Order of 14 March 2000, JO 03/23/2000).

Subcutaneous toxicity studies after 14 days

The aim of this study was to determine the toxicity of the tobacco leaf extract obtained in example 1 in rats after subcutaneous administration (once a week for 2 weeks).

The studies were carried out as follows: _____________________

no. groups Dose expressed as protein (mcg/kg/abd.) Dose volume (ml/kg/abd.) Dose concentration expressed as protein (µg/mL) Number of animals males females 1 0 2 0 6 6 2 11.2 0.4 28 6 6 3 56 2 28 6 6

abdominus = abdominal

The results of the study showed:

no mortality was noted at any of the dosages;

no treatment-related clinical events were observed, and subcutaneous administration was locally well tolerated (clinical manifestations and local tolerability were observed before and after injection and once daily in the absence of therapy);

does not adversely affect body weight and food intake (compared to control animals);

there are biologically significant differences between white blood cell counts in treated and untreated rodents, but these differences are not toxic;

observed differences in protein and cholesterol concentrations between treated and untreated groups, but these differences remained within or close to those of control animals. These changes had no detrimental effect;

no differences were observed in organ masses between animals in the treated group and animals in the control group.

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Thus, subcutaneous administration of the tobacco leaf extract obtained in Example 1 once a week for 2 weeks at dosages of 11.2 and 56 μg protein/kg/abdominal. well tolerated by Wistar rats.

Four-Week Subcutaneous Toxicity Study

The objective of the study was to determine the toxicity of the tobacco leaf extract obtained in example 1, according to the results of subcutaneous administration to Wistar rats once a week, as well as to determine the regression of any sign of toxicity within a 4-week period in the absence of therapy.

Particular attention was paid to possible immunological phenomena. The study was carried out as follows: __________________________________________________________

Group/therapy Dose ⁽¹⁾ (mcg/kg/abd.) Dose volume (ml/kg/abd.) Dose Concentration (mcg/mL) Number of animals After therapy ⁽²⁾ After observation period ⁽³⁾ males females males females 1. Control (vehicle: 0.9% NaCl) 0.0 4.0 0.0 10 10 5 5 2. Low dosage 16.8 0.6 28.0 10 10 / / 3. Intermediate dosage 33.6 1.2 28.0 10 10 / / 4. High dosage 112.0 4.0 28.0 10 10 5 5

(1) Expressed as protein concentration (2) Rats were sacrificed 2 days after the last injection (day 23) (3) Rats were sacrificed 4 weeks after the last injection (day 49)

During the treatment period, rats were observed before and at least 3 times after administration. During the observation period, rats were observed once a day.

A complete clinical examination was performed once a week. Local tolerance was noted before and after each injection, once a day during the first week and then twice a week until the end of the observation period.

Body temperature was measured on the next day after each injection (days 1, 8, 15, and 22).

An ophthalmological examination was performed before the studies, as well as on the next day after the first and last days of administration (days 1 and 22).

Animal body weights and individual food intake were measured twice a week.

Clinical analysis of hematological parameters, blood clotting, serum chemistry and lymphocytic analyzes were performed on days 23 and 49/48 (male/female, respectively). Urinalysis was performed on the 23rd day.

All animals were sacrificed 2 days after the last injection or after a four-week observation period and autopsy was performed. Some organs were weighed and their histopathological examinations were carried out.

The results of the study showed that:

no mortality was noted at any of the dosages;

subcutaneous administration of dosages, expressed in protein content and comprising 16.8, 33.6 and 112.0 μg/kg/abdominal, is well tolerated when administered topically;

did not observe any clinical manifestations associated with therapy;

therapy does not affect body temperature;

no treatment-related ophthalmic effects were observed;

body weight and food intake did not change;

did not observe significant changes in hematological parameters, blood clotting and urine composition;

by the end of the therapy period, the activity of the creatine kinase enzyme decreases in females of groups 3 and 4 (intermediate and high dosages; 33.6 and 112 mcg / kg / abdom., respectively). These changes are not considered harmful as long as they are not associated with histopathology;

observed a slight decrease in the number of NK cells in the bloodstream of females and males treated with intermediate and high dosages (groups 3 and 4);

at the end of the observation period, the average number of NK cells in the bloodstream of males treated with high dosage therapy (group 4) was lower;

no changes in organ mass were observed;

during histopathological examination, a dark spot caused by subcutaneous bleeding was observed at the injection site. This observation was not considered to be related to the administration procedure.

At the end of therapy at the injection site, groups 3 and 4 experienced minimal or mild inflammation.

- 19 040213 literal changes in the skin, while in groups 1 and 2, inflammatory changes were practically not noted.

At the end of the observation period, the inflammatory changes previously observed in group 4 almost completely disappeared.

subcutaneous injections of the tobacco leaf extract obtained in example 1 at dosages up to 112 μg/kg/abdominal. were well tolerated and only minimal and reversible inflammatory changes were observed in treated rats compared to untreated rats.

In conclusion, under the experimental conditions described above, subcutaneous administration of the tobacco leaf extract obtained in Example 1 to Wistar rats once a week for 4 weeks at doses of 16.8, 33.6 and 112 μg/kg/abdominal . well tolerated and did not cause any negative side effects.

Example 7 Clinical studies after administration of the tobacco leaf extract obtained in Example 1.

A pharmaceutical composition comprising the tobacco leaf extract obtained in Example 1 containing 100 μg or 200 μg of proteins and mannitol was administered by subcutaneous injection for the first time on day 0 and the second time on day 29 (each time 1 ml in each hand) twenty-four (24) smokers. Inclusions were performed according to the principle of group expansion, 6 initial patients receiving a dosage of 100 μg were included first, then 6 initial patients receiving a dosage of 200 μg were included and then 6 additional patients were included receiving a dosage of 100 μg. The group of subjects receiving therapy consisted of 24 people, 14 men and 10 women, aged 30 to 65 years, that is, their average age was 44.5 years.

It should be noted that none of the doses tested reached toxicity level 3-4 on the National Cancer Institute's Common Terminology Criteria for Adverse Events toxicity scale v4.0.

The subjects' baseline characteristics describing their tobacco use are presented in the table below.

N % Average Range Consumer status Smoker 24 100 Consumption Finished cigarettes, qty/day 18 75 20 (15; 30) Rolled cigarettes, number/day 3 12.5 20 (15; 30) Cigarillos, number/day 3 12.5 20 (15; 20) Addiction level according to Fagerström 5 eleven 46 6 5 21 7 2 8 8 3 12.5 9 3 12.5 Measured amount of exhaled CO2 (ppm) 24 24.5 (9; 45) Would you like to quit smoking? No Yes 24 100

In FIG. 8 and 9 show the change in tobacco consumption.

Below is the formula for calculating the reduction in the number of cigarettes:

(Number of cigarettes, visit n - Number of cigarettes at inclusion in the study) / Number of cigarettes at inclusion in the study

A survey conducted 4 weeks later showed that 16 smokers (67% of smokers) had quit or reduced their daily cigarette consumption by at least 50% and that 24 smokers had reduced their daily cigarette consumption by 60%.

A survey conducted 12 weeks later showed that 15 smokers (63% of smokers) had quit or reduced their daily cigarette consumption by at least 50% and that 24 smokers had reduced their daily cigarette consumption by 60%.

It is interesting to note that the last 12 smokers (12 out of 24) had more time (sometimes a month or more) between their first contact with the Clinical Research Center and receiving the tobacco extract injection according to Example 1, while the first 12 smokers had this period

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Claims (20)

shorter (often only a few days). For those 12 smokers who had more time, the percentage of smokers who reduced tobacco use by at least 50% or stopped smoking at 3 and 4 weeks was 83% (10 smokers out of 12), while it was only 50% (6 smokers out of 12) for those 12 smokers for whom this period was shorter. This increased efficacy is due to the better preparation of those smokers who had more time to increase motivation before receiving the tobacco extract injection according to example 1. Of the last 12 smokers, 5 were consistently abstinent from the first week after the end of therapy until week 12 (end of follow-up), and 8 have consistently abstained from smoking for the last 7 days of week 12. Treated smokers reported reduced cigarette attraction and urge to smoke, as well as some indifference to cigarettes. They noted a decrease in the attractiveness of cigarettes. Some talked about changing the taste of the cigarette. Their motivation to quit smoking increased. Prior to administration of the pharmaceutical composition, IgG antibodies directed to the pharmaceutical composition were found in all patients. Before receiving therapy, the average concentrations of IgG directed to the pharmaceutical composition were 6.07 μg/ml (standard deviation: 2.98 μg/ml). After administration of the pharmaceutical composition, a significant increase in the concentrations of IgG antibodies to the pharmaceutical composition was observed. Thus, the IgG concentrations measured on day 29 after administration of the pharmaceutical composition were 7.19 μg/ml (SD: 4.04 μg/ml), and these concentrations increased to 7.5 μg/ml (SD 4, 28 μg/ml), 8 weeks after the first administration of the pharmaceutical composition. The variation in IgG concentrations was significant (significance threshold t-test p<0.05) in the range of day 29 - day 0 (p = 0.02) and in the range between measurements of week 8 - day 0 ( p=0.006). A significant difference was also observed between the concentrations of IgG antibodies to the pharmaceutical composition measured at week 8 and day 29 (p=0.029). Statistical analysis linking concentrations of anti-pharmaceutical IgG antibodies and smoking cessation revealed a correlation (Wilcoxon test, p<0.05 significance threshold) between higher levels of anti-pharmaceutical IgG antibodies and smoking cessation. Patients who reduced their tobacco use by at least 50% one week after the first administration of the pharmaceutical composition had higher levels of IgG antibodies to the pharmaceutical composition at day 29 (p=0.034) and at week 8 (p=0.044 ). Patients who abstained from smoking for 7 consecutive days (predominant abstinence) had higher levels of IgG antibodies to the pharmaceutical composition at day 29 (p<0.001) and at week 8 (p <0.001). Patients who abstained from smoking for 7 consecutive days (predominant abstinence) had higher levels of anti-pharmaceutical IgG antibodies at day 29 (p=0.013) and at week 8 by week 12 (p=0.016). Patients who consistently abstained from smoking until week 12 had higher levels of IgG antibodies to the pharmaceutical composition at day 29 (p=0.007) and at week 8 (p=0.009). After administration of the pharmaceutical composition, IgE antibodies were not detected. CLAIM 1. Tobacco leaf extract for the treatment of tobacco dependence, containing at least 5 wt.% proteins based on the total weight of the dry extract, where the molecular weight of the proteins exceeds 10 kDa and the proteins essentially do not contain molecules with a molecular weight of less than 10 kDa, and: the content of proteins, the molecular weight of which exceeds 500 kDa, is less than 15 wt.% of the total mass of proteins in the extract; and the content of molecules with a molecular weight of less than 10 kDa is less than 5 wt.% of the total weight of the extract. 2. The tobacco leaf extract of claim 1, wherein the proteins are selected from the group consisting of the following protein families: lignin-forming anionic peroxidase, glucan-endo-1,3-beta-glucosidase, endochitinase, pathogenesis-associated protein, osmotin, and proteinase inhibitor , as well as their mixtures. 3. Tobacco leaf extract according to claim 1 or 2, wherein the content of proteins whose molecular weight exceeds 500 kDa, preferably whose molecular weight exceeds 400 kDa, more preferably whose molecular weight exceeds 300 kDa, more preferably whose molecular weight exceeds 200 kDa, more preferably having a molecular weight greater than 150 kDa, even more preferably having a molecular weight greater than 100 kDa and even more preferably having a molecular weight greater than 50 kDa, less than 15 wt% of the total weight of proteins in the extract, preferably less than 10 wt% of the total weight proteins in the extract, more preferably - 21 040213 specifically less than 7.5 wt.% of the total weight of the proteins in the extract, more preferably less than 5 wt.% of the total weight of the proteins in the extract, preferably less than 2.5 wt.% of the total weight of the proteins in the extract, more preferably less than 1 wt.% of the total weight of the proteins in the extract, and even more preferably less than 0.5 wt.% of the total weight of the proteins in the extract. 4. Tobacco leaf extract according to any one of the preceding claims, wherein the content of molecules with a molecular weight of less than 10 kDa is less than 2.5 wt%, preferably less than 1 wt% of the total weight of the extract. 5. Tobacco leaf extract according to any of the preceding claims, which is obtained by a solvent extraction method, for example an aqueous solvent, of ground dried tobacco leaves, followed by separation of solid residues from the ground dried tobacco leaf extract solution and subsequent diafiltration at a constant volume of the ground dried tobacco leaf extract solution, which does not contain a solid residue, in the presence of an aqueous solvent taken in an amount of 2 to 12 volumes, preferably 3 to 10 volumes, preferably 4 to 8 volumes, preferably 6 volumes, where the volume is equal to the volume of the extract, using a membrane with limit value of transmission 10 kDa. 6. Tobacco leaf extract according to any one of the preceding claims, comprising at least one protein belonging to the glucan-endo-1,3-beta-glucosidase family and preferably selected from the acid isoform PR-Q' of beta-1,3-endoglucanase (PR36401 according to the UniProt database), the main GLB vacuolar isoform of beta-1,3-endoglucanase (P27666 according to the UniProt database) and mixtures thereof. 7. Tobacco leaf extract according to any of the preceding claims, comprising at least one protein belonging to the endochitinase family and preferably selected from acid endochitinase P (P17513 according to the UniProt database), acid endochitinase Q (P17514 according to the UniProt database), endochitinase B ( P24091 according to the UniProt database) and their mixtures. 8. Tobacco leaf extract according to any of the preceding claims, comprising at least osmotin (P14170 according to the UniProt database). 9. Tobacco leaf extract according to any one of the preceding claims, comprising at least one lignin-forming anionic peroxidase (P11965 according to the UniProt database). 10. Tobacco leaf extract according to one of the preceding claims, comprising at least one pathogenesis-associated protein, preferably selected from pathogenesis-associated protein R (P13046 according to the UniProt database), pathogenesis-related protein PR-4A (PR29062 according to the UniProt database ) associated with the pathogenesis of the PR-4B protein (PR29063 according to the UniProt database) and mixtures thereof. 11. Tobacco leaf extract according to any one of the preceding claims, comprising at least one protein belonging to the family of proteinase inhibitors and preferably selected from proteinase inhibitor I-B (Q03199 according to the UniProt database), proteinase inhibitor I-A (Q03198 according to the UniProt database) and mixtures thereof . 12. Tobacco leaf extract according to at least one of the preceding claims, where the protein content in the dry extract is at least 10 wt.%, preferably at least 15 wt.%, preferably at least 20 wt.% of the total dry weight extract. 13. A pharmaceutical composition for the treatment of tobacco dependence, comprising, as an active ingredient, the tobacco leaf extract according to any one of the preceding paragraphs, as well as a pharmaceutically acceptable excipient. 14. Pharmaceutical composition according to claim 13, wherein the proteins found in the tobacco leaf extract are present in an amount of 1 to 1000 μg/ml, preferably 10 to 500 μg/ml, preferably 50 to 300 μg/ml, preferably 60 to 200 μg/ml, preferably 80 to 150 μg/ml. 15. Pharmaceutical composition according to claim 13 or 14, additionally containing proteins extracted from marijuana. 16. Pharmaceutical composition according to any one of claims 13-15, made in a form suitable for administration by subcutaneous injection, in a form suitable for administration through an adhesive transdermal therapeutic system, such as a patch, or in a form suitable for administration by spraying or evaporation . 17. The use of a pharmaceutical composition according to any one of claims 13 to 16 for the treatment of tobacco dependence. 18. The use of a pharmaceutical composition according to claim 15 for the combined treatment of tobacco dependence and addiction to marijuana. 19. Use according to claim 17 or 18, wherein the pharmaceutical composition is in the form of a dosage form with a volume of 0.03 to 10 ml, preferably 0.1 to 5 ml, preferably 0.5 to 2 ml. 20. A method for producing a tobacco leaf extract according to any one of claims 1 to 12, including the following steps: -