Treatment methods including tobacco leaf extract to aid in smoking cessation

Abstract

The present disclosure generally relates to a treatment method for helping a subject quit smoking, the method comprising at least one step of administering to the subject an effective dose of an aqueous solution of protein-enriched tobacco leaf extract while the subject continues to smoke freely.Optionally, the second step comprises providing the subject with at least one product capable of controlling nicotine withdrawal symptoms, such as a nicotine agonist or partial agonist, and / or a nicotine substitute, by one or several optional complementary administrations.The present disclosure also relates to a kit comprising a protein-enriched tobacco leaf extract and at least one product capable of controlling nicotine withdrawal symptoms, such as a nicotine agonist or partial agonist, and / or a nicotine substitute.

Description

[Background technology]

[0001] The present disclosure generally relates to a treatment method for helping a subject quit smoking, the method comprising at least one step of administering to the subject an effective dose of an aqueous solution of protein-enriched tobacco leaf extract while the subject continues to smoke freely.Optionally, the second step comprises providing the subject with at least one product capable of controlling nicotine withdrawal symptoms, such as a nicotine agonist or partial agonist, and / or a nicotine substitute, by one or several optional complementary administrations.The present disclosure also relates to a kit comprising a protein-enriched tobacco leaf extract and at least one product capable of controlling nicotine withdrawal symptoms, such as a nicotine agonist or partial agonist, and / or a nicotine substitute.

[0002] Tobacco addiction is one of the greatest public health threats the world has ever faced, killing approximately 8 million people each year. There are over 1 billion smokers worldwide, up to half of whom ultimately die from tobacco-related diseases. Tobacco causes one death approximately every 6 seconds and accounts for one in 10 adult deaths. Tobacco was responsible for 100 million deaths in the 20th century and could be responsible for one billion deaths in the 21st century if current trends continue.

[0003] According to India's National Tobacco Control Programme, the tobacco epidemic is one of the major public health threats, with one million tobacco-related deaths in India each year.

[0004] According to the Global Adult Tobacco Survey-2 (GATS-2), one in ten adults (100 million) in India smokes tobacco, and 28.6% of adults aged 15 and above (267 million) use tobacco in some form. 199 million rural adults and 68 million urban adults use tobacco.

[0005] Tobacco smoke contains more than 4,000 chemicals, at least 250 of which are known to be harmful and more than 50 of which are known to be carcinogenic. Tobacco is a risk factor for six of the top eight causes of death. It can cause cancer almost anywhere in the body. In industrialized countries, it is estimated to cause more than 90% of lung cancers in men, approximately 70% of lung cancers in women, and approximately 22% of all cardiovascular disease. Smoking is responsible for approximately 90% of all deaths from chronic obstructive pulmonary disease. Smoking harms almost every organ in the body, affecting a person's overall health.

[0006] Nicotine is believed to be involved in the mechanism of tobacco addiction, particularly in smoking addiction. Therefore, to date, nicotine has been considered the primary reinforcing psychoactive and addictive substance. Nicotine can actually bind to nicotinic acetylcholine receptors, proteins present on the surface of nerve cells. In the presence of nicotine, these receptors (actually channels) open. A series of events then ensues, resulting in the release of the hormone dopamine. Nicotine stimulates the "reward circuit," producing a feeling of satisfaction. Therefore, when people quit smoking, their bodies "crave" nicotine doses to satisfy this euphoric sensation. This is the nicotine withdrawal symptom. For example, the goal of nicotine replacement is to provide a smoker's brain with enough nicotine to prevent withdrawal symptoms. In summary, nicotine targets specific brain receptors, resulting in the release of neurotransmitters such as dopamine in the central nervous system, producing positive sensations such as pleasure, relaxation, and appetite suppression. Over time, these receptors come to expect nicotine, and when it is deprived, smokers experience withdrawal symptoms.

[0007] Smoking also reduces levels of monoamine oxidase (MAO), an enzyme involved in the breakdown of dopamine, resulting in higher dopamine levels and tobacco dependence. Because nicotine itself does not interact with MAO, this change is due to other tobacco smoke components, another cause of tobacco dependence. These components also disrupt the coupling between the noradrenergic and serotonergic systems through desensitization of 5-HT(1A) autoreceptors, increasing the reactivity of dopaminergic neurons. Recent studies have shown that glutamatergic and gamma-aminobutyric acid (GABA) receptors in the medial prefrontal cortex (mPFC) may also be involved through GABAergic plasticity mechanisms in the dorsal mPFC. Several other publications have cited multiple mechanisms involved in tobacco dependence. Smoking behavior also varies depending on genetic and environmental factors. Therefore, the mechanisms of tobacco dependence are highly complex, involving several pathways in the CNS and other molecules besides nicotine.

[0008] Dependence or addiction is defined by the World Health Organization as "a syndrome in which the consumption of a product becomes a greater requirement than other, previously more important, behaviors." In its extreme form, the dependent state is characterized by an irresistible desire for the product, forcing the individual suffering from this addiction to seek it compulsively.

[0009] For all these reasons and / or others, many smokers want to quit.

[0010] Smokers can choose to quit smoking in one step (abrupt quitting) by setting a quit date, or to reduce their consumption before the quit date (gradual quitting). For gradual quitting, a reduction period of 10 days to 2 weeks is recommended before the quit date to avoid any changes in motivation.

[0011] The first 2 weeks after a target quit date (TQD) are significantly correlated with 6-month abstinence. Smokers who do not relapse after a TQD are more likely to become long-term abstinent.

[0012] Current smoking cessation medications include nicotine replacement therapy (NRT) and the nicotine-free smoking cessation medications bupropion hydrochloride (ZYBAN™) and varenicline tartrate (CHAMPIX™ in France, CHANTIX™ in the United States). Several nicotine vaccines are in development.

[0013] Nicotine replacement therapy (NRT) is the first pharmacological treatment approved for use in smoking cessation. It is typically used in conjunction with behavioral support. It includes nicotine gum, transdermal patches, tablets, nasal sprays, and inhalation systems. It acts as a nicotine substitute to reduce withdrawal symptoms and cravings. A 2012 meta-analysis of 117 trials from the Cochrane Tobacco Dependence Group showed that all commercially available NRTs help increase the likelihood of quitting smoking by 50–70% at 6 months. Despite this, the absolute quit rates for these medications are modest, and most smokers relapse. The 6-month abstinence rate for smokers using NRT is 20–30%, and 70–80% of smokers who use these products relapse. NRT can be used for both sudden and gradual quitting. In sudden quitting, NRT is used 8–12 weeks after the TQD. In gradual quitting, NRT is used 6 weeks to 6 months before the TQD to help smokers reduce their consumption, and then 8–12 weeks after the TQD.

[0014] ZYBAN® (bupropion hydrochloride), marketed by GlaxoSmithKline, was the first nicotine-free smoking cessation medication approved in 1997. According to French guidelines, bupropion is superior to placebo, not superior to NRT, and inferior to varenicline for smoking cessation at 6 months. Bupropion has a high level of side effects, including a risk of depression and / or self-aggressive suicidal behavior, allergic skin reactions, neuropsychiatric and neurological disorders, cardiovascular effects including hypertension, angina pectoris and / or myocardial infarction, dry mouth, and nausea. It is recommended that bupropion be used as a last resort to treat specific cases, with close monitoring of patients.

[0015] CHAMPIX™ (varenicline tartrate), marketed by Pfizer, was approved in 2006. Varenicline is a selective partial agonist of the α4β2 nicotinic acetylcholine receptor subtype, inhibiting nicotine's ability to stimulate the central mesolimbic dopamine system. Pfizer evaluated the efficacy of CHAMPIX™ in six clinical trials involving a total of 3,659 chronic smokers. The maximum 1-year benefit of CHAMPIX™ was below 30%, except when treatment was maintained for 6 months (>50%). A 2013 meta-analysis by Cahill et al. demonstrated the superiority of varenicline over placebo, bupropion, and NRT, but not over NRT combinations (transdermal patch plus oral NRT). This product is associated with serious side effects, including neuropsychiatric symptoms, including depression and suicidal / self-injurious behavior, behavioral changes, hostility, and agitation. It has also been associated with sleep disorders, gastrointestinal disorders, and cardiovascular problems, although these last observations remain to be confirmed. Because of these side effects, particularly the association with suicide and depression, it is recommended as a second-line treatment after the failure of nicotine replacement therapy.

[0016] Vaccine therapies are also being evaluated. Nicotine is the target. Nicotine molecules are chemically attached to a carrier protein or particle, stimulating the immune system to produce antibodies that bind to nicotine from tobacco smoke and prevent its entry into the brain. The NIC002 (Cytos / Novartis) vaccine and the NicVAX™ (Nabi / GSK) vaccine failed to demonstrate efficacy in phase II and III clinical trials, respectively. Nic7, a bioconjugate vaccine developed by Pfizer, was evaluated in a phase I clinical trial involving 200 people and discontinued in 2016. SEL-068 (Selecta Biosciences) is a nanoparticle-based vaccine that was in phase I clinical development from 2012 to 2014, but no results or recent developments have been published.

[0017] Several other products under evaluation for smoking cessation include the D3R partial agonists BP1.4979 (Phase II, Bioprojet), nadolol (Phase II, Invion, Inc.), the reversible MAO-A inhibitor moclobemide (Phase II, Duke University Medical Center / Philip Morris), and the Alpha-7 nicotinic acetylcholine receptor agonist EVP-6124 (Forum Pharmaceuticals Inc).

[0018] It is also important to note that despite the growing popularity of e-cigarettes, they are not approved for smoking cessation in many regulatory jurisdictions, creating an even greater need for the development of safe and effective alternatives for this indication.

[0019] As described in WO2017174787, the present inventors have found that injection of an aqueous solution of tobacco leaf extract can reduce addiction. More specifically, they have demonstrated that a single injection of tobacco extract according to WO2017174787 is generally sufficient to reduce or eliminate a smoker's tobacco addiction. This feature represents a significant benefit to the subject, considering that currently available products on the market only provide treatment in a few doses over a long period of time. Conversely, the treatment proposed by the present invention is a shock therapy, i.e., a treatment consisting preferably of a single injection of tobacco extract, optionally followed by at least one second injection several days or weeks later if the treated subject feels the need.

[0020] The inventors have discovered that their previously discovered solutions, under certain circumstances, can allow for easier withdrawal from tobacco addiction, especially when used in conjunction with other products that can control nicotine withdrawal symptoms.

[0021] overview The disclosed invention relates to a method for conditioning a subject to quit smoking, the method comprising administering to the subject, transdermally or subcutaneously, an effective dose of an aqueous solution of a protein-enriched tobacco leaf extract that is substantially free of nicotine, while the subject continues to smoke freely.

[0022] This method advantageously allows the subject to quit smoking more easily compared to existing methods, and indeed, preconditioning the subject to the unpleasant or aversive taste produced by administering an aqueous solution of protein-enriched tobacco leaf extract before the subject actually quits smoking makes it easier for the subject to quit smoking more effectively.

[0023] Therefore, the presently disclosed invention also relates to a method for treating smoking cessation in a subject, comprising the following two consecutive steps: i) a preconditioning step of administering to the subject, transdermally or subcutaneously, an effective dose of at least one aqueous solution of a protein-enriched tobacco leaf extract that is substantially free of nicotine, while the subject continues to smoke freely; and ii) A second step after the preconditioning step i), in which the subject completely quits smoking by one or several optional complementary administrations of at least one product capable of controlling nicotine withdrawal symptoms within 2 to 180 days, preferably within 2 to 30 days, of said administrations administered to the subject during the preconditioning step i) (also referred to as "initial step i)").

[0024] By "product capable of controlling nicotine withdrawal symptoms" is meant within the scope of the present invention a product that is not an aqueous solution of protein-enriched tobacco leaf extract as found in step i). For example, such a product may be a nicotine agonist or nicotine itself, administered, for example, in the form of a nicotine patch or gum or in other forms as known to those skilled in the art.

[0025] The conditioning step with the aqueous solution of protein-enriched tobacco leaf extract, substantially free of nicotine (i.e., step (i) in the treatment method and the method for conditioning the subject to quit smoking) means that on the day the subject decides to quit smoking, i.e., the day the subject no longer receives nicotine from tobacco, this nicotine intake can be replaced, if necessary, with at least one product capable of controlling nicotine withdrawal symptoms, thus facilitating tobacco withdrawal.

[0026] The second step (ii) is to help the subject to completely quit smoking, particularly by weaning them from nicotine addiction.

[0027] The invention of the present disclosure also relates to a kit comprising: - an aqueous solution of protein-enriched tobacco leaf extract, and separately - At least one product capable of controlling nicotine withdrawal symptoms.

[0028] Such kits can assist a subject in applying a treatment method according to the present invention by providing the necessary products, potential instructions for their use, and / or safety precautions.

[0029] Detailed Description While the inventive concepts of the present disclosure are susceptible to various modifications and alternative constructions, specific illustrated embodiments thereof are described in detail below.

[0030] <Method> It has been found that a subject can be preconditioned to quit smoking by administering to said subject an effective dose of an aqueous solution of a protein-enriched tobacco leaf extract, substantially free of nicotine, either transdermally or subcutaneously, while the subject continues to smoke ad libitum.

[0031] To enhance the effect of this method, if necessary, while the subject continues to smoke freely, an effective dose of a second aqueous solution of protein-enriched tobacco leaf extract, substantially free of nicotine, can be administered transdermally or subcutaneously. Although it has been found that such a second administration is not usually necessary, it can be advised, just in case, that if the subject does not experience the intended unpleasant taste or aversion caused by the first administration of the aqueous solution of protein-enriched tobacco leaf extract, to re-administer an effective dose of the aqueous solution of protein-enriched tobacco leaf extract.

[0032] One method of helping the subject determine whether they require an effective dose of the second aqueous solution of protein-enriched tobacco leaf extract, substantially free of nicotine, can be provided by administering a scoring questionnaire that asks questions that allow for a subjective assessment of the need and / or pleasure of smoking. Such a questionnaire can be provided, for example, by asking one or several of the following questions (or similar questions): (1) "Was smoking satisfying?"; (2) "Did the cigarette taste good?"; (3) "Did you enjoy the sensation in your throat and chest?"; (4) "Did smoking calm you down?"; (5) "Did smoking make you more awake?"; (6) "Did smoking make you less irritable?"; (7) "Did smoking help you concentrate?"; (8) "Did smoking reduce your appetite?"; (9) "Did smoking make you dizzy?"; (10) "Did smoking make you nauseous?"; (11) "Did smoking immediately relieve your craving for cigarettes?"; (12) "Did you enjoy smoking?".

[0033] For example, each question can be answered by giving a score from 1 to 7 depending on the subject's feelings, with 4 out of 7 being the middle point, indicating whether each question is not met at all (score equals 1) or completely met (score equals 7).

[0034] The questionnaire may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or all 12 of the questions described herein above.

[0035] In one embodiment, the questionnaire is the so-called "modified Tobacco Evaluation Questionnaire (mCEQ)," which includes all 12 questions described above. The mCEQ uses three multi-item subscales and two single items: "Smoking Satisfaction" (items 1, 2, and 12); "Psychological Reward" (items 4-8); "Aversion" (items 9 and 10); "Respiratory Sensation Enjoyment" (item 3), and "Craving Reduction" (item 11). The score for each subscale is calculated as the average of the individual item responses or single items. Higher scores indicate greater intensity on that scale.

[0036] Preferably, the effective dose of the second aqueous solution of protein-enriched tobacco leaf extract, substantially free of nicotine, can be administered within 2 to 15 days, preferably within 5 days, of the first administration.

[0037] Preferably, the present invention relates to a method for preconditioning a subject to quit smoking, wherein the tobacco leaf extract contains at least 5% by weight of proteins with a molecular weight greater than 10 kDa, based on the total weight of the dried extract, and is substantially free of molecules with a molecular weight less than 10 kDa and RuBisCO protein, and the content of proteins with a molecular weight greater than 500 kDa in the tobacco leaf extract is less than 15% by weight, based on the total protein weight of the dried extract.

[0038] In one embodiment, the present invention relates to a method of preconditioning a subject to quit smoking, wherein the protein is selected from the group consisting of the following protein families: lignin-forming anionic peroxidases, glucan endo-1,3-beta-glucosidases, endochitinases, disease-associated proteins, osmotins, protease inhibitors, and mixtures thereof.

[0039] Preferably, the present invention relates to a method for preconditioning a subject to quit smoking, wherein the content of proteins with a molecular weight of more than 100 kDa in a tobacco leaf extract is less than 15% by weight, based on the total protein weight of the dried extract.

[0040] Preferably, the present invention relates to a method for preconditioning a subject to quit smoking, wherein the tobacco leaf extract contains at least one protein belonging to the family of glucan endo-1,3-beta-glucosidases selected from the group consisting of beta-1,3-endoglucanase acidic isoform PR-Q' (PR36401 according to UniProt database), beta-1,3-endoglucanase basic vacuolar isoform GLB (P27666 according to UniProt database), and mixtures thereof.

[0041] Preferably, the present invention relates to a method for preconditioning a subject to quit smoking, wherein the tobacco leaf extract comprises at least one protein belonging to the family of endochitinases selected from the group consisting of acidic endochitinase P (P17513 according to UniProt database), acidic endochitinase Q (P17514 according to UniProt database), endochitinase B (P24091 according to UniProt database), and mixtures thereof.

[0042] Preferably, the present invention relates to a method for preconditioning a subject to quit smoking, wherein the tobacco leaf extract contains at least osmotin (P14170 according to the UniProt database).

[0043] Preferably, the present invention relates to a method for preconditioning a subject to quit smoking, wherein the tobacco leaf extract comprises at least one lignin-forming anionic peroxidase (P11965 according to the UniProt database).

[0044] Preferably, the present invention relates to a method for preconditioning a subject to quit smoking, wherein the tobacco leaf extract comprises at least one disease-related protein selected from the group consisting of disease-related protein R (P13046 according to UniProt database), disease-related protein PR-4A (PR29062 according to UniProt database), disease-related protein PR-4B (PR29063 according to UniProt database), and mixtures thereof.

[0045] Preferably, the present invention relates to a method for preconditioning a subject to quit smoking, wherein the tobacco leaf extract comprises at least one protein belonging to the family of protease inhibitors selected from the group consisting of protease inhibitor IB (Q03199 according to UniProt database), protease inhibitor IA (Q03198 according to UniProt database), and mixtures thereof.

[0046] Preferably, the present invention relates to a method for preconditioning a subject to quit smoking, wherein the protein content in the dry matter of the tobacco leaf extract is at least 10% by weight, based on the total weight of the dry matter.

[0047] Preferably, the present invention relates to a method for preconditioning a subject to quit smoking, wherein the protein present in the tobacco leaf extract is present in an amount ranging from 1 to 1000 μg / mL.

[0048] Preferably, the present invention relates to a method for preconditioning a subject to quit smoking, wherein the tobacco leaf extract or composition is provided in a form suitable for administration by subcutaneous injection, by an adhesive transdermal therapeutic system such as a patch, or by administration by spray or vaporization.

[0049] Preferably, the present invention relates to a method for preconditioning a subject to quit smoking, wherein the tobacco leaf extract or composition is provided in a dosage form of 0.03 mL to 10 mL.

[0050] As mentioned above, another aspect of the present disclosure relates to a method for treating smoking cessation in a subject, comprising the following two sequential steps: i) a preconditioning step of administering to the subject, transdermally or subcutaneously, an effective dose of at least one aqueous solution of a protein-enriched tobacco leaf extract that is substantially free of nicotine, while the subject continues to smoke freely; and ii) a second step after preconditioning step i), in which the subject completely quits smoking by one or several optional complementary administrations of at least one product capable of controlling nicotine withdrawal symptoms within 2 to 180 days, preferably within 2 to 30 days, of said administrations carried out to the subject during preconditioning step i).

[0051] As mentioned above, the purpose of administering an effective dose of a protein-enriched tobacco leaf extract solution that is substantially free of nicotine is to induce a distaste or aversion to tobacco. However, as also mentioned above, tobacco contains many addictive components, particularly nicotine. Therefore, if a subject experiences one or several side effects associated with nicotine withdrawal, administering a product that can control nicotine withdrawal symptoms, such as a nicotine patch, may be particularly helpful in helping the subject overcome nicotine addiction. One way to help a subject determine whether they require such an alternative treatment for nicotine can be by administering a scoring questionnaire using known symptoms of nicotine withdrawal.

[0052] Nicotine withdrawal can cause the following adverse events: depression, insomnia, irritability / frustration / anger, anxiety, difficulty concentrating, restlessness, increased appetite / weight gain, constipation, bradycardia...

[0053] For example, questions such as "Have you noticed a change in your mood over the last 2, 4, etc. days?", "Have people around you described you as being in a bad mood since you quit smoking?", or "Have you noticed that you have been hungrier than usual over the last 2, 4, etc. days?" could be given a score to help participants determine their nicotine need.

[0054] A method for helping a subject determine whether they are able to proceed to step (ii) of the methods described hereinabove and quit smoking completely can be provided by administering a scoring questionnaire as described above, in which questions are asked that allow for a subjective assessment of the need and / or pleasure of smoking. Such a questionnaire can be provided, for example, by asking one or several of the following questions (or similar questions): (1) "Was smoking satisfying?"; (2) "Did the cigarette taste good?"; (3) "Did you enjoy the sensation in your throat and chest?"; (4) "Did smoking calm you down?"; (5) "Did smoking make you more awake?"; (6) "Did smoking make you less irritable?"; (7) "Did smoking help you concentrate?"; (8) "Did smoking reduce your appetite?"; (9) "Did smoking make you dizzy?"; (10) "Did smoking make you nauseous?"; (11) "Did smoking immediately relieve your craving for cigarettes?"; (12) "Did you enjoy smoking?".

[0055] For example, each question can be answered by giving a score from 1 to 7 depending on the subject's feelings, with 4 out of 7 being the middle point, indicating whether each question is not met at all (score equals 1) or completely met (score equals 7).

[0056] The questionnaire may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or all 12 of the questions described herein above.

[0057] In one embodiment, the questionnaire is the so-called "modified Tobacco Evaluation Questionnaire (mCEQ)," which includes all 12 questions described above. The mCEQ uses three multi-item subscales and two single items: "Smoking Satisfaction" (items 1, 2, and 12); "Psychological Reward" (items 4-8); "Aversion" (items 9 and 10); "Respiratory Sensation Enjoyment" (item 3), and "Craving Reduction" (item 11). The score for each subscale is calculated as the average of the individual item responses or single items. Higher scores indicate greater intensity on that scale.

[0058] This questionnaire can be completed before, during and / or after step (i) and the results can be compared with each other to monitor the effect of the administrations given and consequently adapt the treatments given (e.g. by adding more and / or different products capable of controlling nicotine withdrawal symptoms).

[0059] In one embodiment, the present invention relates to a method of treatment wherein the preconditioning step comprises two or three transdermal administrations of effective doses of two or three aqueous solutions of protein-enriched tobacco leaf extracts, substantially free of nicotine, while the subject continues to smoke ad libitum.

[0060] Preferably, the present invention relates to a method of treatment in which two or three transdermal administrations of effective doses of two or three aqueous solutions of protein-enriched tobacco leaf extracts, substantially free of nicotine, are administered at least 10 days apart, preferably at least 20 days apart, while the subject continues to smoke freely.

[0061] Preferably, the present invention relates to a method of treatment wherein the preconditioning step comprises a single transdermal administration of an effective dose of an aqueous solution of a protein-enriched tobacco leaf extract, substantially free of nicotine, while the subject continues to smoke freely.

[0062] Preferably, the present invention relates to a treatment method in which, in a second step after the preconditioning step i), the subject completely quits smoking by one or several optional complementary administrations of at least one product capable of controlling nicotine withdrawal symptoms within 2 to 120 days, preferably within 2 to 60 days, of said administrations carried out to the subject during the preconditioning step i).

[0063] More preferably, the present invention relates to a method of treatment in which, in a second step after the preconditioning step i), the subject completely quits smoking by one or several optional complementary administrations of at least one product capable of controlling nicotine withdrawal symptoms within 2 to 30 days of said administrations carried out to the subject during the preconditioning step i).

[0064] In one embodiment, the present invention relates to a method of treatment, wherein said optional one or several supplementary administrations of at least one product capable of controlling nicotine withdrawal symptoms are carried out in a manner that involves accompanying the subject to gradually withdraw from nicotine, thereby controlling the costs of nicotine withdrawal over a period of time. Preferably, the accompanying is characterized by consulting the subject monthly, preferably every 2 weeks, every week, every 6 days, every 5 days, every 4 days, every 3 days, every 2 days, or every day, for a period of 1 week to 12 months, preferably 2 weeks to 6 months, more preferably 1 month to 4 months (e.g., 3 months).

[0065] In one embodiment, the present invention relates to a treatment method in which, when several products capable of controlling nicotine withdrawal symptoms are administered, they are administered temporally independent of each other, which allows a more precise and adaptable treatment of smoking cessation, in particular of withdrawal from nicotine.

[0066] Preferably, the present invention relates to a method of treatment, wherein said at least one product capable of controlling nicotine withdrawal symptoms is selected from a nicotine agonist or partial agonist, a nicotine replacement, or a combination thereof.

[0067] Preferably, the present invention relates to a method of treatment wherein said nicotinic agonist or partial agonist is selected from varenicline, cytisine, lobeline, or a combination thereof.

[0068] In one embodiment, the present invention relates to a method of treatment, wherein the second step is carried out within 3 to 10 days of said administration carried out to the subject during preconditioning step i).

[0069] Preferably, the present invention relates to a method of treatment, wherein the second step is carried out within 3 to 4 days of said administration carried out to the subject during preconditioning step i).

[0070] In one embodiment, the present invention relates to a method of treatment, wherein the tobacco leaf extract contains at least 5% by weight of proteins having a molecular weight greater than 10 kDa, based on the total weight of the dried extract, and is substantially free of molecules having a molecular weight less than 10 kDa and RuBisCO protein, and the content of proteins having a molecular weight greater than 500 kDa in the tobacco leaf extract is less than 15% by weight, based on the total protein weight of the dried extract.

[0071] Preferably, the present invention relates to a method of treatment, wherein the protein is selected from the group consisting of the following protein families: lignin-forming anionic peroxidases, glucan endo-1,3-beta-glucosidases, endochitinases, disease-associated proteins, osmotin, protease inhibitors, and mixtures thereof.

[0072] In one embodiment, the present invention relates to a method of treatment, wherein the content of proteins with a molecular weight of more than 100 kDa in the tobacco leaf extract is less than 15% by weight based on the total protein weight of the dried extract.

[0073] In one embodiment, the present invention relates to a method of treatment, wherein the tobacco leaf extract contains at least one protein belonging to the family of glucan endo-1,3-beta-glucosidases selected from the group consisting of beta-1,3-endoglucanase acidic isoform PR-Q' (PR36401 according to UniProt database), beta-1,3-endoglucanase basic vacuolar isoform GLB (P27666 according to UniProt database), and mixtures thereof.

[0074] In one embodiment, the present invention relates to a method of treatment, wherein the tobacco leaf extract contains at least one protein belonging to the family of endochitinases selected from the group consisting of acidic endochitinase P (P17513 according to UniProt database), acidic endochitinase Q (P17514 according to UniProt database), endochitinase B (P24091 according to UniProt database), and mixtures thereof.

[0075] In one embodiment, the present invention relates to a method of treatment, wherein the tobacco leaf extract comprises at least osmotin (P14170 according to the UniProt database).

[0076] In one embodiment, the present invention relates to a method of treatment, wherein the tobacco leaf extract comprises at least one lignin-forming anionic peroxidase (P11965 according to the UniProt database).

[0077] In one embodiment, the present invention relates to a method of treatment, wherein a tobacco leaf extract comprises at least one disease-related protein selected from the group consisting of disease-related protein R (P13046 according to UniProt database), disease-related protein PR-4A (PR29062 according to UniProt database), disease-related protein PR-4B (PR29063 according to UniProt database), and mixtures thereof.

[0078] In one embodiment, the present invention relates to a method of treatment, wherein the tobacco leaf extract contains at least one protein belonging to the family of protease inhibitors selected from the group consisting of protease inhibitor IB (Q03199 according to UniProt database), protease inhibitor IA (Q03198 according to UniProt database), and mixtures thereof.

[0079] In one embodiment, the present invention relates to a method of treatment, wherein the protein content in the dry extract of tobacco leaf extract is at least 10% by weight, based on the total weight of the dry extract.

[0080] In one embodiment, the present invention relates to a method of treatment, wherein the protein present in the tobacco leaf extract is present in an amount ranging from 1 to 1000 g / mL.

[0081] In one embodiment, the present invention relates to a method of treatment, wherein the tobacco leaf extract or composition is provided in a form suitable for administration by subcutaneous injection, by an adhesive transdermal therapeutic system such as a patch, or by administration by spray or vaporization.

[0082] In one embodiment, the present invention relates to a method of treatment, wherein the tobacco leaf extract or composition is provided in a dosage form of 0.03 mL to 10 mL.

[0083] <Parts kit> As noted above, a second aspect of the present disclosure relates to a kit comprising: - an aqueous solution of protein-enriched tobacco leaf extract; and - At least one product capable of controlling nicotine withdrawal symptoms.

[0084] As described above for the treatment method, preferably, the tobacco leaf extract contains at least 5% by weight of proteins with a molecular weight of more than 10 kDa, based on the total weight of the dry extract, and is substantially free of molecules with a molecular weight of less than 10 kDa.

[0085] More preferably, said protein is selected from the group consisting of the following protein families: lignin-forming anionic peroxidases, glucan endo-1,3-beta-glucosidases, endochitinases, disease-associated proteins, osmotin and protease inhibitors and mixtures thereof.

[0086] The method for preparing the tobacco leaf extract is as described above.

[0087] In one embodiment, the present invention relates to a kit suitable for transdermal or subcutaneous administration of an effective dose of an aqueous solution of protein-enriched tobacco leaf extract.

[0088] In one embodiment, the present invention relates to a kit, wherein said at least one product capable of controlling nicotine withdrawal symptoms is selected from a nicotine agonist or partial agonist, a nicotine substitute, or a combination thereof.

[0089] In one embodiment, the present invention relates to a kit, wherein said nicotinic agonist or partial agonist is selected from varenicline, cytisine, lobeline, or a combination thereof.

[0090] In one embodiment, the present invention relates to a kit comprising: the tobacco leaf extract contains at least 5% by weight of proteins having a molecular weight of greater than 10 kDa, based on the total weight of the dried extract, and is substantially free of molecules having a molecular weight of less than 10 kDa and RuBisCO protein; and The content of proteins having a molecular weight of more than 500 kDa in the tobacco leaf extract is less than 15% by weight based on the total protein weight of the dried extract.

[0091] In one embodiment, the invention is a kit wherein the protein is selected from the group consisting of the following protein families: Lignin-forming anionic peroxidases, glucan endo-1,3-beta-glucosidases, endochitinases, disease-related proteins, osmotins, protease inhibitors, and mixtures thereof.

[0092] In one embodiment, the present invention relates to a kit, wherein the content of proteins having a molecular weight of more than 100 kDa in a tobacco leaf extract is less than 15% by weight, based on the total protein weight of the dried extract.

[0093] In one embodiment, the present invention relates to a kit, wherein a tobacco leaf extract comprises at least one protein belonging to the family of glucan endo-1,3-beta-glucosidases selected from the group consisting of beta-1,3-endoglucanase acidic isoform PR-Q' (PR36401 according to UniProt database), beta-1,3-endoglucanase basic vacuolar isoform GLB (P27666 according to UniProt database), and mixtures thereof.

[0094] In one embodiment, the present invention relates to a kit, wherein the tobacco leaf extract comprises at least one protein belonging to the family of endochitinases selected from the group consisting of acidic endochitinase P (P17513 according to UniProt database), acidic endochitinase Q (P17514 according to UniProt database), endochitinase B (P24091 according to UniProt database), and mixtures thereof.

[0095] In one embodiment, the present invention relates to a kit, wherein the tobacco leaf extract comprises at least osmotin (P14170 according to the UniProt database).

[0096] In one embodiment, the present invention relates to a kit, wherein the tobacco leaf extract comprises at least one lignin-forming anionic peroxidase (P11965 according to the UniProt database).

[0097] In one embodiment, the present invention relates to a kit, wherein a tobacco leaf extract comprises at least one disease-related protein selected from the group consisting of disease-related protein R (P13046 according to UniProt database), disease-related protein PR-4A (PR29062 according to UniProt database), disease-related protein PR-4B (PR29063 according to UniProt database), and mixtures thereof.

[0098] In one embodiment, the present invention relates to a kit, wherein the tobacco leaf extract comprises at least one protein belonging to a family of protease inhibitors selected from the group consisting of protease inhibitor IB (Q03199 according to the UniProt database), protease inhibitor IA (Q03198 according to the UniProt database), and mixtures thereof.

[0099] In one embodiment, the present invention relates to a kit, wherein the protein content in the dried extract of the tobacco leaf extract is at least 10% by weight, based on the total weight of the dried extract.

[0100] In one embodiment, the present invention relates to a kit, wherein the proteins present in the tobacco leaf extract are present in an amount ranging from 1 to 1000 μg / mL.

[0101] In one embodiment, the present invention relates to a kit in which the tobacco leaf extract or composition is provided in a form suitable for administration by subcutaneous injection, by an adhesive transdermal therapeutic system such as a patch, or by administration by spray or vaporization.

[0102] In one embodiment, the present invention relates to a kit in which the tobacco leaf extract or composition is provided in a dosage form of 0.03 mL to 10 mL.

[0103] In one embodiment, the kit comprises one or more doses of tobacco leaf extract, preferably in lyophilized form, and further comprises one or more doses of saline or WFI for preparing the pharmaceutical compositions described herein immediately prior to administration.

[0104] In one embodiment, the kit contains one or more doses of the pharmaceutical composition ready to be administered to a patient.

[0105] The kit may optionally include one or more syringes for administering the pharmaceutical composition by subcutaneous injection.

[0106] The kit may optionally include one or more patches for transdermal administration of the pharmaceutical composition.

[0107] <Tobacco leaf extract and pharmaceutical composition thereof> The protein-enriched tobacco leaf extract for use in the treatment methods and kits described herein above can be obtained as disclosed in WO2017174787.

[0108] That is, the tobacco leaf extract can be obtained by a preparation method including the following steps: a. Drying tobacco leaves, b. Grinding the dried tobacco leaves to obtain ground dried tobacco leaves; c. Extracting the ground dried tobacco leaves with an aqueous solvent, preferably an aqueous buffer solution having a pH of 6.0 to 8.5, under mechanical agitation; d. Separating the solid residue from the ground dried tobacco extract solution by filtration or centrifugation to obtain a ground dried tobacco extract solution free of solid residue; e. The solution obtained in step d is subjected to constant volume filtration using a 10 kDa cutoff membrane with a solvent, preferably an aqueous solvent, in an amount of 2 to 12 times, preferably 3 to 10 times, preferably 4 to 8 times, preferably 6 times the volume of the extract; f. Optionally, lyophilize the protein solution obtained in step e.

[0109] The dried extract of the tobacco leaf extract of the present invention can be obtained before freeze-drying, by placing it in a vacuum bell jar, preferably in the presence of P2O5, until the extract reaches a constant mass.

[0110] The drying in step a is carried out outdoors, known as air drying. In this case, drying occurs outdoors or indoors in the presence of natural air. Drying can be carried out in open or closed, covered or uncovered structures. Air drying can be carried out, for example, in an air drying shed. In this case, tobacco leaves immediately after harvest or after preliminary drying are allowed to dry naturally in a natural environment. The tobacco leaves can be hung, for example, in an unheated, ventilated shed. The tobacco leaves can be air-dried, for example, until brown. At this stage, substantially no sugars remain in the leaves. Advantageously, drying is carried out for a period suited to the local climatic conditions, preferably at least one month. For example, for harvests in central France, drying can be carried out between September and December. To ensure uniform drying and to avoid the formation of condensation and the rotting or deterioration of the tobacco leaves, the tobacco leaves may be turned one or more times during drying. The air drying method can be used, for example, for burley tobacco. In a specific embodiment, the drying in step a is carried out in an air drying shed.

[0111] For the purposes of this invention, "air drying" or "natural drying" refers to drying performed in the presence of natural outdoor or indoor air. Air drying can be performed in open or closed, covered or uncovered structures. Air drying can be performed, for example, in natural drying sheds. In this case, tobacco leaves immediately after harvest or after preliminary drying are allowed to dry naturally in a natural environment. The tobacco leaves can be hung, for example, in unheated, ventilated sheds. The tobacco leaves can be naturally dried, for example, until brown. At this stage, substantially no sugars remain in the leaves. Advantageously, drying is performed for a period suited to the local climatic conditions, preferably at least one month. For example, for harvests in central France, drying can be performed between September and December. To ensure uniform drying and to avoid the formation of condensation and the rotting or deterioration of the tobacco leaves, the tobacco leaves may be turned or ventilated one or more times during drying. The air drying method can be used, for example, with burley tobacco.

[0112] Tobacco leaf extract can be obtained from tobacco leaves that have been dried in an air drying shed.

[0113] Tobacco leaf extracts can be obtained from tobacco leaves that have been dried in a heated drying shed, where the drying can take place in a shed or structure that is heated to a suitable temperature.

[0114] Tobacco leaf extract can be obtained from flame-cured tobacco leaves. In this case, the curing can be carried out in a structure or shed heated to a suitable temperature. Heat can be introduced into the structure or shed through a conduit connected to an external heat source. This controlled heating produces yellow-orange leaves. These leaves therefore contain a high sugar content. Virginia tobacco, for example, can be cured by this method.

[0115] Tobacco leaf extract can be obtained from sun-dried tobacco leaves. In this case, the tobacco leaves are laid out on shelves and exposed to sunlight for 12 to 30 days. Under the direct light and heat from the sun, the leaves turn yellow or orange and retain a high sugar content. Oriental tobaccos are commonly dried using this method.

[0116] Tobacco leaf extract can be obtained from flue-cured tobacco leaves, where small pieces of wood are burned underneath the tobacco, allowing the leaves to dry while absorbing the "smoky" flavor.

[0117] In one embodiment, the tobacco leaf is of the species Nicotiana tabacum or Nicotiana rustica. The tobacco leaf may be from brown or blond tobacco and may be selected from Virginia tobacco, Burley tobacco, Oriental tobacco, Latakia tobacco, Perique tobacco, Maryland tobacco, Kentucky tobacco, California tobacco, Tex-Mex tobacco, and mixtures thereof.

[0118] Of course, the extract does not necessarily consist of a pure extract of tobacco leaves, but may also contain added proteins extracted from cannabis to treat both tobacco and cannabis addiction.

[0119] In a particular embodiment, the tobacco leaf extract is obtained from a 1 / 1 / 1 blend of brown tobacco, Virginia tobacco, and burley tobacco.

[0120] In certain embodiments, the tobacco leaf extract is obtained from burley tobacco.

[0121] In one embodiment, the tobacco leaf extract is obtained from dried tobacco leaves.

[0122] In certain embodiments, the tobacco leaf extract is obtained from air-cured tobacco leaves.

[0123] In certain embodiments, the tobacco leaf extract is obtained from tobacco leaves that have been air-dried for a period suitable for the local climatic conditions, preferably at least one month.

[0124] Steps c to e correspond to extraction, filtration and diafiltration processes of the type described in U.S. Patent No. 5,770,698 (column 6, line 47 to column 7, line 7) and patent application US 2009 / 0162403 (paragraphs

[0032] to

[0040] ).

[0125] Preferably, the extraction in step c is carried out at a temperature of 4 to 20°C, preferably 4 to 10°C.

[0126] Preferably, the extraction in step c is carried out for 12 to 36 hours, preferably 22 to 26 hours, preferably 24 hours.

[0127] In certain embodiments, the solvent used in step c is an aqueous solvent. Preferably, the aqueous solvent used in step c is an aqueous buffer solution of ammonium bicarbonate, preferably at a concentration of 2-6 g / L, preferably 4 g / L.

[0128] Preferably, the tobacco leaf extract can be obtained by a solvent extraction process, for example, an aqueous solvent extraction process, of ground dried tobacco leaves, followed by separating the solid residue from the ground dried tobacco leaf extract solution, and then subjecting the ground dried tobacco leaf extract solution free of the solid residue to constant volume diafiltration using a 10 kDa cutoff membrane with an aqueous solvent having a volume that is 2 to 12 times, preferably 3 to 10 times, preferably 4 to 8 times, and preferably 6 times the volume of the extract.

[0129] Of course, other types of solvents can also be used to carry out the extraction process of tobacco leaf extract in step c. The extraction in step c can be carried out with an organic solvent or an inorganic solvent, or a mixture of organic and / or inorganic solvents.

[0130] To ensure good extraction efficiency and yield and to obtain a quality extract with the desired purity profile and composition, a person skilled in the art will readily know how to select a suitable solvent according to the following criteria: - polarity of the solvent or solvent mixture (polar or non-polar); - the physical state of the solvent or solvent mixture (e.g., liquid, solid, supercritical or gas); - Chemical nature of the solvent or solvent mixture (e.g., organic or inorganic); - the charge of the solvent or solvent mixture (e.g., ionic or non-ionic); - the origin of the solvent or solvent mixture; - the miscibility of the solvent or solvent mixture; and / or - Solubility in the solvent or solvent mixture.

[0131] Those skilled in the art will know which techniques to use to select a solvent that corresponds to that criteria, based on the desired extraction yield and the purity and composition of the extract.

[0132] Preferably, the extraction in step c is carried out by suspending the ground dried tobacco leaves in a buffer solution, preferably at a concentration of 30-70 g / L, preferably 40-60 g / L, more preferably 50 g / L. The suspended solid residue is then removed by filtration, for example, by Buchner filtration (step d), to obtain ground dried tobacco leaves free of solid residue. Preferably, the aqueous solvent used in step d is water for injection (WFI).

[0133] Preferably, the tobacco leaf extract may contain at least 5% by weight, preferably at least 10% by weight, preferably at least 15% by weight, preferably about 20% by weight of proteins having a molecular weight greater than 10 kDa, based on the total weight of the dried extract, and may be substantially free of molecules having a molecular weight less than 10 kDa.

[0134] For purposes of the present invention, "about" means plus or minus 1% due to measurement uncertainty.

[0135] Advantageously, step e removes more than 99% of molecules with a molecular weight of less than 10 kDa, in particular free amino acids resulting from the degradation of the proteins of step a, proteins and peptides with a molecular weight of less than 10 kDa, and protein residues with a molecular weight of less than 10 kDa.

[0136] Advantageously, the content of molecules with a molecular weight of less than 10 kDa is less than 5% by weight, preferably less than 2.5% by weight, and even more preferably less than 1% by weight, based on the total weight of the extract.

[0137] The content of proteins with a molecular weight of more than 10 kDa in the tobacco leaf extract is usually measured at about 1%. Therefore, the tobacco leaf extract for the present invention contains proteins with a molecular weight of more than 10 kDa.

[0138] The proteins in the protein-enriched tobacco leaf extract are preferably selected from the group consisting of the following protein families: lignin-forming anionic peroxidases, glucan endo-1,3-beta-glucosidases, endochitinases, disease-related proteins, osmotin and protease inhibitors and mixtures thereof.

[0139] The names given for the proteins present in the extracts for the purposes of the present invention correspond to the names given in the Swiss-Prot database, a biological database that lists protein sequences.

[0140] In one embodiment, the tobacco leaf extract for the present invention comprises at least one protein belonging to the family of glucan endo-1,3-beta-glucosidases, preferably selected from beta-1,3-endoglucanase acidic isoform PR-Q' (PR36401 according to the UniProt database), beta-1,3-endoglucanase basic vacuolar isoform GLB (P27666 according to the UniProt database), and mixtures thereof.

[0141] In one embodiment, the tobacco leaf extract for the present invention comprises at least one protein belonging to the family of endochitinases, preferably selected from acidic endochitinase P (P17513 according to the UniProt database), acidic endochitinase Q (P17514 according to the UniProt database), endochitinase B (P24091 according to the UniProt database), and mixtures thereof.

[0142] In one embodiment, the tobacco leaf extract for the present invention comprises at least osmotin (P14170 according to the UniProt database).

[0143] In one embodiment, tobacco leaf extracts for the present invention contain at least one lignin-forming anionic peroxidase (P11965 according to the UniProt database).

[0144] In one embodiment, the tobacco leaf extract for the present invention comprises at least one disease-related protein, preferably selected from disease-related protein R (P13046 according to the UniProt database), disease-related protein PR-4A (PR29062 according to the UniProt database), disease-related protein PR-4B (PR29063 according to the UniProt database), and mixtures thereof.

[0145] In one embodiment, the tobacco leaf extract for the present invention comprises at least one protein belonging to the family of protease inhibitors, preferably selected from protease inhibitor IB (Q03199 according to the UniProt database), protease inhibitor IA (Q03198 according to the UniProt database), and mixtures thereof.

[0146] In one embodiment, tobacco leaf extracts for the present invention also contain polysaccharides with a molecular weight above 10 kDa, preferably water-soluble.

[0147] Preferably, the tobacco leaf extract for the present invention comprises at least 5% by weight, preferably at least 10% by weight, preferably at least 15% by weight, preferably about 20% by weight, of proteins selected from the group consisting of the following protein families: lignin-forming anionic peroxidases, glucan endo-1,3-beta-glucosidases, endochitinases, disease-related proteins, osmotin and protease inhibitors and mixtures thereof, based on the total weight of the dry extract.

[0148] In one embodiment, tobacco leaf extracts for the present invention are substantially free of high molecular weight proteins.

[0149] For the purposes of the present invention, "high molecular weight protein" means a protein having a molecular weight of more than 500 kDa, preferably a protein having a molecular weight of more than 400 kDa, more preferably a protein having a molecular weight of more than 300 kDa, preferably a protein having a molecular weight of more than 200 kDa, even more preferably a protein having a molecular weight of more than 150 kDa, and even more preferably a protein having a molecular weight of more than 100 kDa.

[0150] In one embodiment, tobacco leaf extracts for purposes of the present invention are substantially free of proteins with a molecular weight above 50 kDa.

[0151] For purposes of this disclosure, "substantially free" means a molecular content of less than 15% by weight based on the total protein weight of the extract, preferably less than 10% by weight based on the total protein weight of the extract, more preferably less than 7.5% by weight based on the total protein weight of the extract, more preferably less than 5% by weight based on the total protein weight of the extract, preferably less than 2.5% by weight based on the total protein weight of the extract, more preferably less than 1% by weight based on the total protein weight of the extract, and even more preferably less than 0.5% by weight based on the total protein weight of the extract.

[0152] Advantageously, the content of high molecular weight proteins is less than 15% by weight based on the total protein weight of the extract, preferably less than 10% by weight based on the total protein weight of the extract, more preferably less than 7.5% by weight based on the total protein weight of the extract, more preferably less than 5% by weight based on the total protein weight of the extract, preferably less than 2.5% by weight based on the total protein weight of the extract, more preferably less than 1% by weight based on the total protein weight of the extract, and even more preferably less than 0.5% by weight based on the total protein weight of the extract.

[0153] In one embodiment, the tobacco leaf extract for the present invention is substantially free of RuBisCO protein. Advantageously, the drying of step a promotes the degradation of high molecular weight tobacco proteins, such as RuBisCO, e.g., by a hydrolytic mechanism.

[0154] Preferably, the protein solution obtained in step e is subjected to sterile filtration in step e' prior to step f.

[0155] The tobacco leaf extract for the present invention obtained at the end of the above process can be stored as obtained at the end of step d' of sterile filtration or freeze-dried as obtained at the end of step f.

[0156] The tobacco leaf extract for the purposes of the present invention is advantageously an aqueous extract.

[0157] The tobacco leaf extract for the purposes of the present invention is advantageously a brown aqueous extract.

[0158] The present disclosure also relates to a pharmaceutical composition containing the concentrated tobacco leaf extract for the present invention, which contains the above-mentioned tobacco leaf extract as an active ingredient.

[0159] Pharmaceutical compositions for the present invention may further contain at least one pharmaceutically acceptable excipient such as a pharmaceutically acceptable solvent, eg water.

[0160] Advantageously, the pharmaceutical composition for the present invention may contain proteins present in tobacco leaf extract in a content 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.

[0161] The pharmaceutical composition for the present invention is preferably an aqueous composition.

[0162] According to one embodiment, the pharmaceutical composition of the present invention further contains an adjuvant. Examples of adjuvants include sugars such as lactose, sucrose, trehalose, sorbitol, glucose, raffinose, and mannitol (preferably lactose, sucrose, trehalose, glucose, or mannitol), amino acids such as arginine, glycine, and histidine (preferably glycine), and swelling agents such as dextran or polyethylene glycol-type polymers, or mixtures thereof. According to this embodiment, the pharmaceutical composition contains 50 to 99 wt. %, preferably 80 to 97 wt. %, of the adjuvant based on the total weight of the pharmaceutical composition.

[0163] According to certain embodiments, the pharmaceutical composition for the present invention may further contain proteins extracted from cannabis.

[0164] Preferably, the pharmaceutical composition can be manufactured by a process comprising the following steps: a1. Preparation of the tobacco leaf extract described above b1. Adjust the protein concentration to obtain a protein concentration of 100–200 μg / mL c1. Addition of pharmaceutically acceptable excipients, and d1. Optionally, adding an adjuvant to the extract (preferably adding mannitol)

[0165] The tobacco leaf extract used in step b1 may be obtained immediately after a diafiltration step using a 10 kDa cut-off membrane, or after a sterile diafiltration step, or may be a tobacco leaf extract that has been lyophilized and then reconstituted, for example in saline or water.

[0166] Advantageously, the concentration of step b1 can be adjusted in the following way: - Dilution with water to reduce protein concentration - Diafiltration to increase protein concentration

[0167] A particularly preferred pharmaceutically acceptable excipient for step c1 is water.

[0168] Adjuvants that can be added in step d1 can be, in particular, sugars such as lactose, sucrose, trehalose, sorbitol, glucose, raffinose, mannitol, etc. (preferably lactose, sucrose, trehalose, glucose, or mannitol), amino acids such as arginine, glycine, or histidine, (preferably glycine), swelling agents such as dextran or polyethylene glycol type polymers, or mixtures thereof.

[0169] According to a particular embodiment, step d1 comprises the addition of at least mannitol.

[0170] According to one embodiment, the pharmaceutical composition for the present invention is provided in a form suitable for subcutaneous administration.

[0171] According to one embodiment, the pharmaceutical composition of the present invention comprises an adhesive transdermal therapeutic system containing the tobacco leaf extract described above. According to this embodiment, the pharmaceutical composition of the present invention is provided in a form suitable for administration by the adhesive transdermal therapeutic system. Advantageously, the pharmaceutical composition of the present invention is provided in the form of a patch.

[0172] The patch may be in the form of a reservoir-type patch or a matrix-type patch having one or more compartments. The actual implementation of the patch will be determined by those skilled in the art based on their general knowledge of the subject, to achieve sustained and controlled systemic administration of the tobacco leaf extract throughout the application period of the patch, for example, for about 2 to 24 hours.

[0173] Reservoir-type patches contain one or more independent reservoirs containing active ingredients (including tobacco leaf extract) in solution or suspended in a polymer matrix, and are in contact with the skin via a semipermeable polymeric membrane that can control the release rate of the active ingredient.

[0174] Matrix-type patches contain polymeric materials in which active ingredients are dissolved or dispersed in suitable proportions, and these active ingredients are released by diffusing through the polymer chains of the matrix.

[0175] According to certain embodiments of this type of patch, the adhesive covers the entire release surface of the matrix and is an integral part of the matrix, thus making it an active adhesive type patch well known to those skilled in the art, which simplifies manufacturing and allows for the creation of a thin, reasonably flexible patch that can be comfortably applied to a patient's skin.

[0176] To effectively penetrate these active ingredients into the subcutaneous or bloodstream at an appropriate dosage, at an appropriate diffusion rate, and for an appropriate period of time, the following parameters can be readily determined by a person skilled in the art: - The ratio of the surface area to the volume of each section of the patch; - optional addition of one or more hydrophilic additives; - optional addition of one or more diffusion activators or inhibitors; - optional addition of one or more solubilizers; - optional addition of one or more stabilizers; - the optional addition of one or more absorption enhancers; and More generally, any type of additive familiar to those skilled in the art that allows good control of the flowability and stability of the tobacco leaf extract.

[0177] Those skilled in the art will know what techniques to use to set the above parameters based on the desired solubility and stability.

[0178] The various manufacturing parameters of the patch will be readily adapted by one skilled in the art to arrive at the desired dosage.

[0179] In a manner known per se, such patches are provided with a removable protective film for protecting the adhesive surface applied to the skin after production of the patch and throughout its storage period: in a manner known per se to those skilled in the art, a polyester film can be used, for example, one side of which can be treated with a release silicone.

[0180] According to one embodiment, the composition for the present invention is administered by subcutaneous injection. According to this embodiment, the pharmaceutical composition is provided in a form suitable for subcutaneous administration.

[0181] According to one embodiment, the pharmaceutical composition is provided in a dosage form of 0.03 mL to 10 mL, preferably 0.1 mL to 5 mL, preferably 0.5 mL to 2 mL. Thus, the pharmaceutical composition is administered by subcutaneous injection in a volume of preferably 0.03 mL to 10 mL, preferably 0.1 mL to 5 mL, preferably 0.5 mL to 2 mL.

[0182] The pharmaceutical composition may be provided to be administered by a single subcutaneous injection.

[0183] The pharmaceutical composition may be provided to be administered by subcutaneous injection several times, preferably two or three times.

[0184] In the latter case, the pharmaceutical composition is preferably administered by subcutaneous injection on days 0 and 10, or on days 0, 10 and 30.

[0185] The second and optionally third injected doses may be the same as the first injected dose. [Brief explanation of the drawings]

[0186] [Figure 1] FIG. 1 graphically depicts the reduction in smoking satisfaction with NFL-101 and placebo from the PRECESTO study (see Examples). [Figure 2]FIG. 2 graphically depicts the difference in reduction in smoking satisfaction between NFL-101 and placebo, as well as exploratory p-values, obtained in the PRECESTO study (see Examples). [Figure 3] FIG. 3 graphically depicts the results of a comparative study of reduced smoking satisfaction between Champix™ (ie, the commercially available alternative solution, varenicline) and NFL-101. [Example]

[0187] NFL-101 is an aqueous extract of tobacco leaves prepared according to Good Manufacturing Practice guidelines. This subcutaneous injection solution is available as a lyophilized powder (140 μg protein per vial) and is accompanied by a sterile diluent. To ensure product stability, vials are reconstituted in the laboratory on the day of injection.

[0188] The manufacturing process of NFL-101 is in accordance with the teachings of WO2017174787. Therefore, in order to complete the disclosure of the present invention, Example 1 of WO2017174787 is disclosed herein below for the purpose of reproducing the present invention.

[0189] <Example 1: Preparation of tobacco leaf extract according to the present invention (WO2017174787)> Burley tobacco leaves are used to prepare the extract.

[0190] 100 g of burley tobacco leaves that had been air-dried for approximately 3 months (September to December in central France) were ground. The ground leaves were suspended in 1,892 g of WFI with 8 g of ammonium bicarbonate at 4-10°C for 24 hours. The suspended solid residue was then removed by Buchner filtration.

[0191] The extract is then subjected to 0.2 μm clarification filtration. The resulting extract is brown in color. The extract is then weighed to determine the volume of WFI to be used in the constant volume diafiltration step. In this case, the mass of the liquid extract is 1680 g.

[0192] Next, proteins are extracted from the pulverized and dried tobacco leaf extract from which the solid residue has been removed: 10,080 g of WFI is added to the pulverized and dried tobacco leaf extract from which the solid residue has been removed. The 11,760 g of solution thus obtained is diafiltered against 6 volumes at a constant volume using a 10 kDa cut-off (Merck Millipore) until the mass of the retentate decreases to 1,680 g.

[0193] The retentate obtained after diafiltration is a protein mixture in which proteins with a molecular weight of less than 10 kDa are detected at a concentration 99% lower than their initial concentration. The color of the obtained retentate is between B2 and B3 according to the measurement scale defined in EUROPEAN PHARMACOPOEIA 5.0, 2.2.2, Liquid Color Degree.

[0194] This diafiltrate can be lyophilized using a SMH150 lyophilizer, for example after adding mannitol, to obtain a lyophilized tobacco leaf extract.

[0195] NFL-101 is an aqueous extract of tobacco leaves.

[0196] NFL-101 (the extract of Example 1) is provided in the form of one vial of 140 μg of sterile lyophilized powder and one vial of 5 mL of 0.9% sodium chloride.

[0197] The vial must be reconstituted with 1.4 mL or 2.8 mL of 0.9% sodium chloride to obtain a solution at a concentration of 50 μg / mL or 100 μg / mL. The reconstituted NFL-101 solution is pale yellowish-brown.

[0198] <Example 2: Clinical Development> <a. Phase I Clinical Trial (CESTO Trial)> A phase I study was conducted in 24 smoking subjects (mean Fagerstrom score: 6.25). The primary objective was to evaluate the safety of NFL-101. Secondary objectives were to evaluate the immunogenicity and efficacy of NFL-101 in these smoking subjects. This was a prospective, single-center, open-label, non-randomized phase I study conducted at the Institut Regional du Cancer (ICM) in Montpellier, France.

[0199] NFL-101 was administered subcutaneously twice, four weeks apart, to smoking subjects who wanted to quit smoking. Each subject received two injections (one in each arm) on days 1 and 29. The study investigated two protein levels (100 μg or 200 μg protein) to determine whether both dose levels were safe and which dose was most effective. Subjects were enrolled according to a 6 + 6 dose-escalation design. The first 12 subjects (six doses of 100 μg protein + six doses of 200 μg protein) received treatment several days after the recruitment visit. The final 12 subjects received a longer period of preparation before treatment administration, ensuring they were better psychologically prepared to quit smoking.

[0200] Dose-limiting toxicities (DLTs) in this study were defined as grade 3-4 toxicities according to the NCI-CTCAE V4.0 classification. No DLTs were observed in any of the subjects. Only one case of grade 2 toxicity (anxiety) was reported at dose level 2 (high dose) that was considered related or likely related to NFL-101 treatment. Other adverse events observed were the following grade 1 toxicities: n=20: injection site pain, deltoid muscle pain, deltoid muscle edema, bilateral pain (10 cases in dose 1 [low dose], 10 cases in dose 2 [high dose]), n=7: dry mouth (3 cases at dose 1, 4 cases at dose 2), n=6: fatigue (3 cases at dose 1, 3 cases at dose 2), n=5: nausea (1 case at dose 1, 4 cases at dose 2), n=4: dry eyes (3 cases at dose 1, 1 case at dose 2), n = 3: Insomnia (2 cases at dose 1 and 1 case at dose 2), n = 3: Deltoid erythema (1 case at dose 1 and 2 cases at dose 2), n = 2: Chills (1 case at dose 1 and 1 case at dose 2), n = 2: Irritability (1 case at dose 1 and 1 case at dose 2), n = 2: Headache (1 case at dose 1 and 1 case at dose 2), n = 2: Dysgeusia (2 cases at dose 1 and 0 cases at dose 2), n = 2: Restlessness (1 case at dose 1 and 1 case at dose 2), n = 1: Bradycardia, photosensitivity, diarrhea, itching of the tongue, gastroesophageal reflux, weight gain, calf muscle spasm, pain (distal part of deltoid), anxiety, depression, obvious rhinitis, nasal dryness, cough with phlegm, itching (wrists and feet), itching (nose).

[0201] During the test period, among 24 smokers, 67% reduced their cigarette consumption by at least 50% at the 4 - week mark and 63% at the 12 - week mark. Among 24 smokers, 33% had not smoked for at least the past 7 days at the 12 - week mark. Among 24 smokers, 21% maintained continuous smoking cessation at the 12 - week mark. Among the last 12 smokers who had better psychological preparation for the smoking cessation test, 80% reduced their cigarette consumption by at least 50%, and among them, 5 continued to smoke cessation continuously 8 weeks after the end of treatment (12 - week mark).

[0202] Immunological evaluation showed a significant increase in anti - NFL - 101 IgG (already present on day 1), and a correlation was shown between the higher value of anti - NFL - 101 IgG (after treatment administration) and the reduction in cigarette consumption, the 7 - day smoking cessation rate, and continuous smoking cessation.

[0203] All subjects spontaneously reported that NFL - 101 reduced the craving for smoking and the attractiveness of tobacco, but experienced typical withdrawal symptoms during the attempt to quit smoking.

[0204] <Conclusion of the CESTO test> People attempting to quit smoking typically experience urges to smoke (often described as cravings), often in response to environmental cues associated with smoking (e.g., alcohol). These urges decrease in intensity and frequency over time. The key to quitting smoking is resisting these urges. Effective smoking cessation medications reduce the intensity of urges, which is likely their mechanism of action.

[0205] There are three medications approved for smoking cessation: bupropion, varenicline, and nicotine replacement therapy (NRT). Bupropion has slightly more contraindications and more severe adverse effects than the other two medications. Therefore, when smokers use medications to help them quit, they usually use either varenicline or NRT.

[0206] Traditionally, people who quit smoking start NRT on the day they quit.

[0207] The most effective drug for helping people quit smoking is varenicline, and because varenicline is a nicotine partial agonist, it was surprising to find that it was more effective than nicotine, a full agonist.

[0208] One study, which examined possible mechanisms of action, compared varenicline, bupropion, and placebo and found that varenicline reduced smoking urges to a lower level than bupropion.

[0209] Furthermore, varenicline reduces the satisfaction of relapse (smoking episodes) after quitting smoking more than bupropion, but many other mood-related symptoms associated with withdrawal are of similar intensity. This suggests that the primary mechanism of action of smoking cessation pharmacotherapies, directly related to efficacy, is smoking urge control and reduced smoking satisfaction.

[0210] Unlike NRT, varenicline is used starting 1-2 weeks before the quit date, which may explain its superior effectiveness. In clinical trials, nicotine preloading (i.e., use of NRT before quitting) has been studied for its ability to increase quit rates when implemented in smokers who continue to smoke the same amount or smoke freely.

[0211] In a meta-analysis of nicotine preloading for smoking cessation that included 8 related studies (2,813 participants), a weak non-significant effect of nicotine preloading on smoking cessation was concluded. In another study of 1,792 smokers, use of nicotine patch preloading before attempting to quit was concluded to be able to increase the proportion of people who succeed in quitting, but the benefit is impaired because it reduces the use of varenicline after preloading.

[0212] The question to be answered is whether using NFL-101 before quitting can significantly reduce the satisfaction with smoking.

[0213] <b.Combination of NFL-101 Preconditioning and NRT Treatment> NRT is one of two drugs preferably used for smoking cessation. Currently, it is not used as preconditioning before the quit date, and its effectiveness as preconditioning has not been clearly shown. Therefore, it is interesting to examine whether smoking cessation can be improved by using NFL-101 as preconditioning before the quit date and starting NRT.

[0214] By understanding the onset of tobacco dependence, it may be possible to hypothesize the potential mechanisms of NFL-101 preconditioning and subsequent NRT treatment.

[0215] Smoking generates positive rewards such as feelings of pleasure, which lead to the reinforcement of subsequent smoking. After regular smoking for a certain period, as the brain adapts to the regular administration of nicotine and the nicotine concentration in the brain decreases, smokers experience negative moods. These feelings are alleviated by smoking, and thus smoking undergoes negative reinforcement.

[0216] Irrespective of the rewards a person may experience, the mechanisms in the nucleus accumbens build associative learning that gives rise to the impulse to smoke. These mechanisms produce the acquired impulse to smoke and tobacco dependence. This manifests as the regular consumption of tobacco, the withdrawal symptoms that occur when smoking is not possible, and the difficulty of quitting smoking when one chooses to stop. The net effect of the change in the impulse to smoke means not smoking when one would normally smoke, usually when receiving cues to smoke from the environment. Not smoking when receiving cues to smoke begins to erase the learned association between smoking behavior and reinforcement in the brain.

[0217] Apart from the mechanism based on this dependence, three other mediating hypotheses can be presented regarding the mechanism of action of NFL-101 in preconditioning.

[0218] The first hypothesis is that preconditioning may improve medication adherence after quitting smoking.

[0219] The second hypothesis is that the reduction in the amount of smoking that occurs upon administration of NFL-101 may increase confidence in achieving smoking cessation after the quit date. Confidence in achieving smoking cessation is associated with achieving smoking cessation.

[0220] The third hypothesis is that the aversion reaction to tobacco after administration of NFL-101 may increase the smoking cessation rate. Aversion smoking is the process of smoking excessively until nausea and vomiting are induced. Aversion smoking is rarely used as a treatment to promote smoking cessation, but it is effective in increasing the smoking cessation rate.

[0221] <c.PRECESTO test> <c.1. Historical Background> Therefore, to evaluate the effect of NFL-101, which reduces the reward of smoking, compared with placebo, it was proposed to conduct a Phase IIa trial (so-called PRECESTO trial). A crossover design was used with 34 freely smoking patients, and the "mCEQ" questionnaire was used as the primary evaluation criterion. The subjects were informed that they would be administered both the active drug (200 μg of NFL-101) and the placebo test drugs, but during the first and second periods, which test drug, NFL-101 or placebo, was administered remained blinded. By setting a period of 4 weeks, it became possible to clearly observe the change in the effect over time. A washout period of more than 30 days was considered long enough to avoid residual effects at the start of the second period.

[0222] The primary evaluation item should have been to succeed in achieving a 1-point decrease in the "smoking satisfaction" subscale of mCEQ measured on the 4th day (D4) after the first administration of NFL-101. mCEQ should include all of the above 12 questions and be on a 7-point scale.

[0223] The secondary criteria to be evaluated should have been to achieve the following 1-point deductions: - The subscale of "smoking satisfaction" of mCEQ measured on D7, D14, D21 and D28, and / or - The subscales of "psychological reward", "aversion", "pleasure of respiratory sensation" and "relief of craving" of mCEQ measured on D4, D7, D14, D21 and D28.

[0224] In addition to this, other questions should have been presented to the subjects to evaluate the effectiveness of the treatment: - At the time points of D4, D7, D14, D21 and D28, "Was the craving to smoke in the past 24 hours stronger or weaker than usual?" (Answer options were "much stronger", "slightly stronger", "the same as before", "slightly weaker" and "much weaker"). - At D4, D7, D14, D21, and D28, "Was your enjoyment of tobacco in the past 24 hours greater or less than usual?" (Answer options were "Much more enjoyable", "Somewhat more enjoyable", "The same as before", "Somewhat less enjoyable", and "Much less enjoyable"). - What was your motivation to quit smoking scale? (From 01 to 10. 10 represents a strong desire to quit smoking.) - How many cigarettes did you smoke in the past 24 hours? Furthermore, it should have been possible to evaluate the criteria due to nicotine withdrawal (depression, insomnia, irritability / frustration / anger, anxiety, difficulty concentrating, restlessness, increased appetite / weight gain, constipation, bradycardia...). These were the milestones initially planned in the completion of the CESTO study.

[0225] <c.2. Conditions and implementation of the study conducted> Specifically, PRECESTO was the second study following CESTO and clarified the effect of NFL-101 on reducing smoking satisfaction. The clinically meaningful effect lasted longer than expected.

[0226] These results confirm the therapeutic interest of NFL-101, administered alone or in combination with nicotine replacement therapy, in reducing withdrawal symptoms. Therefore, they support the results of the previous CESTO study.

[0227] <Summary: What is PRECESTO?> PRECESTO was a single-center, placebo-controlled, randomized, double-blind, exploratory Phase 2a study with two 28-day periods, each with a crossover design, in 34 smokers who did not wish to quit and had high smoking satisfaction. Each participant served as their own control and received NFL-101 or placebo at the beginning of each of the two periods, randomly and alternately. At the end of the study, all participants received one dose of NFL-101 and one dose of placebo. The administration order of NFL-101 and placebo for each participant was kept confidential. In each period, participants received treatment on Day 1 (D1) and then independently completed the modified Cigarette Smoking Assessment Questionnaire (mCEQ) on Days 4 (D4), 7 (D7), 14 (D14), 21 (D21), and 28 (D28).

[0228] The objective of PRECESTO was "to evaluate the effectiveness of NFL-101 compared with placebo in reducing the positive reinforcement of tobacco as measured by the smoking satisfaction subscale of the mCEQ (questions 1, 2, and 12)." The primary endpoint resulting from this objective was selected as "the smoking satisfaction subscale of the mCEQ measured on D4 (items 1, 2, and 12)."

[0229] The mCEQ: a leading international questionnaire for measuring the effects of smoking The mCEQ is an independently completed international questionnaire. Study participants, who are smokers with high smoking satisfaction and no desire to quit, answer 12 questions by marking the number that best describes how smoking makes them feel (1-not at all, 2-very little, 3-a little, 4-moderately, 5-quite a bit, 6-very much, 7-extremely). The questions are as follows: 1. Was smoking satisfying? 2. Did the tobacco taste good? 3. Did you enjoy the sensations in your throat and chest? 4. Did smoking calm you down? 5. Did smoking help you wake up better? 6. Did smoking make you less irritable? 7. Did smoking help you concentrate? 8. Does smoking reduce appetite? 9. Did smoking cause dizziness? 10. Did smoking cause nausea? 11. Did smoking immediately relieve the craving for tobacco? 12. Did you enjoy smoking? This questionnaire uses three multiple-item subscales and two single items: "smoking satisfaction" (items 1, 2, and 12); "psychological reward" (items 4 - 8); "aversion" (items 9 and 10); "enjoyment of respiratory sensations" (item 3), and "relief of craving" (item 11). The score for each subscale is calculated as the average of the responses to the individual items or as a single item. Thus, each score can vary from 1 to 7. A higher score indicates a greater intensity on that scale.

[0230] Participants in the PRECESTO trial completed this questionnaire independently and remotely using a computer. To standardize, as much as possible, the changes in each subject's perception over time, they were asked to refer to the tobacco smoked after dinner the previous day.

[0231] <Reasons for conducting the PRECESTO trial> The PRECESTO trial can provide a deeper understanding of the activity of NFL - 101, particularly the effectiveness of this product in reducing smoking satisfaction.

[0232] In smoking cessation, it is essential to reduce smoking satisfaction. To successfully quit smoking, it is important to address both smoking satisfaction and nicotine withdrawal. Regarding withdrawal, there is nicotine replacement therapy, but there is no effective drug without side effects for addressing satisfaction.

[0233] <c.3 Results> <PRECESTO Phase 2a Exploratory Clinical Trial> It was previously verified that statistical analysis could be performed jointly across all subjects and the two sequences (carryover effect: p > 0.10). Data were processed by calculating least-squares means according to the PRECESTO protocol, taking into account the crossover design of the study.

[0234] Smoking satisfaction was the average of responses to questionnaire questions 1, 2, and 12. The mean smoking satisfaction score at enrollment was 6.12 (standard deviation 0.77).

[0235] The PRECESTO results are as follows: Regarding the reduction in smoking satisfaction with NFL-101: Observations from the CESTO Phase 1 clinical trial predicted a rapid effect, reaching a peak level on D4 and then dissipating over the next 10 days. The effect observed in PRECESTO was different (Figure 1): there was an effect on D4 (mean reduction -0.810; CI (-1.139; -0.481)), and this effect strengthened relatively linearly until the final measurement on D28 (mean reduction -1.261; CI (-1.590; -0.932)). The mean level of smoking satisfaction, which was between "quite a bit" and "extremely" at enrollment, decreased to "moderately" and "quite a bit" by D28, representing a clinically meaningful reduction.

[0236] Regarding the effect of NFL-101 compared to placebo and the reduction in smoking satisfaction: The placebo effect is significant but lower than the effect of NFL-101 throughout the entire observation period (Figure 1, bottom). The increase slows from D7 and declines from D21 to D28 (mean decrease -0.947; CI (-1.277; -0.618)). The difference between the effects of NFL-101 and placebo (Figure 2) did not reach significance (> 0.05) at D4, with exploratory p-values ​​of 0.0003 (mean difference -0.188; CI (-0.289, -0.087)) for the entire observation period from D4 to D28 and 0.007 (mean difference -0.314; CI (-0.540, -0.088)) at D28.

[0237] Regarding specific effects on other mCEQ subscales and items for secondary outcomes: Exploratory p-values ​​for these outcomes were not calculated. Study limitations detailed below may explain this, particularly for the craving outcome.

[0238] Figure 1 graphically illustrates the reduction in smoking satisfaction with NGL-101 and placebo. Smoking satisfaction (vertical axis) is the least squares mean (LSM) change from screening (pooled Period 1 and Period 2) with 95% CI from MMRM. The graph clearly shows that while placebo has an effect on smoking satisfaction, NGL-101 (active ingredient) has a greater effect on reducing smoking satisfaction compared to placebo.

[0239] Figure 2 graphically illustrates the difference in reduction in smoking satisfaction with NFL-101 versus placebo, including exploratory p-values. Smoking satisfaction (vertical axis) is the placebo-adjusted least squares mean (LSM) change from screening (pooled Period 1 and Period 2), with 95% CI and exploratory p-values ​​from the MMRM including period, sequence, all visits, and gender and screening values ​​as covariates. This graph demonstrates a significant reduction in smoking satisfaction over the 28-day period, even after accounting for the placebo effect.

[0240] <Comparison with varenicline (Chantix / Champix (trademark))> To better understand the therapeutic potential of NFL-101, it is interesting to compare the reduction in smoking satisfaction obtained with PRECESTO with that obtained with varenicline under pre-treatment conditions, i.e., before attempting to quit smoking (Bohadana A, Freier-Dror Y, Peles V, Babai P, Izbicki G. Extending varenicline preloading to 6 weeks facilitates smoking cessation: A single-site, randomised controlled trial. EClinicalMedicine. 2020 Feb 3;19:100228. doi: 10.1016 / j.eclinm.2019.11.021. PMID: 32055787; PMCID: PMC7005428). Over a 4-week period, a single dose of NFL-101 reduced smoking satisfaction more than 53 doses of varenicline, as shown in Figure 3. Thus, in Figure 3, the reduction in smoking satisfaction during the pre-treatment period comparing NFL to placebo is directly compared to that when comparing varenicline (Champix™) to placebo, based on the PRECESTO clinical trial and the literature cited above in this specification (Bohadana A et al.). The Champix™ vs placebo results show a difference of 0.3 (p = 0.04) based on 53 doses of Champix™. The NFL-101™ vs placebo results show a difference of 0.36 (p = 0.007) under the above conditions. The reduction in smoking satisfaction from baseline is greater for NFL-101 (-1.26) than for Champix™ (-0.9).

[0241] <Explanation regarding p-value> For p-values of 0.0003 and 0.007, it means that the probability of obtaining data as different as or more different than the observed data, when there is no actual effect or difference in the data, is only 0.03% and 0.7% respectively.

[0242] The exploratory p-value calculations performed in the post hoc analysis were limited to the primary objectives of the trial. This approach reduces the risk of obtaining spuriously low p-values that can occur when calculating p-values for multiple evaluation items or when relating evaluation items to each other. These calculations allow for adaptation when unexpectedly long-term effects are observed while respecting the spirit of the protocol and its primary objectives.

[0243] <Limitations of the PRECESTO Trial> In the PRECESTO trial, the mCEQ was completed by smokers who did not desire to quit smoking and smoked freely (''as they wished''). Reducing tobacco consumption or quitting smoking is known to have a major impact on the cognitive and behavioral responses associated with tobacco consumption. In a crossover design, it was not possible to include subjects who attempted to reduce consumption or quit smoking. This is because it led to an overly large carry-over effect of the effect from period 1 to period 2. Furthermore, recruiting subjects who desired to quit smoking carried the risk of information loss if they did quit. This is because subjects who quit smoking can no longer be evaluated for their level of smoking satisfaction and thus cannot complete the questionnaire. Recruiting smokers who did not desire to quit smoking made it more difficult to show the effect of NFL-101 on certain evaluation items such as cravings that regularly decreased with the tobacco smoked without restriction.

[0244] <c.4. Conclusion: The First Placebo-Controlled Clinical Trial with Promising Results> · This exploratory trial showed that a single dose of NFL-101 decreased smoking satisfaction in a clinically meaningful way and with an unexpectedly long-term effect. This confirms the therapeutic interest in administering NFL-101 alone or in combination with nicotine replacement therapy to reduce withdrawal symptoms.

[0245] This exploratory study is the second clinical trial following CESTO, which demonstrated efficacy in reducing smoking satisfaction, and this time it was placebo-controlled, strengthening the level of evidence for NFL-101's efficacy and supporting the aforementioned clinical results (CESTO study).

Claims

1. 1. A method for preconditioning a subject to quit smoking, comprising: A method comprising the step of administering to a subject an effective dose of an aqueous solution of a protein-enriched tobacco leaf extract that is substantially free of nicotine transdermally or subcutaneously while the subject continues to smoke ad libitum.

2. 10. The method of claim 1, wherein the subject is administered an effective dose of a second aqueous solution of protein-enriched tobacco leaf extract substantially free of nicotine transdermally or subcutaneously while the subject continues to smoke ad libitum.

3. 3. The method of claim 2, wherein an effective dose of a second aqueous solution of protein-enriched tobacco leaf extract substantially free of nicotine is administered within 2 to 15 days, preferably within 5 days, of the first administration of claim 1.

4. The tobacco leaf extract contains at least 5% by weight of proteins having a molecular weight greater than 10 kDa, based on the total weight of the dry extract, and is substantially free of molecules having a molecular weight less than 10 kDa and RuBisCO protein; and 4. The method of claim 1, wherein the content of proteins with a molecular weight of more than 500 kDa in the tobacco leaf extract is less than 15% by weight, based on the total protein weight of the dried extract.

5. 5. The method of claim 4, wherein the protein is selected from the group consisting of the following protein families: Lignin-forming anionic peroxidases, glucan endo-1,3-beta-glucosidases, endochitinases, disease-related proteins, osmotin, protease inhibitors, and mixtures thereof.

6. 6. The method of claim 1, wherein the content of proteins with a molecular weight of more than 100 kDa in the tobacco leaf extract is less than 15% by weight based on the total protein weight of the dried extract.

7. 7. The method of treatment according to any one of claims 1 to 6, wherein the tobacco leaf extract comprises at least one protein belonging to the family of glucan endo-1,3-beta-glucosidases selected from the group consisting of beta-1,3-endoglucanase acidic isoform PR-Q' (PR36401 according to UniProt database), beta-1,3-endoglucanase basic vacuolar isoform GLB (P27666 according to UniProt database), and mixtures thereof.

8. 8. The method of treatment according to any one of claims 1 to 7, wherein the tobacco leaf extract comprises at least one protein belonging to the family of endochitinases selected from the group consisting of acidic endochitinase P (P17513 according to UniProt database), acidic endochitinase Q (P17514 according to UniProt database), endochitinase B (P24091 according to UniProt database), and mixtures thereof.

9. 9. The method of treatment according to any one of claims 1 to 8, wherein the tobacco leaf extract contains at least osmotin (P14170 according to the UniProt database).

10. 10. The method of treatment according to any one of claims 1 to 9, wherein the tobacco leaf extract comprises at least one lignin-forming anionic peroxidase (P11965 according to the UniProt database).

11. 11. The method of any one of claims 1 to 10, wherein the tobacco leaf extract comprises at least one disease-related protein selected from the group consisting of disease-related protein R (P13046 according to UniProt database), disease-related protein PR-4A (PR29062 according to UniProt database), disease-related protein PR-4B (PR29063 according to UniProt database), and mixtures thereof.

12. 12. The method of treatment according to any one of claims 1 to 11, wherein the tobacco leaf extract comprises at least one protein belonging to the family of protease inhibitors selected from the group consisting of protease inhibitor I-B (Q03199 according to UniProt database), protease inhibitor IA (Q03198 according to UniProt database), and mixtures thereof.

13. 13. The method of treatment according to any one of claims 1 to 12, wherein the protein content in the dry matter of the tobacco leaf extract is at least 10% by weight, based on the total weight of the dry matter.

14. 14. The method of treatment according to any one of claims 1 to 13, wherein the proteins present in the tobacco leaf extract are present in an amount ranging from 1 to 1000 μg / mL.

15. 15. The method of treatment according to any one of claims 1 to 14, wherein the tobacco leaf extract or composition is provided in a form suitable for administration by subcutaneous injection, by an adhesive transdermal therapeutic system such as a patch, or by administration by spray or vaporization.

16. 16. The method of treatment according to any one of claims 1 to 15, wherein the tobacco leaf extract or composition is provided in a dosage form of 0.03 mL to 10 mL.

17. 1. A method for treating smoking cessation in a subject, comprising the following two consecutive steps: i) a preconditioning step of administering to the subject an effective dose of at least one aqueous solution of a protein-enriched tobacco leaf extract that is substantially free of nicotine, transdermally or subcutaneously, while the subject continues to smoke freely; and ii) a second step after preconditioning step i), in which the subject completely quits smoking with one or several optional complementary administrations of at least one product capable of controlling nicotine withdrawal symptoms within 2 to 180 days of said administrations administered to the subject during preconditioning step i).

18. 18. The method of claim 17, wherein the preconditioning step comprises two or three transdermal administrations of effective doses of two or three aqueous solutions of protein-enriched tobacco leaf extracts substantially free of nicotine while the subject continues to smoke freely.

19. 19. The method of treatment according to claim 18, wherein the two or three transdermal administrations of effective doses of two or three aqueous solutions of protein-enriched tobacco leaf extracts substantially free of nicotine are spaced at least 10 days apart, preferably at least 20 days apart, from each other while the subject continues to smoke freely.

20. 18. The method of claim 17, wherein the preconditioning step comprises a single transdermal administration of an effective dose of an aqueous solution of protein-enriched tobacco leaf extract substantially free of nicotine while the subject continues to smoke freely.

21. 21. A method of treatment according to any one of claims 17 to 20, wherein in a second step after preconditioning step i), the subject completely quits smoking by one or several optional complementary administrations of at least one product capable of controlling nicotine withdrawal symptoms within 2 to 30 days of the administrations carried out to the subject during preconditioning step i).

22. 22. The method of treatment according to any one of claims 17 to 21, wherein said one or several optional complementary administrations of at least one product capable of controlling nicotine withdrawal symptoms are carried out in an accompanying manner to gradually withdraw the subject from nicotine.

23. 23. A method of treatment according to any one of claims 17 to 22, wherein if several products capable of controlling nicotine withdrawal symptoms are administered, they are administered temporally independently of each other.

24. 24. The method of treatment according to claim 22 or 23, wherein the at least one product capable of controlling nicotine withdrawal symptoms is selected from a nicotine agonist or partial agonist, a nicotine replacement, or a combination thereof.

25. 25. The method of treatment of any one of claims 17 to 24, wherein the nicotinic agonist or partial agonist is selected from varenicline, cytisine, lobeline, or a combination thereof.

26. 26. The method of treatment according to any one of claims 17 to 25, wherein the second step is carried out within 3 to 10 days of the administration carried out to the subject during preconditioning step i).

27. 27. The method of treatment according to any one of claims 17 to 26, wherein the second step is carried out within 3 to 4 days of the administration carried out to the subject during preconditioning step i).

28. The tobacco leaf extract contains at least 5% by weight of proteins having a molecular weight greater than 10 kDa, based on the total weight of the dry extract, and is substantially free of molecules having a molecular weight less than 10 kDa and RuBisCO protein; and 28. The method of treatment according to any one of claims 17 to 27, wherein the content of proteins with a molecular weight of more than 500 kDa in the tobacco leaf extract is less than 15% by weight based on the total protein weight of the dried extract.

29. 29. The method of treatment of any one of claims 17 to 28, wherein the protein is selected from the group consisting of the following protein families: Lignin-forming anionic peroxidases, glucan endo-1,3-beta-glucosidases, endochitinases, disease-related proteins, osmotin, protease inhibitors, and mixtures thereof.

30. 30. The method of treatment according to any one of claims 17 to 29, wherein the content of proteins with a molecular weight of more than 100 kDa in the tobacco leaf extract is less than 15% by weight based on the total protein weight of the dried extract.

31. 31. The method of treatment according to any one of claims 17 to 30, wherein the tobacco leaf extract comprises at least one protein belonging to the family of glucan endo-1,3-beta-glucosidases selected from the group consisting of beta-1,3-endoglucanase acidic isoform PR-Q' (PR36401 according to UniProt database), beta-1,3-endoglucanase basic vacuolar isoform GLB (P27666 according to UniProt database), and mixtures thereof.

32. 32. The method of treatment according to any one of claims 17 to 31, wherein the tobacco leaf extract comprises at least one protein belonging to the family of endochitinases selected from the group consisting of acidic endochitinase P (P17513 according to UniProt database), acidic endochitinase Q (P17514 according to UniProt database), endochitinase B (P24091 according to UniProt database), and mixtures thereof.

33. 33. The method of treatment according to any one of claims 17 to 32, wherein the tobacco leaf extract contains at least osmotin (P14170 according to the UniProt database).

34. 34. The method of treatment according to any one of claims 17 to 33, wherein the tobacco leaf extract comprises at least one lignin-forming anionic peroxidase (P11965 according to the UniProt database).

35. 35. The method of any one of claims 17 to 34, wherein the tobacco leaf extract comprises at least one disease-related protein selected from the group consisting of disease-related protein R (P13046 according to UniProt database), disease-related protein PR-4A (PR29062 according to UniProt database), disease-related protein PR-4B (PR29063 according to UniProt database), and mixtures thereof.

36. 36. The method of treatment according to any one of claims 17 to 35, wherein the tobacco leaf extract comprises at least one protein belonging to a family of protease inhibitors selected from the group consisting of protease inhibitor I-B (Q03199 according to UniProt database), protease inhibitor IA (Q03198 according to UniProt database), and mixtures thereof.

37. 37. The method of treatment according to any one of claims 17 to 36, wherein the protein content in the dry matter of the tobacco leaf extract is at least 10% by weight, based on the total weight of the dry matter.

38. 38. The method of treatment according to any one of claims 17 to 37, wherein the protein present in the tobacco leaf extract is present in an amount ranging from 1 to 1000 μg / mL.

39. 39. The method of treatment of any one of claims 17 to 38, wherein the tobacco leaf extract or composition is provided in a form suitable for administration by subcutaneous injection, by an adhesive transdermal therapeutic system such as a patch, or by administration by spray or vaporization.

40. 40. The method of treatment according to any one of claims 17 to 39, wherein the tobacco leaf extract or composition is provided in a dosage form of 0.03 mL to 10 mL.

41. Kit includes: - an aqueous solution of protein-enriched tobacco leaf extract, and separately - At least one product capable of controlling nicotine withdrawal symptoms.

42. 42. The kit of claim 41, wherein the aqueous solution of protein-enriched tobacco leaf extract is suitable for transdermal or subcutaneous administration in an effective dose.

43. 43. The kit of claim 41 or 42, wherein the at least one product capable of controlling nicotine withdrawal symptoms is selected from a nicotine agonist or partial agonist, a nicotine replacement, or a combination thereof.

44. 44. The kit of claim 43, wherein the nicotinic agonist or partial agonist is selected from varenicline, cytisine, lobeline, or a combination thereof.

45. The tobacco leaf extract contains at least 5% by weight of proteins having a molecular weight greater than 10 kDa, based on the total weight of the dry extract, and is substantially free of molecules having a molecular weight less than 10 kDa and RuBisCO protein; and 45. The kit of any one of claims 41 to 44, wherein the content of proteins with a molecular weight of more than 500 kDa in the tobacco leaf extract is less than 15% by weight based on the total protein weight of the dried extract.

46. 46. ​​The kit of any one of claims 41 to 45, wherein the protein is selected from the group consisting of the following protein families: Lignin-forming anionic peroxidases, glucan endo-1,3-beta-glucosidases, endochitinases, disease-related proteins, osmotin, protease inhibitors, and mixtures thereof.

47. 47. The kit of any one of claims 41 to 46, wherein the content of proteins with a molecular weight of more than 100 kDa in the tobacco leaf extract is less than 15% by weight based on the total protein weight of the dried extract.

48. 48. The kit of any one of claims 41 to 47, wherein the tobacco leaf extract comprises at least one protein belonging to the family of glucan endo-1,3-beta-glucosidases selected from the group consisting of beta-1,3-endoglucanase acidic isoform PR-Q' (PR36401 according to UniProt database), beta-1,3-endoglucanase basic vacuolar isoform GLB (P27666 according to UniProt database), and mixtures thereof.

49. 49. The kit of any one of claims 41 to 48, wherein the tobacco leaf extract comprises at least one protein belonging to the family of endochitinases selected from the group consisting of acidic endochitinase P (P17513 according to UniProt database), acidic endochitinase Q (P17514 according to UniProt database), endochitinase B (P24091 according to UniProt database), and mixtures thereof.

50. 50. The kit of any one of claims 41 to 49, wherein the tobacco leaf extract comprises at least osmotin (P14170 according to the UniProt database).

51. 51. The kit of any one of claims 41 to 50, wherein the tobacco leaf extract comprises at least one lignin-forming anionic peroxidase (P11965 according to the UniProt database).

52. 52. The kit of any one of claims 41 to 51, wherein the tobacco leaf extract comprises at least one disease-related protein selected from the group consisting of disease-related protein R (P13046 according to UniProt database), disease-related protein PR-4A (PR29062 according to UniProt database), disease-related protein PR-4B (PR29063 according to UniProt database), and mixtures thereof.

53. 53. The kit of any one of claims 41 to 52, wherein the tobacco leaf extract comprises at least one protein belonging to a family of protease inhibitors selected from the group consisting of protease inhibitor I-B (Q03199 according to UniProt database), protease inhibitor IA (Q03198 according to UniProt database), and mixtures thereof.

55. 55. The kit of any one of claims 41 to 54, wherein the protein present in the tobacco leaf extract is present in an amount ranging from 1 to 1000 μg / mL.

56. 56. The kit of any one of claims 41 to 55, wherein the tobacco leaf extract or composition is provided in a form suitable for administration by subcutaneous injection, by an adhesive transdermal therapeutic system such as a patch, or by administration by spray or vaporization.

57. 57. The kit of any one of claims 41 to 56, wherein the tobacco leaf extract or composition is provided in a dosage form of 0.03 mL to 10 mL.

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