Therapeutic method for assisting smoking cessation by using tobacco leaf extract

Abstract

The present disclosure is generally directed to a method of treatment to aid in smoking cessation of a subject, comprising: at least one step during which an effective dose of an aqueous solution of a protein-rich tobacco extract is administered to the subject during which the subject continues to freely smoke; and optionally a second step during which at least one product capable of controlling the symptom of nicotine withdrawal, such as a nicotine agonist or a nicotine partial agonist, and / or a nicotine substitute, is administered to the subject optionally in a supplemental manner once or several times. The present disclosure also relates to a kit comprising a protein-rich tobacco extract and at least one product capable of controlling nicotine withdrawal symptoms of nicotine, such as a nicotine agonist or a nicotine partial agonist and / or a nicotine substitute.

Description

[0001] background

[0002] The present disclosure generally relates to methods of treating a subject for quitting smoking, the methods comprising:

[0003] at least one step during which an effective dose of an aqueous solution of a protein-rich tobacco leaf extract is administered to the subject, during which the subject continues to smoke freely; and optionally a second step during which the subject is provided with one or more optional supplemental administrations of at least one product capable of controlling nicotine withdrawal symptoms, such as a nicotine agonist or a nicotine partial agonist, and

[0004] The present disclosure also relates to a kit comprising a protein-rich tobacco leaf extract and at least one product capable of controlling nicotine withdrawal symptoms, such as a nicotine agonist or a nicotine partial agonist and / or a nicotine substitute.

[0005] Tobacco addiction is one of the greatest public health threats the world has ever faced, killing nearly 8 million people each year. There are over 1 billion smokers worldwide, and up to half will eventually die from tobacco-related diseases. Approximately every six seconds, tobacco kills one person, accounting for one in ten adult deaths. Tobacco killed 100 million people in the 20th century. If current trends continue, it could kill 1 billion people in the 21st century.

[0006] According to India's National Tobacco Control Program, the tobacco epidemic is one of the major public health threats, killing one million people in India every year.

[0007] According to the Global Adult Tobacco Survey-2 (GATS-2), in India, one in ten adults (100 million) smokes and 28.6% of adults aged 15 years and above (267 million) smoke in any form; 199 million adults in rural areas smoke and 68 million adults in urban areas smoke.

[0008] There are more than 4,000 chemicals in tobacco smoke, at least 250 of which are known to be harmful.

[0009] Over 50 types of tobacco are known to cause cancer. Tobacco is a risk factor for six of the eight leading causes of death. It can cause cancer almost anywhere in the body. It is estimated to cause over 90% of lung cancer cases in men, about 70% of lung cancer cases in women, and about 22% of all cardiovascular disease in industrialized countries. Approximately 90% of deaths from chronic obstructive pulmonary disease are caused by smoking. It harms nearly every organ in the body and affects a person's overall health.

[0010] Specifically, nicotine is hypothesized to be involved in the mechanisms of tobacco dependence during smoking addiction. Therefore, until now, nicotine has been considered the primary psychoactive and addictive substance. Nicotine can indeed bind to proteins on the surface of nerve cells, known as nicotinic acetylcholine receptors. In the presence of nicotine, these receptors (actually, channels) open. Subsequently, a series of events unfolds, leading to the release of the hormone dopamine. Nicotine stimulates the "reward circuit," producing a feeling of satisfaction. Therefore, when people quit smoking, their bodies "crave" a certain dose of nicotine to satisfy this feeling of well-being: these are the symptoms of nicotine withdrawal. For example, nicotine replacement products aim to provide the smoker's brain with a sufficient amount of nicotine to prevent withdrawal symptoms. Generally speaking, nicotine targets specific brain receptors, causing the central nervous system to release neurotransmitters such as dopamine, which in turn produce pleasant sensations such as pleasure, relaxation, and appetite suppression. Over time, these receptors become conditioned to anticipate nicotine, and when the nicotine supply is interrupted, smokers experience withdrawal symptoms.

[0011] Smoking also reduces levels of monoamine oxidase (MAO), an enzyme responsible for the breakdown of dopamine, leading to elevated dopamine levels and tobacco dependence. Since nicotine itself does not interact with MAO, this change may be due to other tobacco smoke components, another contributing factor to tobacco addiction. These components also disrupt the coupling between noradrenergic and serotonergic systems through desensitization of 5-HT(1A) autoreceptors, increasing the responsiveness of dopaminergic neurons. A recent study suggests that glutamatergic and gamma-aminobutyric acid (GABAergic) receptors in the medial prefrontal cortex (mPFC) may also be involved through GABAergic plasticity mechanisms in the dorsal mPFC. Several other publications have mentioned multiple mechanisms involved in tobacco addiction. Smoking behavior also varies depending on genetic and environmental factors. Therefore, the mechanisms of tobacco addiction are very complex, involving several pathways in the central nervous system and other molecules in addition to nicotine.

[0012] The World Health Organization (WHO) defines dependence or addiction as "a syndrome in which the need for a substance overrides the need for other, previously more important behaviors. In extreme forms, the state of dependence is characterized by an irresistible need for the substance, compelling the afflicted individual to seek the substance impulsively."

[0013] For all of these reasons and / or others, many smokers therefore seek to quit smoking.

[0014] Smokers can choose to quit smoking suddenly in one step by setting a quit date (abrupt quitting) or by reducing their smoking intake before the quit date (stepwise quitting). In the case of stepwise quitting, a 10-day to 2-week reduction phase is recommended before the quit date to avoid changing motivation.

[0015] Abstinence during the first two weeks after the target quit date (TQD) was significantly associated with abstinence at six months. Smokers who did not relapse after the TQD had an increased probability of becoming long-term abstainers.

[0016] Current medications for smoking cessation include nicotine replacement therapy (NRT), nicotine-free smoking cessation medication, and bupropion hydrochloride. and varenicline tartrate (French or American ). Several nicotine vaccines are in development.

[0017] Nicotine replacement therapy (NRT) was the first pharmacological treatment approved for smoking cessation. It is typically used in combination with behavioral support. NRT includes nicotine gum, transdermal patches, tablets, nasal sprays, and inhalation systems that act as nicotine substitutes to reduce withdrawal symptoms and the urge to smoke. A 2012 Cochrane Tobacco Addiction Group meta-analysis of 117 trials showed that all commercial NRTs can help people increase their chances of successfully quitting smoking by 50-70% within six months. However, absolute abstinence rates with these medications are low, and most smokers relapse: while the six-month abstinence rate for smokers using NRT is 20-30%, 70-80% of smokers who use these products relapse. NRT can be used for both abrupt and gradual smoking cessation. For abrupt cessation, NRT is used within eight to twelve weeks after quitting smoking completely. In a gradual smoking cessation approach, NRT can be used for six weeks to six months before TQD to help smokers reduce their smoking and continued for eight to twelve weeks from the start of TQD.

[0018] GlaxoSmithKline sells Bupropion (bupropion hydrochloride) was the first nicotine-free smoking cessation medication approved for marketing in 1997. According to French guidelines, bupropion is superior to placebo in achieving six-month smoking cessation, but not superior to NRT or varenicline. It has a high risk of side effects: depression and / or risk of self-aggressive suicidal behavior; allergic skin reactions, neuropsychiatric and neurological disorders; and cardiovascular effects, including hypertension, angina and / or myocardial infarction, dry mouth, and nausea. Bupropion is recommended as a last resort in selected cases, with close monitoring of subjects.

[0019] Pfizer sells Varenicline (tartrate) was approved for marketing in 2006. Varenicline is a selective partial agonist of the α4β2 nicotinic acetylcholine receptor subtype and has the ability to block nicotine from stimulating the central nervous system mesolimbic dopamine system. Pfizer evaluated the effect of varenicline in six clinical trials. The trials included a total of 3659 long-term smokers. Unless treatment is continued for six months (>50%), The maximum efficacy achieved within one year is less than 30%. A 2013 meta-analysis by Cahill et al. showed that varenicline was superior to placebo, bupropion, and NRT, but not to a combination of NRT (transdermal patch + oral NRT). This product is associated with serious side effects such as neuropsychiatric symptoms, including depression and suicidal / self-injurious behavior, behavioral changes, hostility, and agitation. It has also been described as being associated with sleep, gastrointestinal disorders, and cardiovascular disease, but these latter observations have not been confirmed. Due to these adverse effects, particularly those related to suicidality and depression, it is recommended as a second-line treatment after failure of nicotine replacement therapy.

[0020] Vaccine treatments are also being evaluated. Nicotine is the target. Nicotine molecules are chemically bound to a carrier protein or particle to stimulate the immune system to produce antibodies that bind to the nicotine in the smoke and prevent it from entering the brain. NIC002 (Cytos / Novartis) and The Nabi / GSK vaccine failed to demonstrate efficacy in Phase II and Phase III clinical trials. Nic7, a bioconjugate vaccine developed by Pfizer, was evaluated in a Phase I clinical trial of 200 individuals and discontinued in 2016. SEL-068 (Selecta Biosciences, Inc.), a nanoparticle-based vaccine, was in Phase I clinical development from 2012 to 2014, but no results or updates have been released.

[0021] Several other products are also being evaluated for smoking cessation, including BP1.4979, a D3R partial agonist (Phase II, Bioprojet); nadolol (Phase II, Invion Inc.); molobemide, a reversible MAO-A inhibitor (Phase II: Duke University Medical Center / Philip Morris); and EVP-6124, an alpha-7 nicotinic acetylcholine receptor agonist (Forum Pharmaceuticals Inc.).

[0022] It should also be mentioned that e-cigarettes are growing in popularity but are not approved for smoking cessation in many regulatory jurisdictions. The need to develop safe and effective alternatives for this indication is greater than ever.

[0023] As described in WO2017174787, the present inventors have found that injecting an aqueous solution of tobacco leaf extract can reduce dependence. More specifically, it can be demonstrated that a single injection of tobacco extract according to WO2017174787 is often sufficient to reduce or even eliminate tobacco dependence in smokers.

[0024] This feature is a major advantage for the subject, given that currently available products on the market offer only long-term and multiple-dose treatments. Conversely, the treatment according to the invention provides a shock therapy, i.e. a treatment preferably consisting of a single injection of tobacco extract, which can optionally be followed by at least one second injection a few days or even weeks later, if the subject feels the need.

[0025] The inventors have now discovered that their previously discovered solution can make it easier to break free from cigarette addiction when used in certain circumstances, particularly when used in conjunction with other products that can manage nicotine withdrawal symptoms.

[0026] Summary of the Invention

[0027] The disclosed invention relates to a method for preconditioning a subject to quit smoking, comprising the steps of administering to the subject transdermally or subcutaneously an effective amount of an aqueous solution of a substantially nicotine-free and protein-rich tobacco leaf extract, during which the subject continues to smoke freely.

[0028] This method advantageously makes it easier for the subject to quit smoking than existing methods. In fact, the subject's preconditioning makes him quit smoking more effectively because the administration of the aqueous solution of the protein-rich tobacco extract prior to actual smoking cessation produces a feeling of aversion, or even disgust, in the subject.

[0029] Therefore, the disclosed invention also relates to a method for treating a subject to quit smoking, comprising the following two consecutive steps:

[0030] i) a preconditioning step during which at least one effective dose of an aqueous solution of a substantially nicotine-free and protein-rich tobacco leaf extract is administered percutaneously or subcutaneously to the subject, during which the subject continues to smoke tobacco freely, and

[0031] ii) a second step: which follows the pre-adaptation step i), within 2 to 180 days, preferably 2 to 30 days, after the administration to the subject during the pre-adaptation step i) (also referred to as "initial step i)"), during which the subject completely quits smoking by optionally supplementing with one or more of the at least one product capable of controlling nicotine withdrawal symptoms.

[0032] A "product capable of controlling nicotine withdrawal symptoms" is understood within the scope of the present invention to mean a product which is not the aqueous solution of the protein-rich tobacco leaf extract found in step i).

[0033] For example, such a product may be a nicotine agonist or nicotine itself, administered as a nicotine patch or chewing gum or other form as is known in the art.

[0034] The preconditioning phase with the aqueous solution of the substantially nicotine-free and protein-rich tobacco leaf extract (i.e., step (i) of the method of treatment and the preconditioning method for the subject to quit smoking) is the day on which the subject decides to quit smoking, that is, the day on which he / she will no longer consume any nicotine from cigarettes and, if necessary, replace this intake with at least one product capable of controlling nicotine withdrawal symptoms. The problem of quitting smoking is thereby alleviated.

[0035] The second step (ii) is to help the subject to quit smoking completely, in particular by quitting nicotine addiction.

[0036] The disclosed invention also relates to a kit comprising:

[0037] - an aqueous solution of a protein-rich tobacco leaf extract; and a separate

[0038] - At least one product that can manage nicotine withdrawal symptoms.

[0039] Such a kit may assist a subject in applying the treatment method according to the present invention by providing the necessary products, possible instructions for their use, and / or safety measures. Detailed Description of the Invention

[0041] While various modifications and additional understandings of the inventive concepts of this disclosure are permitted, certain illustrative embodiments of the invention will now be described in detail.

[0042] ·method

[0043] It has been found that subjects can be preconditioned to quit smoking by administering to the subject transdermally or subcutaneously an effective dose of an aqueous solution of a substantially nicotine-free, protein-rich tobacco leaf extract, during which time the subject continues to smoke freely.

[0044] To enhance the effect of the method, if desired, a second effective dose of the aqueous solution of protein-rich tobacco leaf extract, substantially free of nicotine, may be administered to the subject transdermally or subcutaneously, during which time the subject continues to smoke freely. Although this second administration has been found to be generally unnecessary, to avoid problems, if the subject does not experience the aversion or disgust expected from the first administration of the aqueous solution of protein-rich tobacco leaf extract, a second effective dose of the aqueous solution of protein-rich tobacco leaf extract may be recommended.

[0045] A method for assisting a subject in determining whether he / she needs a second effective dose of an aqueous solution of a substantially nicotine-free and protein-rich tobacco leaf extract is provided by administering a rating questionnaire, wherein the rating questionnaire asks questions that allow for a subjective assessment of the desire and / or pleasure of smoking. Such a questionnaire may be provided by asking one or more of the following questions (or similar questions): (1) "Does smoking provide satisfaction?", (2) "Do cigarettes taste good?", (3) "Do you enjoy the sensation in your throat and chest?", (4) "Does smoking calm you down?", (5) "Does smoking make you feel more alert?", (6) "Does smoking make you feel less irritable?", (7) "Does smoking help you concentrate?", (8) "Does smoking reduce your cravings for food?", (9) "Does smoking make you dizzy?", (10) "Does smoking make you nauseous?", (11) "Does smoking immediately relieve your craving for cigarettes?", (12) "Do you enjoy smoking?"

[0046] For example, each question can be answered by giving a score from 1 to 7, depending on how the subject feels, whether the answer to each question is not satisfied at all (score equal to 1) or completely satisfied (score equal to 7), with the middle score being 4 out of 7.

[0047] The questionnaire may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or all 12 questions.

[0048] In one embodiment, the questionnaire includes all 12 questions listed herein and is referred to as the "modified Cigarette Evaluation Questionnaire (mCEQ)". The modified Cigarette Evaluation Questionnaire (mCEQ) uses three multi-item subscales and two single items: "Smoking satisfaction" (items 1, 2, and 12); "Psychological rewards" (items 4 to 8); "Aversion" (items 9 and 10);

[0049] "Enjoy the sensation of breathing" (item 3); and "Reduce cravings" (item 11). The score for each subscale is calculated as the individual item answers or the average of the individual items. Higher scores indicate greater strength of the scale.

[0050] Preferably, the second effective dose of the aqueous solution of tobacco leaf extract that is substantially free of nicotine and rich in protein may be administered within 2 to 15 days, preferably within 5 days, after the first administration of the aqueous solution.

[0051] 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 having a molecular weight greater than 10 kDa, based on the total weight of the dry extract thereof, and is substantially free of molecules having a molecular weight less than 10 kDa and RuBisCO protein; and

[0052] 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 dry extract.

[0053] In one embodiment, the present invention relates to a method for 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-β-glucosidases, endochitinases, pathogenesis-related proteins, osmomodulins, protease inhibitors, and mixtures thereof.

[0054] Preferably, the present invention relates to a method for preconditioning a subject to quit smoking, wherein the content of proteins with a molecular weight greater than 100 kDa in the tobacco leaf extract is less than 15% by weight based on the total protein weight of the dry extract.

[0055] Preferably, the present invention relates to a preconditioning method for enabling a subject to quit smoking, wherein the tobacco leaf extract comprises at least one protein belonging to the glucan endo-1,3-β-glucosidase family, which is selected from the group consisting of β-1,3-endoglucanase acidic isoform PR-Q′ (according to PR36401 of the UniProt database), β-1,3-endoglucanase alkaline vacuolar isoform GLB (according to P27666 of the UniProt database) and mixtures thereof.

[0056] Preferably, the present invention relates to a preconditioning method for a subject to quit smoking, wherein the tobacco leaf extract comprises at least one protein belonging to the endochitinase family, which is selected from the group consisting of acid endochitinase P (according to P17513 of the UniProt database), acid endochitinase Q (according to P17514 of the UniProt database), endochitinase B (according to P24091 of the UniProt database) and mixtures thereof.

[0057] Preferably, the present invention relates to a method for preconditioning a subject to quit smoking, wherein the tobacco leaf extract comprises at least osmomodulin (P14170 according to the UniProt database).

[0058] 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).

[0059] Preferably, the present invention relates to a preconditioning method for enabling a subject to quit smoking, wherein the tobacco leaf extract comprises at least one pathogenesis-related protein selected from the group consisting of pathogenesis-related protein R (according to P13046 of the UniProt database), pathogenesis-related protein PR-4A (according to PR29062 of the UniProt database), pathogenesis-related protein PR-4B (according to PR29063 of the UniProt database) and a mixture thereof.

[0060] 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 protease inhibitor family 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.

[0061] Preferably, the present invention relates to a method for preconditioning a subject to quit smoking, wherein the protein content of the dry extract of the tobacco leaf extract is at least 10 wt. %, based on the total weight of the dry extract.

[0062] Preferably, the present invention relates to a method for preconditioning a subject to quit smoking, wherein the amount of protein present in the tobacco leaf extract ranges from 1 μg / mL to 1000 μg / mL.

[0063] 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, in a form suitable for administration by an adhesive transdermal therapeutic system (such as a patch), or in a form suitable for administration by spray or vaporization.

[0064] 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.

[0065] As mentioned above, another aspect of the present disclosure relates to a method for treating a subject to quit smoking, comprising the following two consecutive steps:

[0066] i) a preconditioning step during which at least one effective dose of an aqueous solution of a substantially nicotine-free and protein-rich tobacco leaf extract is administered percutaneously or subcutaneously to the subject, during which the subject continues to smoke tobacco freely, and

[0067] ii) a second step, which is performed after the pre-adaptation step i), within 2 to 180 days, preferably 2 to 30 days, after the administration to the subject during the pre-adaptation step i), during which the subject completely quits smoking by optionally supplementing with one or more of the at least one product capable of controlling nicotine withdrawal symptoms.

[0068] As mentioned above, the purpose of administering an effective dose of an aqueous solution of a tobacco leaf extract that is substantially free of nicotine and rich in protein is to cause aversion or disgust to cigarettes. However, as mentioned above, cigarettes contain many addictive ingredients, particularly nicotine. Therefore, if a subject encounters one or more side effects related to nicotine withdrawal, administering a product (such as a nicotine patch) that can control nicotine withdrawal symptoms will specifically help the subject overcome nicotine addiction. A method for helping a subject determine whether he / she needs such a replacement therapy for nicotine can be provided by providing a scoring questionnaire of the effects of known nicotine withdrawal symptoms.

[0069] Nicotine withdrawal may cause the following adverse events: depression, insomnia, irritability / frustration / anger, anxiety, difficulty concentrating, restlessness, appetite / weight gain, constipation, bradycardia...

[0070] For example, questions such as “Have you noticed a change in your mood in the past two days, four days, etc.?”, “Have people around you commented on your bad mood since you quit smoking?”, or “Have you noticed being hungrier than usual in the past two days, four days, etc.?”, along with a rating sheet, were used to help the subject identify his need for nicotine.

[0071] By providing a scoring questionnaire as disclosed above, a method can be provided to help a subject determine whether he / she can proceed to step (ii) of the above method to completely quit smoking, wherein the questions asked can provide a subjective assessment of the desire and / or pleasure of smoking. Such a questionnaire can be provided by asking one or more of the following questions (or similar questions): (1) "Is smoking satisfying?", (2) "Do cigarettes taste good?", (3) "Do you enjoy the feeling in your throat and chest?", (4) "Does smoking calm you down?", (5) "Does smoking make you feel more alert?", (6) "Does smoking make you feel less irritable?", (7) "Does smoking help you concentrate?", (8) "Does smoking reduce your desire for food?", (9) "Does smoking make you dizzy?", (10) "Does smoking make you nauseous?", (11) "Does smoking immediately relieve your craving for cigarettes?", (12) "Do you enjoy smoking?".

[0072] For example, each question can be answered by giving a score from 1 to 7, depending on how the subject feels, whether the answer to each question is not satisfied at all (score equal to 1) or completely satisfied (score equal to 7), with the middle score being 4 out of 7.

[0073] The questionnaire may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or all 12 questions.

[0074] In one embodiment, the questionnaire includes all 12 questions listed herein and is referred to as the "modified Cigarette Evaluation Questionnaire (mCEQ)". The modified Cigarette Evaluation Questionnaire (mCEQ) uses three multi-item subscales and two single items: "Smoking satisfaction" (items 1, 2, and 12); "Psychological rewards" (items 4 to 8); "Aversion" (items 9 and 10);

[0075] "Enjoy the sensation of breathing" (item 3); and "Reduce cravings" (item 11). The score for each subscale is calculated as the individual item answers or the average of the individual items. Higher scores indicate greater strength of the scale.

[0076] The questionnaire can be answered before, during and / or after step (i) and the results compared with each other to monitor the effectiveness of the administration and thereby adjust the treatment to be performed (e.g. by adding more and / or another product capable of controlling nicotine withdrawal symptoms).

[0077] In one embodiment, the 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 substantially nicotine-free and protein-rich tobacco leaf extracts, during which time the subject continues to smoke freely.

[0078] Preferably, the present invention relates to a method of treatment wherein an effective dose of an aqueous solution of two or three substantially nicotine-free and protein-rich tobacco leaf extracts is administered transdermally two or three times, said administrations being separated from each other by at least 10 days, preferably at least 20 days, during which time the subject continues to smoke freely.

[0079] 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 substantially nicotine-free and protein-enriched tobacco leaf extract, during which time the subject continues to smoke tobacco freely.

[0080] Preferably, the present invention relates to a method of treatment, wherein the second step after the preconditioning step i) is: within 2 to 120 days, preferably 2 to 60 days after the administration to the subject during the preconditioning step i), during which the subject completely quits smoking by one or more optional supplementary administrations of at least one product capable of controlling nicotine withdrawal symptoms.

[0081] More preferably, the present invention relates to a method of treatment, wherein the second step after the preconditioning step i) is: within 2 to 30 days after the administration to the subject during the preconditioning step i), during this period, the subject completely quits smoking by one or more optional supplementary administrations of at least one product capable of controlling nicotine withdrawal symptoms.

[0082] In one embodiment, the present invention relates to a method of treatment, wherein the one or more optional supplemental administrations of at least one product capable of controlling nicotine withdrawal symptoms are carried out in a manner that accompanies the subject to gradually wean the subject off nicotine. This enables controlled compensation of nicotine withdrawal over a period of time. Preferably, the accompanying is characterized in that the subject is asked monthly, preferably every two weeks, weekly, every 6 days, every 5 days, every 4 days, every 3 days, every 2 days or even every day over a period of one week to 12 months, preferably two weeks to 6 months, more preferably one month to four months (such as 3 months).

[0083] In one embodiment, the present invention relates to a method of treatment in which, when several products capable of controlling nicotine withdrawal symptoms are administered, they are administered at independent times. This allows for a more precise and adaptable treatment of smoking cessation, in particular nicotine withdrawal.

[0084] Preferably, the present invention relates to a method of treatment, wherein the at least one product capable of controlling nicotine withdrawal symptoms is selected from nicotine agonists or partial agonists, nicotine substitutes or combinations thereof.

[0085] Preferably, the present invention relates to a method of treatment, wherein the nicotine agonist or partial agonist is selected from varenicline, cytisine, lobeline or a combination thereof.

[0086] In one embodiment, the invention relates to a method of treatment, wherein the second step is performed within 3 to 10 days after said administration to the subject during the preconditioning step i).

[0087] Preferably, the present invention relates to a method of treatment, wherein the second step is performed within 3 to 4 days after said administration to the subject during the preconditioning step i).

[0088] 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 dry extract thereof, and is substantially free of molecules having a molecular weight less than 10 kDa and RuBisCO protein; and

[0089] 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 dry extract.

[0090] 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-β-glucosidases, endochitinases, pathogenesis-related proteins, osmomodulins, protease inhibitors and mixtures thereof.

[0091] In one embodiment, the present invention relates to a method of treatment wherein the tobacco leaf extract contains less than 15% by weight of proteins having a molecular weight greater than 100 kDa, based on the total protein weight of the dry extract.

[0092] In one embodiment, the present invention relates to a method of treatment, wherein the tobacco leaf extract comprises at least one protein belonging to the glucan endo-1,3-β-glucosidase family, which is selected from the group consisting of β-1,3-endoglucanase acidic isoform PR-Q′ (PR36401 according to the UniProt database), β-1,3-endoglucanase alkaline vacuolar isoform GLB (P27666 according to the UniProt database) and mixtures thereof.

[0093] In one embodiment, the present invention relates to a method of treatment, wherein the tobacco leaf extract comprises at least one protein belonging to the endochitinase family, which is selected from the group consisting of acid endochitinase P (P17513 according to the UniProt database), acid endochitinase Q (P17514 according to the UniProt database), endochitinase B (P24091 according to the UniProt database) and mixtures thereof.

[0094] In one embodiment, the present invention relates to a method of treatment, wherein the tobacco leaf extract comprises at least osmomodulin (P14170 according to the UniProt database).

[0095] 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).

[0096] In one embodiment, the present invention relates to a method of treatment, wherein the tobacco leaf extract comprises at least one pathogenesis-related protein selected from the group consisting of pathogenesis-related protein R (according to P13046 of the UniProt database), pathogenesis-related protein PR-4A (according to PR29062 of the UniProt database), pathogenesis-related protein PR-4B (according to PR29063 of the UniProt database) and a mixture thereof.

[0097] In one embodiment, the present invention relates to a method of treatment, 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 the UniProt database), protease inhibitor IA (Q03198 according to the UniProt database) and mixtures thereof.

[0098] In one embodiment, the invention relates to a method of treatment wherein the protein content of the dry extract of the tobacco leaf extract is at least 10% by weight, based on the total weight of the dry extract.

[0099] In one embodiment, the present invention relates to a method of treatment wherein the protein is present in the tobacco leaf extract in an amount ranging from 1 μg / mL to 1000 μg / mL.

[0100] In one embodiment, the 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, in a form suitable for administration by an adhesive transdermal therapeutic system (e.g., a patch), or in a form suitable for administration by spray or vaporization.

[0101] 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.

[0102] Component Kit

[0103] As mentioned above, the second aspect of the present disclosure relates to a kit comprising:

[0104] - an aqueous solution of a protein-rich tobacco leaf extract; and

[0105] - At least one product that can manage nicotine withdrawal symptoms.

[0106] As described above, for the methods of treatment, preferably 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.

[0107] More preferably, the protein is selected from the group consisting of the following protein families: lignin-forming anionic peroxidases, glucan endo-1,3-β-glucosidases, endochitinases, pathogenesis-related proteins, osmomodulins and protease inhibitors, and mixtures thereof.

[0108] The method for preparing the tobacco leaf extract is as described above.

[0109] In one embodiment, the present invention relates to a kit wherein the aqueous solution of protein-rich tobacco leaf extract is suitable for transdermal or subcutaneous administration in an effective dose.

[0110] In one embodiment, the present invention relates to a kit, wherein the at least one product capable of managing nicotine withdrawal symptoms is selected from a nicotine agonist or partial agonist, a nicotine substitute, or a combination thereof.

[0111] In one embodiment, the present invention relates to a kit wherein the nicotine agonist or partial agonist is selected from varenicline, cytisine, lobeline, or a combination thereof.

[0112] In one embodiment, the present invention relates to a kit, 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 dry extract thereof, and is substantially free of molecules having a molecular weight less than 10 kDa and RuBisCO protein; and

[0113] 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 dry extract.

[0114] In one embodiment, the present invention relates to a kit wherein the protein is selected from the group consisting of the following protein families: lignin-forming anionic peroxidases, glucan endo-1,3-β-glucosidases, endochitinases, pathogenesis-related proteins, osmomodulins, protease inhibitors, and mixtures thereof.

[0115] In one embodiment, the present invention relates to a kit wherein the tobacco leaf extract contains less than 15% by weight of proteins having a molecular weight greater than 100 kDa, based on the total protein weight of the dry extract.

[0116] In one embodiment, the present invention relates to a kit, wherein the tobacco leaf extract comprises at least one protein belonging to the glucan endo-1,3-β-glucosidase family, which is selected from the group consisting of β-1,3-endoglucanase acidic isoform PR-Q′ (PR36401 according to the UniProt database), β-1,3-endoglucanase alkaline vacuolar isoform GLB (P27666 according to the UniProt database) and mixtures thereof.

[0117] In one embodiment, the present invention relates to a kit, wherein the tobacco leaf extract comprises at least one protein belonging to the endochitinase family selected from the group consisting of acid endochitinase P (P17513 according to the UniProt database), acid endochitinase Q (P17514 according to the UniProt database), endochitinase B (P24091 according to the UniProt database) and mixtures thereof.

[0118] In one embodiment, the present invention relates to a kit, wherein the tobacco leaf extract comprises at least osmomodulin (P14170 according to the UniProt database).

[0119] In one embodiment, the present invention relates to a kit, wherein the tobacco leaf extract comprises at least one lignin-forming anionic peroxidase (according to the UniProt database P11965).

[0120] In one embodiment, the present invention relates to a kit, wherein the tobacco leaf extract comprises at least one pathogenesis-related protein selected from the group consisting of pathogenesis-related protein R (according to P13046 of the UniProt database), pathogenesis-related protein PR-4A (according to PR29062 of the UniProt database), pathogenesis-related protein PR-4B (according to PR29063 of the UniProt database) and mixtures thereof.

[0121] In one embodiment, the present invention relates to a kit, wherein the tobacco leaf extract comprises at least one protein belonging to the protease inhibitor family 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.

[0122] In one embodiment, the present invention relates to a kit wherein the protein content of the dry extract of the tobacco leaf extract is at least 10 wt. %, based on the total weight of the dry extract.

[0123] In one embodiment, the present invention relates to a kit wherein the amount of protein present in the tobacco leaf extract ranges from 1 μg / mL to 1000 μg / mL.

[0124] In one embodiment, the present invention relates to a kit, wherein the tobacco leaf extract or composition is provided in a form suitable for administration by subcutaneous injection, in a form suitable for administration by an adhesive transdermal therapeutic system (e.g., a patch), or in a form suitable for administration by spray or vaporization.

[0125] In one embodiment, the present invention relates to a kit wherein the tobacco leaf extract or composition is provided in a dosage form of 0.03 mL to 10 mL.

[0126] According to one embodiment, the kit comprises one or more doses of tobacco leaf extract, preferably in lyophilized form, and one or more doses of saline solution or WFI, to prepare the pharmaceutical composition described herein just prior to administration.

[0127] According to one embodiment, the kit comprises one or more doses of the pharmaceutical composition ready for administration to a patient.

[0128] The kit may optionally contain one or more syringes for administering the pharmaceutical composition by subcutaneous injection.

[0129] The kit may optionally contain one or more patches for transdermal administration of the pharmaceutical composition.

[0130] Tobacco leaf extract and its pharmaceutical composition

[0131] The protein-rich tobacco leaf extract used in the above-mentioned treatment method and component kit is obtained according to the disclosure of WO2017174787.

[0132] That is, the tobacco leaf extract can be obtained by a preparation method comprising the following steps:

[0133] a. Curing tobacco leaves,

[0134] b. grinding the dried tobacco leaves to obtain ground dried tobacco leaves,

[0135] c. extracting the ground dried tobacco leaves with a solvent under mechanical stirring, the solvent being, for example, an aqueous solvent, preferably an aqueous buffer solution having a pH of 6.0-8.5,

[0136] d. separating the solid residue from the ground dried tobacco leaf extract solution by filtration or centrifugation to obtain a ground dried tobacco leaf extract solution free of solid residues,

[0137] e. filtering the solution obtained in step d with a solvent (preferably an aqueous solvent) in an amount ranging from 2 to 12 volumes, preferably 3 to 10 volumes, preferably 4 to 8 volumes, preferably 6 volumes, based on the volume of the extract, using a 10 kDa cut-off membrane,

[0138] f. Optionally lyophilizing the protein solution obtained in step e.

[0139] The dry extract may be obtained before freeze-drying the tobacco leaf extract according to the invention and then placed under a vacuum bell jar, preferably in the presence of P2O5, until a constant quality of extract is obtained.

[0140] The drying in step a is carried out in the open air, which is called natural drying. In this case, the drying is carried out in a natural open air environment, outdoors or indoors. The drying can be carried out in an open or closed, covered or uncovered structure. For example, air-curing can be carried out in a natural drying bin. In this case, freshly collected or pre-dried tobacco leaves are naturally dried under the action of the open air. For example, the tobacco leaves can be hung in a ventilated bin that is not heated. For example, the tobacco leaves can be naturally dried until they turn brown. At this stage, there is almost no sugar left in the leaves. Advantageously, the duration of the drying is adapted to the local climatic conditions and preferably lasts at least one month. For example, the harvest in central France can be dried between September and December. During the drying process, the tobacco leaves can be turned over once or multiple times to ensure uniform drying and to avoid condensation and rotting or degradation of the tobacco leaves. The air-curing method can be used for example for burley tobacco. According to a specific embodiment, the drying in step a is carried out in a natural drying bin.

[0141] For the purposes of the present invention, "air-curing" or "natural curing" means drying in the presence of natural outdoor or indoor air. Air-curing can be carried out in open or closed, covered or uncovered structures. For example, air-curing can be carried out in a natural curing silo. In this case, freshly collected or previously cured tobacco leaves are naturally dried under the influence of the open air. For example, the tobacco leaves can be hung in a ventilated silo that is not heated. For example, the tobacco leaves can be naturally dried until they turn brown. At this stage, there is almost no sugar left in the leaves. Advantageously, the duration of the drying process is adapted to the local climatic conditions and preferably lasts at least one month. For example, the harvest in central France can be dried between September and December. During the drying process, the tobacco leaves can be turned or aired one or more times to ensure uniform drying and to avoid the formation of condensation and the decay or degradation of the tobacco leaves. The air-curing method can be used for example for burley tobacco.

[0142] Tobacco leaf extracts can be obtained from tobacco leaves that have been cured in natural curing barns.

[0143] Tobacco extract can be obtained from tobacco leaves dried in heated drying chambers. In this case, drying can be carried out in a chamber or structure heated to a suitable temperature.

[0144] Tobacco extract can be obtained from cured tobacco leaves. In this case, curing occurs in a structure or silo heated to a suitable temperature. Heat can be introduced into the structure or silo via pipes connected to an external heat source. This controlled heating produces yellow-orange leaves. Consequently, these leaves have a high sugar content. For example, Virginia tobacco can be cured using this method.

[0145] Tobacco extract can be obtained from tobacco leaves that have been sun-cured. In this process, the leaves are laid out on racks and exposed to the sun for 12 to 30 days. The direct sunlight and heat of the sun cause the leaves to turn yellow or orange and retain their high sugar content. Oriental tobacco is often cured this way.

[0146] Tobacco extract can be obtained from flue-cured tobacco leaves. In this case, small pieces of wood are burned under the leaves, allowing them to dry while absorbing the "smoky" aroma.

[0147] According to one embodiment, the tobacco leaf is 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.

[0148] Of course, the extract does not necessarily consist of pure tobacco leaf extract, and proteins extracted from cannabis may be added to treat tobacco and cannabis dependence (the proteins extracted from cannabis described in this specification are for medical purposes only, in particular for the treatment of cannabis dependence).

[0149] According to a particular embodiment, the tobacco leaf extract is obtained from a 1 / 1 / 1 mixture of brown tobacco, Virginia tobacco and burley tobacco.

[0150] According to a particular embodiment, the tobacco leaf extract is obtained from Burley tobacco.

[0151] According to one embodiment, the tobacco leaf extract is obtained from cured tobacco leaves.

[0152] According to a particular embodiment, the tobacco leaf extract is obtained from air-cured tobacco leaves.

[0153] According to a particular embodiment, the tobacco leaf extract is obtained from tobacco leaves air-cured for a duration adapted to the local climatic conditions, preferably for at least one month.

[0154] Steps c to e correspond to extraction, filtration and diafiltration steps of the type described in patent US 5,770,698 (column 6, line 47 to column 7, line 7) and patent application US 2009 / 0162403 (paragraphs

[0032] to

[0040] ).

[0155] Preferably, the extraction in step c is carried out at a temperature of 4°C to 20°C, preferably 4°C to 10°C.

[0156] Preferably, the extraction in step c is carried out for 12 to 36 hours, preferably 22 to 26 hours, preferably 24 hours.

[0157] According to a particular embodiment, the solvent used in step c is an aqueous solvent. Preferably, the aqueous solvent used in step c is an aqueous buffered solution of ammonium bicarbonate, preferably at a concentration between 2 g / L and 6 g / L, preferably 4 g / L.

[0158] Preferably, the tobacco leaf extract can be obtained by subjecting the ground dried tobacco leaves to a solvent (e.g., an aqueous solvent) extraction method, 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 solid residues to constant volume diafiltration with an aqueous solvent, wherein the amount of the aqueous solvent is 2 to 12 volumes, preferably 3 to 10 volumes, preferably 4 to 8 volumes, preferably 6 volumes, based on the volume of the extract, and a 10 kDa cut-off membrane is used.

[0159] Of course, other types of solvents can be used to carry out the extraction method of the tobacco leaf extract in step c. The extraction in step c can be carried out with an organic or inorganic solvent, or a mixture of an organic and / or inorganic solvent.

[0160] In order to ensure good extraction efficiency and yield, and obtain high-quality extracts with desired purity properties and composition, those skilled in the art will readily know how to select a suitable solvent based on the following criteria:

[0161] - polarity of the solvent or solvent mixture (polar or non-polar);

[0162] - the physical state of the solvent or solvent mixture (e.g. liquid, solid, supercritical or gaseous);

[0163] - the chemical nature of the solvent or solvent mixture (e.g. organic or inorganic);

[0164] - the charge of the solvent or solvent mixture (e.g. ionic or non-ionic);

[0165] - the source of the solvent or solvent mixture;

[0166] - miscibility of the solvent or solvent mixture; and / or

[0167] -Solubility of the solvent or solvent mixture.

[0168] Based on the desired extraction yield and extract purity and composition, one skilled in the art will know which technique to use to select a solvent corresponding to these criteria.

[0169] Preferably, the extraction in step c is carried out by suspending the ground dried tobacco leaves in a buffer solution, wherein the concentration of the ground dried tobacco leaves in the buffer solution is preferably 30 g / L to 70 g / L, preferably 40 g / L to 60 g / L, and more preferably 50 g / L. The suspended solid residues are then removed by filtration, for example by Büchner filtration (step d) to obtain ground dried tobacco leaves free of solid residues. Preferably, the aqueous solvent used in step d is water for injection (WFI).

[0170] 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 dry extract, and is essentially free of molecules having a molecular weight less than 10 kDa.

[0171] For purposes of this invention, "about" means plus or minus 1% due to measurement uncertainty.

[0172] Advantageously, step e removes more than 99% of molecules with a molecular weight of less than 10 kDa, in particular free amino acids, proteins and peptides with a molecular weight of less than 10 kDa, and protein residues with a molecular weight of less than 10 kDa resulting from the protein degradation of step a.

[0173] 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.

[0174] The content of proteins with a molecular weight greater than 10 kDa in the obtained extract is usually measured in tobacco leaf extracts and is about 1%. Therefore, the tobacco leaf extract of the present invention contains proteins with a molecular weight greater than 10 kDa.

[0175] The protein in the protein-rich tobacco leaf extract is preferably selected from the group consisting of the following protein families: lignin-forming anionic peroxidases, glucan endo-1,3-β-glucosidases, endochitinases, pathogenesis-related proteins, osmomodulins and protease inhibitors and mixtures thereof.

[0176] The names mentioned for the proteins present in the extracts according to the invention correspond to the names given in the Swiss-Prot database, a biological database listing protein sequences.

[0177] According to one embodiment, the tobacco leaf extract of the present invention contains at least one protein belonging to the glucan endo-1,3-β-glucosidase family, and is preferably selected from the group consisting of β-1,3-endoglucanase acidic isoform PR-Q′ (according to PR36401 of the UniProt database), β-1,3-endoglucanase alkaline vacuolar isoform GLB (according to P27666 of the UniProt database) and mixtures thereof.

[0178] According to one embodiment, the tobacco leaf extract of the present invention contains at least one protein belonging to the endochitinase family, and is preferably selected from acid endochitinase P (according to P17513 of the UniProt database), acid endochitinase Q (according to P17514 of the UniProt database), endochitinase B (according to P24091 of the UniProt database) and mixtures thereof.

[0179] According to one embodiment, the tobacco leaf extract of the present invention comprises at least osmomodulin (P14170 according to the UniProt database).

[0180] According to one embodiment, the tobacco leaf extract of the invention comprises at least one lignin-forming anionic peroxidase (according to the UniProt database P11965).

[0181] According to one embodiment, the tobacco leaf extract of the present invention contains at least one pathogenesis-related protein, and is preferably selected from pathogenesis-related protein R (according to P13046 of the UniProt database), pathogenesis-related protein PR-4A (according to PR29062 of the UniProt database), pathogenesis-related protein PR-4B (according to PR29063 of the UniProt database) and a mixture thereof.

[0182] According to one embodiment, the tobacco leaf extract of the present invention comprises at least one protein belonging to the protease inhibitor family, and is preferably selected from protease inhibitor IB (Q03199 according to UniProt database), protease inhibitor IA (Q03198 according to UniProt database) and mixtures thereof.

[0183] According to one embodiment, the tobacco leaf extract of the present invention further comprises polysaccharides having a molecular weight greater than 10 kDa and preferably being water-soluble.

[0184] Preferably, the tobacco leaf extract of 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-β-glucosidases, endochitinases, pathogenesis-related proteins, osmomodulins and protease inhibitors and mixtures thereof, based on the total weight of the dry extract.

[0185] According to one embodiment, the tobacco leaf extract of the present invention is substantially free of high molecular weight proteins.

[0186] For the purposes of the present invention, "high molecular weight protein" refers to a protein with a molecular weight greater than 500 kDa, preferably a protein with a molecular weight greater than 400 kDa, more preferably a protein with a molecular weight greater than 300 kDa, preferably a protein with a molecular weight greater than 200 kDa, even more preferably a protein with a molecular weight greater than 150 kDa, and more preferably a protein with a molecular weight greater than 100 kDa.

[0187] According to one embodiment, the tobacco leaf extract of the present invention is substantially free of proteins having a molecular weight greater than 50 kDa.

[0188] For the purposes of the present invention, "substantially free" means a molecular content of less than 15 wt% based on the total protein weight of the extract, preferably a molecular content of less than 10 wt% based on the total protein weight of the extract, more preferably less than 7.5 wt% based on the total protein weight of the extract, more preferably less than 5 wt% based on the total protein weight of the extract, preferably less than 2.5 wt% based on the total protein weight of the extract, more preferably less than 1 wt% based on the total protein weight of the extract, and still more preferably less than 0.5 wt% based on the total protein weight of the extract.

[0189] Advantageously, the content of high molecular weight proteins is less than 15 wt.-% based on the total protein weight of the extract, preferably less than 10 wt.-% based on the total protein weight of the extract, more preferably less than 7.5 wt.-% based on the total protein weight of the extract, more preferably less than 5 wt.-% based on the total protein weight of the extract, more preferably less than 2.5 wt.-% based on the total protein weight of the extract, more preferably less than 1 wt.-% based on the total protein weight of the extract, and still more preferably less than 0.5 wt.-% based on the total protein weight of the extract.

[0190] According to one embodiment, the tobacco leaf extract of 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), for example by a hydrolytic mechanism.

[0191] Preferably, before step f, the protein solution obtained in step e is sterilized and filtered in step e'.

[0192] The tobacco leaf extract of the invention obtained at the end of the above-described process can be stored as obtained at the end of sterile filtration step d′ or can be freeze-dried as obtained at the end of step f.

[0193] The tobacco leaf extract of the present invention is advantageously an aqueous extract.

[0194] The tobacco leaf extract of the present invention is advantageously a brown aqueous extract.

[0195] The present disclosure also relates to a pharmaceutical composition containing the tobacco leaf extract enriched according to the present invention, which comprises the tobacco leaf extract as described above as an active ingredient.

[0196] The pharmaceutical composition of the present invention may further comprise at least one pharmaceutically acceptable excipient, such as a pharmaceutically acceptable solvent, such as water.

[0197] Advantageously, the pharmaceutical composition of the present invention may comprise the protein present in the tobacco leaf extract in an amount ranging from 1 μg / mL to 1000 μg / mL, preferably from 10 μg / mL to 500 μg / mL, preferably from 50 μg / mL to 300 μg / mL, preferably from 60 μg / mL to 200 μg / mL, preferably between 80 μg / mL and 150 μg / mL.

[0198] The pharmaceutical composition of the present invention is preferably an aqueous composition.

[0199] According to one embodiment, the pharmaceutical composition of the present invention further comprises an adjuvant. Examples of adjuvants include: swelling agents, for example sugars such as lactose, sucrose, trehalose, sorbitol, glucose, raffinose, mannitol, preferably lactose, sucrose, trehalose, glucose or mannitol; amino acids such as arginine, glycine or histidine, preferably glycine; or polymers of the dextran or polyethylene glycol type; or mixtures thereof. According to this embodiment, the pharmaceutical composition comprises 50% to 99% by weight, preferably 80% to 97% by weight, of the adjuvant, based on the total weight of the pharmaceutical composition.

[0200] According to a specific embodiment, the pharmaceutical composition of the present invention may further comprise a protein extracted from cannabis (the protein extracted from cannabis described in this specification is only used for medical purposes, in particular for the treatment of cannabis dependence).

[0201] Preferably, the pharmaceutical composition can be prepared by a method comprising the following steps:

[0202] a1. Preparation of tobacco extract as described above,

[0203] b1. Adjust the protein concentration to obtain a protein concentration of 100 μg / mL to 200 μg / mL,

[0204] c1. adding pharmaceutically acceptable excipients, and

[0205] d1. Optionally, adding an adjuvant, preferably mannitol, to the extract.

[0206] The tobacco leaf extract used in step b1 can be the tobacco leaf extract obtained directly after the diafiltration step on a 10 kDa cut-off membrane, or the tobacco leaf extract obtained after a sterilization diafiltration step, or the tobacco leaf extract that is freeze-dried and then reconstituted in saline or water.

[0207] Advantageously, the concentration in step b1 can be adjusted as follows:

[0208] - by diluting with water to reduce the protein concentration, or

[0209] - Increase protein concentration by diafiltration.

[0210] A particularly preferred pharmaceutically acceptable excipient in step c1 is water.

[0211] Auxiliary agents that may be added in step d1 may in particular be: swelling agents, for example sugars such as lactose, sucrose, trehalose, sorbitol, glucose, raffinose, mannitol, preferably lactose, sucrose, trehalose, glucose or mannitol; amino acids such as arginine, glycine or histidine, preferably glycine; or polymers of the dextran or polyethylene glycol type; or mixtures thereof.

[0212] According to a particular embodiment, step d1 comprises at least the addition of mannitol.

[0213] According to one embodiment, the pharmaceutical composition of the present invention is provided in a form suitable for subcutaneous administration.

[0214] According to one embodiment, the pharmaceutical composition of the present invention comprises an adhesive transdermal therapeutic system containing the tobacco leaf extract as defined above. According to this embodiment, the pharmaceutical composition of the present invention is provided in a form suitable for administration via an adhesive transdermal therapeutic system. Advantageously, the pharmaceutical composition of the present invention is provided in the form of a patch.

[0215] The patch can be in the form of a reservoir patch or a matrix patch having one or more compartments. The actual implementation of the patch will be determined by one skilled in the art based on his overall understanding of the subject to obtain controlled and long-term systemic administration of the tobacco leaf extract over the entire application period of the patch (e.g., about 2 hours to 24 hours).

[0216] Reservoir patches will include one or more separate reservoirs containing a solution of the active ingredient (including tobacco leaf extract), or the active ingredient (including tobacco leaf extract) suspended in a polymer matrix, in contact with the skin through a semipermeable polymer membrane so that the release rate of the active ingredient can be adjusted.

[0217] Matrix patches contain a polymeric substance in which the active ingredients are dissolved or dispersed in appropriate proportions. These active ingredients are released by diffusion through the polymer chains of the matrix.

[0218] According to a particular embodiment of this type of patch, the adhesive substance covers the entire release surface of the matrix and is an integral part of the matrix. Therefore, such active adhesive substance type patches are known to those skilled in the art, simplify their manufacture and allow the production of thin, flexible patches that are suitable, thereby allowing comfortable application on the patient's skin.

[0219] In order to ensure that these active ingredients can be well infused subcutaneously or in the blood at an appropriate dose, an appropriate diffusion rate, and an appropriate duration, those skilled in the art can easily set the following parameters:

[0220] - the ratio of the surface area to the volume of each compartment of the patch;

[0221] - optionally adding one or more hydrophilic additives;

[0222] - optionally adding one or more diffusion activators or inhibitors;

[0223] - optionally adding one or more solubilizing agents;

[0224] - optionally adding one or more stabilizers;

[0225] - optionally adding one or more absorption enhancers, and

[0226] - More generally, all types of additives well known to those skilled in the art which allow good control of the flow and stability of tobacco leaf extracts.

[0227] One skilled in the art will know which techniques to use to set the parameters listed above based on the solubility and stability desired.

[0228] Those skilled in the art will readily adjust the various manufacturing parameters of the patch to achieve the desired dosage.

[0229] In a manner known per se, this patch comprises a removable protective film which serves to protect the adhesive side to be applied to the skin after manufacture and throughout storage of the patch. In a manner known per se, a person skilled in the art will, for example, use a polyester film, one side of which may be treated with anti-adhesive silicone.

[0230] According to one embodiment, the composition of the invention is administered by subcutaneous injection.According to this embodiment, the pharmaceutical composition is provided in a form suitable for subcutaneous administration.

[0231] 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. Therefore, the pharmaceutical composition is preferably administered by subcutaneous injection in an amount of 0.03 mL to 10 mL, preferably 0.1 mL to 5 mL, preferably 0.5 mL to 2 mL.

[0232] The pharmaceutical composition may be provided in a form intended for administration only once by subcutaneous injection.

[0233] The pharmaceutical composition may be provided in a form intended for administration several times, preferably two or three times, by subcutaneous injection.

[0234] In the latter case, the pharmaceutical composition is preferably administered on day 0 and day 10, or on day 0, day 10 and day 30 by subcutaneous injection.

[0235] The dose of the second and optional third injections may be the same as the dose of the first injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0236] Figure 1 The graph shows the effect of NFL-101 and placebo on smoking satisfaction reduction obtained in the PRECESTTO study (see Examples).

[0237] Figure 2 The difference between the effects of NFL-101 and placebo on smoking satisfaction reduction obtained in the PRECESTTO study is shown in the graph, including exploratory p-values (see Examples).

[0238] Figure 3 The chart shows the effect of reducing smoking satisfaction. Comparative testing results between varenicline, a commercially available alternative, and NFL-101. Example

[0239] NFL-101 is an aqueous extract of tobacco leaves prepared according to Good Manufacturing Practice guidelines. The subcutaneous injection solution is provided as a lyophilized powder (140 μg protein per vial) with a sterile diluent. On the day of injection, the research team will reconstitute the vial to ensure product stability.

[0240] The manufacturing process of NFL-101 follows the teachings of WO2017174787. Therefore, for the completeness of the disclosure of the present invention, Example 1 of WO2017174787 is disclosed below to reproduce the present invention.

[0241] Example 1: Preparation of tobacco extract according to the present invention (WO2017174787)

[0242] Burley tobacco leaves are used to prepare the extract.

[0243] 100 g of Burley tobacco leaves, naturally dried for about three months (September to December in central France), were ground. The ground leaves were suspended in 1892 g of WFI to which 8 g of ammonium bicarbonate had been added at a temperature of 4°C to 10°C for 24 hours. The suspended solid residue was then removed by Büchner filtration.

[0244] The extract was then clarified by 0.2 μm filtration. The resulting extract was brown in color. The extract was then weighed to determine the volume of WFI used in the constant volume diafiltration step; in this case, the mass of the liquid extract was 1680 g.

[0245] The protein of the ground tobacco extract, which was free of solid residues, was then extracted: 10080 g of WFI were then added to the ground dried tobacco extract, which was free of solid residues. The 11760 g of solution thus obtained were diafiltered at a constant volume against 6 volumes with a cut-off of 10 kDa (Merck Millipore) until the mass of the retentate was reduced to 1680 g.

[0246] The retentate obtained after diafiltration is a protein mixture in which the concentration of proteins with a molecular weight of less than 10 kDa detected is 99% lower than its initial concentration. According to the measurement scale defined in EUROPEAN PHARMACOPOEIA 5.0, 2.2.2, Degree of coloration of liquids, the color of the retentate obtained is between B2 and B3.

[0247] The permeate can be freeze-dried using an SMH 150 freeze dryer and after adding, for example, mannitol, to obtain a freeze-dried tobacco leaf extract.

[0248] NFL-101 is an aqueous extract of tobacco leaves.

[0249] NFL-101 (the extract of Example 1) was presented as one vial of 140 μg sterile lyophilized powder and one vial of 5 mL 0.9% sodium chloride.

[0250] The vial must be reconstituted in 1.4 mL or 2.8 mL of 0.9% sodium chloride to obtain a solution with a concentration of 50 μg / mL or 100 μg / mL. The reconstituted NFL-101 solution is light yellow-brown.

[0251] Example 2: Clinical Development

[0252] Phase aI clinical trial (CESTO trial)

[0253] A Phase I trial was conducted in 24 smoking subjects (average 6.25). The primary objective was to evaluate the safety of NFL-101. Secondary objectives were to assess the immunogenicity and efficacy of NFL-101 in these smokers. This study was a prospective, single-center, open-label, non-randomized Phase I trial conducted at the Institut Régional du Cancer (ICM) in Montpellier, France.

[0254] NFL-101 was administered subcutaneously twice, 4 weeks apart, to smoking subjects willing to quit smoking. Each subject received 2 injections (1 in each arm) on D1 and D29. The study was designed to investigate two dose levels of the protein (100 μg or 200 μg protein) to determine if 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 (6 given 100 μg protein + 6 given 200 μg protein) received treatment a few days after the recruitment visit. The second 12 subjects had more time to prepare for their quit attempt before treatment was administered and therefore were thought to be better psychologically prepared to quit smoking.

[0255] In this study, dose-limiting toxicity (DLT) was defined as any grade 3-4 toxicity according to the NCI-CTCAE V4.0 classification. No DLT was observed in any subject. At dose level 2 (higher dose), only one grade 2 toxicity (anxiety) was reported that was considered related or possibly related to NFL-101 treatment. Other adverse events included the following grade 1 toxicities:

[0256] n = 20: injection site pain, deltoid pain, deltoid edema, bilateral pain (10 patients at dose 1 [low dose] and 10 patients at dose 2 [high dose]);

[0257] n=7: dry mouth (3 patients at dose 1 and 4 patients at dose 2);

[0258] n=6: fatigue (3 patients at dose 1 and 3 patients at dose 2);

[0259] n=5: nausea (1 patient at dose 1 and 4 patients at dose 2);

[0260] n = 4: dry eye (3 patients at dose 1 and 1 patient at dose 2);

[0261] n = 3: Insomnia (2 patients at dose 1 and 1 patient at dose 2);

[0262] n=3: deltoid erythema (1 case at dose 1 and 2 cases at dose 2);

[0263] n = 2: shiver (1 patient at dose 1 and 1 patient at dose 2);

[0264] n = 2: irritability (1 patient at dose 1 and 1 patient at dose 2);

[0265] n = 2: headache (1 patient at dose 1 and 1 patient at dose 2);

[0266] n = 2: dysgeusia (2 cases at dose 1 and 0 cases at dose 2);

[0267] n = 2: agitation (1 patient at dose 1 and 1 patient at dose 2);

[0268] n=1: bradycardia, photophobia, diarrhea, pruritus of the tongue, gastroesophageal reflux, weight gain, calf cramps, pain (deltoid, limbs), anxiety, depression, significant rhinitis, dry nasal cavity, productive cough, pruritus (arms and legs), pruritus (nose).

[0269] During the study, 67% of the 24 smokers reduced their smoking by at least 50% at four weeks and 63% at 12 weeks. 33% of the 24 smokers did not smoke for at least the last seven days of the 12-week period. 21% of the 24 smokers maintained sustained abstinence at 12 weeks. Of the last 12 participants, who were better prepared for the smoking cessation trial, 80% reduced their smoking by at least 50%, and five of them maintained abstinence at week 12, eight weeks after the end of treatment.

[0270] Immunological assessments showed a significant increase in anti-NFL101 IgG (already present at D1), and higher anti-NFL-101 IgG (after treatment administration) was associated with smoking reduction, 7-day point abstinence rate, and sustained abstinence.

[0271] All subjects spontaneously reported that NFL-101 reduced their desire to smoke and cigarette cravings, but reported experiencing typical withdrawal symptoms during their quit attempts.

[0272] CESTO trial conclusions:

[0273] People trying to quit smoking often experience urges to smoke, often described as cravings, which are often in response to environmental cues associated with smoking, such as alcohol. The intensity and frequency of these urges decrease over time. The key to quitting smoking is resisting these urges. Effective smoking cessation medications can reduce the intensity of urges, a possible mechanism of action.

[0274] There are three licensed pharmacotherapy treatments for smoking cessation: bupropion, varenicline, and nicotine replacement therapy (NRT). Bupropion has more contraindications and more severe adverse effects than the other two pharmacotherapy treatments. Therefore, when smokers use pharmacotherapy to help them, they usually use varenicline or NRT.

[0275] Typically, smokers who quit smoking start using NRT on their quit day.

[0276] The most effective medication for supporting smoking cessation is varenicline. Varenicline is a partial nicotine agonist and therefore, surprisingly, more effective than the full agonist (nicotine).

[0277] One study, which considered possible mechanisms of action and compared varenicline, bupropion, and placebo, found that varenicline reduced the urge to smoke to lower levels than bupropion.

[0278] Furthermore, compared with bupropion, varenicline reduced the perceived satisfaction associated with relapse (occasional smoking episodes) after quitting. However, many other mood-related withdrawal symptoms were similarly intense. This suggests that a key mechanism of action of smoking cessation medications is controlling the urge to smoke and reducing the perceived satisfaction associated with smoking, which is directly related to efficacy.

[0279] Unlike NRT, varenicline is used one to two weeks before quitting, which may explain its superior efficacy. Clinical trials have investigated whether nicotine preloading (i.e., continuing to try to smoke the same amount of cigarettes or smoking freely before quitting and then using NRT) improves abstinence rates.

[0280] A meta-analysis of nicotine preloading for smoking cessation included eight studies involving 2,813 participants and concluded that nicotine preloading had a weak and nonsignificant effect on quit rates. Another study of 1,792 smokers concluded that preloading with a nicotine patch before a quit attempt increased the proportion of people who successfully quit, but the benefit was attenuated by reduced utilization of varenicline after preloading.

[0281] The question that needs to be answered is whether NFL-101 can significantly reduce the satisfaction from smoking so that it can be used before quitting smoking.

[0282] b. NFL-101 preconditioning combined with NRT

[0283] Because NRT is one of the two preferred pharmacological treatments for smoking cessation, it has not yet been used for preconditioning before quitting day, and its effectiveness for preconditioning has not been clearly demonstrated. Therefore, it is necessary to study whether using NFL-101 as preconditioning before quitting day and starting NRT can improve smoking cessation outcomes.

[0284] By understanding the development of tobacco addiction, we can hypothesize the potential mechanisms of NRT treatment after NFL-101 preconditioning.

[0285] Smoking produces positive rewards, such as feelings of pleasure, which lead to the reinforcing effects of subsequent smoking. After a period of regular smoking, neural adaptations to regular doses of nicotine mean that smokers experience negative emotions when nicotine concentrations in the brain decrease; smoking can alleviate these emotions, so smoking produces negative reinforcement.

[0286] Independent of any rewards a person may experience, mechanisms in the nucleus accumbens form associative learning that drives smoking behavior. These mechanisms produce a learned drive to smoke and tobacco dependence, manifested by regular smoking behavior, tobacco withdrawal symptoms when unable to smoke, and difficulty quitting if an individual chooses to. The net effect of this change in the drive to smoke means that a person who would not normally smoke will typically smoke in response to environmental cues. When a person does not smoke when prompted to do so, the learned association between smoking behavior and reinforcement in the brain begins to wane.

[0287] In addition to this addiction-based mechanism, three other mediating effect hypotheses can be proposed for the mechanism of action of NFL-101 in preconditioning.

[0288] The first hypothesis was that preconditioning would increase medication adherence after quitting.

[0289] The second hypothesis was that the reduction in smoking levels when people are treated with NFL-101 might increase a person's confidence that they can quit smoking after their quit date; and that a person's confidence that they can achieve abstinence is associated with the outcome of achieving abstinence.

[0290] The third hypothesis is that the aversive reaction to cigarettes after receiving NFL-101 may improve smoking cessation rates. Aversive smoking is a condition in which people smoke excessively to the point of nausea and vomiting. Although it is rarely used as a treatment to enhance smoking cessation, it can effectively increase quit rates.

[0291] c.Precesto study

[0292] c.1. Historical aspects

[0293] Therefore, a Phase IIa trial (the so-called PRECESTTO study) was proposed to evaluate the efficacy of NFL-101 in reducing smoking rewards compared with placebo. The questionnaire "mCEQ" was used as the main assessment criterion in a cross-over design in 34 free-smoking patients. The subjects were informed that they would receive both the active study drug (200 μg NFL-101) and a placebo, but were blinded to whether they received NFL-101 or placebo during the first and second phases. The duration of 4 weeks allowed for a clear observation of the effect over time. A minimum washout period of 30 days was considered sufficient to avoid any residual effects when the second washout period began.

[0294] The primary endpoint is a 1-point reduction in the mCEQ "Smoking Satisfaction" subscale measured on Day 4 (D4) from the first dose of NFL-101. The mCEQ will include all 12 questions listed above and use a 7-point scale.

[0295] Another commendable minor criterion is to reduce the score by 1 point in the following areas:

[0296] - mCEQ "Smoking Satisfaction" subscale measured on D7, D14, D21 and D28, and / or

[0297] - mCEQ “Psychological Reward”; “Aversion”; “Enjoyment of Respiratory Sensation”; and “Desire Reduction” subscales measured on D4, D7, D14, D21, and D28.

[0298] For this purpose, subjects were asked additional questions to assess the effectiveness of the treatment:

[0299] - On Days 4, 7, 14, 21, and 28, “Did you feel your urge to smoke was stronger or weaker than usual in the past 24 hours?” (Response options were “stronger,” “slightly stronger,” “same as before,” “slightly weaker,” and “much weaker”)

[0300] - On D4, D7, D14, D21, and D28, “Did you find smoking in the past 24 hours more or less pleasant than usual?” (response options were “more pleasant,” “somewhat more pleasant,” “the same as usual,” “somewhat less pleasant,” and “not very pleasant”)

[0301] - Quit Smoking Motivation Scale? (Scale from 01 to 10, 10 represents impulsive quitting)?

[0302] -How many cigarettes did you smoke in the past 24 hours?

[0303] Furthermore, the overreaction criteria due to nicotine withdrawal (depression, insomnia, irritability / frustration / anger, anxiety, difficulty concentrating, restlessness, increased appetite / weight gain, constipation, bradycardia…) were understandable and were the initial milestones planned for completion of the CESTO study.

[0304] c.2. Conditions and implementation of the research

[0305] Specifically, PRECESTO is the second study following CESTO to demonstrate that NFL-101 reduces smoking gratification, a clinically relevant effect that lasted longer than expected.

[0306] These results confirm the therapeutic potential of NFL-101, administered alone or in combination with nicotine replacement therapy, to alleviate withdrawal symptoms. They therefore confirm the findings of the previous CESTO study.

[0307] Review: What is PRECESTO?

[0308] PRECESTO is a single-center, placebo-controlled, randomized, double-blind, Phase 2a exploratory study, a crossover study covering two 28-day periods, that included 34 smokers who did not want to quit and had high smoking satisfaction. Each participant served as his or her own control group and was randomized to receive either NFL-101 or placebo at the beginning of both periods. At the end of the study, all participants received one dose of NFL-101 and one dose of placebo. The order of administration of NFL-101 and placebo was kept confidential for each participant. For each period, participants received treatment on Day 1 (D1) and then independently answered the modified Cigarette Evaluation Questionnaire (mCEQ) on Days 4 (D4), 7 (D7), 14 (D14), 21 (D21), and 28 (D28).

[0309] The aim of PRECESTO was to “evaluate the effectiveness of NFL-101 compared with placebo in reducing the positive reinforcing effects of cigarettes as measured on the subscale of the mCEQ assessing smoking gratification (questions 1, 2, and 12)”. The primary criterion arising from this aim was selected as “the smoking gratification subscale of D4 as measured on the mCEQ (items 1, 2, and 12)”.

[0310] mCEQ, a leading international questionnaire measuring the effects of smoking

[0311] The mCEQ is an independently completed international questionnaire. Participants in the study were smokers who reported high smoking satisfaction and did not want to quit. They answered 12 questions, selecting the value that best represented how smoking made them feel (1 = no feeling at all, 2 = very slight, 3 = slight, 4 = moderate, 5 = noticeable, 6 = very noticeable, 7 = extremely strong). The questions were as follows:

[0312] 1. Does smoking bring satisfaction?

[0313] 2. Do cigarettes taste good?

[0314] 3. Do you enjoy the sensations in your throat and chest?

[0315] 4. Does smoking help you calm down?

[0316] 5. Does smoking make you feel more alert?

[0317] 6. Does smoking make you feel less irritable?

[0318] 7. Does smoking help you concentrate?

[0319] 8. Does smoking reduce your desire for food?

[0320] 9. Does smoking make you dizzy?

[0321] 10. Does smoking make you sick?

[0322] 11. Does smoking immediately relieve your craving for cigarettes?

[0323] 12. Do you like smoking?

[0324] The questionnaire uses three multi-item subscales and two single items: "Smoking Satisfaction" (items 1, 2, and 12); "Psychological Rewards" (items 4 to 8); "Aversion" (items 9 and 10); "Enjoyment of Respiratory Sensation" (item 3); and "Reduced Craving" (item 11). Scores for each subscale are calculated as the average of individual item responses or individual item averages. Therefore, each score ranges from 1 to 7. Higher scores indicate greater strength of the scale.

[0325] Participants in the PRECESTO study completed the questionnaire remotely using their computers. They were asked to refer to a cigarette they had smoked after dinner the previous day in order to standardize changes in perception over time as much as possible for each subject.

[0326] Rationale for the PRECESTO study

[0327] The PRECESTO study will provide a better understanding of the activity of NFL-101, particularly its efficacy in reducing smoking gratification.

[0328] Reducing smoking gratification is crucial in the fight against smoking: To successfully quit smoking, addressing both gratification and nicotine withdrawal is crucial. While there are nicotine replacement therapies for withdrawal, there are no effective pharmaceutical products without side effects to address gratification.

[0329] c.3. Results

[0330] PRECESTO Phase 2a exploratory clinical study

[0331] Statistical analysis was previously demonstrated to be feasible for all subjects and for the combined analysis of both series (carryover effect: p>0.10). Data were processed by calculating least squares means, taking into account the crossover design of the study and following the PRECESTO protocol.

[0332] Smoking satisfaction was the average of the responses to Questions 1, 2, and 12. At inclusion, the mean score for smoking satisfaction was 6.12 (SD 0.77).

[0333] The PRECESTO results are as follows:

[0334] Regarding the reduction in smoking satisfaction caused by NFL-101: Based on observations from the CESTO Phase 1 clinical trial, it is expected that after D4 reaches maximum levels, it will quickly subside within 10 days. The effect observed with PRECESTO is different ( Figure 1 ): There was an effect on D4 (mean decrease -0.810; confidence interval (CI) (-1.139; -0.481)), which then increased on a relatively linear basis until the final measurement on D28 (mean decrease -1.261; CI (-1.590; -0.932)). The mean level of smoking satisfaction, which was between "very strong" and "extremely strong" at inclusion, decreased to between "moderate" and "strong" on D28, representing a clinically relevant reduction.

[0335] Regarding the effects of NFL-101 and placebo and the reduction of smoking satisfaction: the placebo effect was significant but remained lower than the effect of NFL-101 throughout the observation period (see below). Figure 1 ). It increased at a slower rate starting from D7 and then decreased from D21 to D28 (mean decrease -0.947; confidence interval (-1.277; -0.618)). Regarding the difference in effect between NFL-101 and placebo ( Figure 2), which did not reach significance (>0.05) at D4, with exploratory p-values of 0.0003 (mean difference -0.188; confidence interval (-0.289; -0.087)) for the entire monitoring period from D4 to D28 and 0.007 (mean difference -0.314; confidence interval (-0.540; -0.088)) at D28.

[0336] Regarding the specific effects of the secondary criteria on other mCEQ subscales and items: Exploratory p-values were not calculated for these criteria. The study limitations detailed below may provide explanations, particularly for the criteria related to desire.

[0337] Figure 1 The figure shows the effect of NGL-101 and placebo on reducing smoking gratification. Smoking gratification (ordinate) is the least squares mean (LSM) change from screening (pooled for phases 1 and 2), with a 95% CI for the MMRM. It is clear from the figure that, despite the effect of placebo on smoking gratification, NFL-101 (active ingredient) did have a greater effect on reducing smoking gratification compared to placebo.

[0338] Figure 2 The difference between NFL-101 and placebo in reducing smoking satisfaction is shown in the chart, including exploratory p-values. Smoking satisfaction (ordinate) is the placebo-corrected least squares mean (LSM) change in screening (pooled for phase 1 and phase 2), with 95% CI and exploratory p-values for the MMRM, which includes phase, sequence, all visits, and gender and screening values as covariates. The figure demonstrates that smoking satisfaction is significantly reduced over a period of up to 28 days, even considering the placebo effect.

[0339] Varenicline Comparison:

[0340] To better understand the therapeutic potential of NFL-101, it will be interesting to compare the reduction in smoking cessation achieved in PRECEST0 with the reduction achieved with varenicline under pre-treatment conditions (that is, before the withdrawal attempt) (Bohadana A, Freier-Dror Y, Peles V, Babai P, Izbicki G. Extending varenicline preloading to 6 weeks facilitates smoking cessation: A single-site, randomized controlled trial. EClinicalMedicine. 2020Feb3;19:100228. doi:10.1016 / j.eclinm.2019.11.021. PMID:32055787; PMCID:PMC7005428). Figure 3 As shown in Figure 2, a single administration of NFL-101 resulted in a greater reduction in smoking satisfaction than 53 administrations of varenicline. Figure 3 The effects of NFL vs. placebo on smoking satisfaction reduction during the pretreatment period were compared with those of varenicline. Compared with placebo. On the basis of The difference with the placebo result was 0.3 (p=0.04). Under the above conditions, The difference from the placebo result was 0.36 (p=0.007). (-0.9) has a greater effect on reducing smoking satisfaction.

[0341] Clarification on p-values:

[0342] For p-values of 0.0003 and 0.007, this means that if no actual effect or difference appeared in the data, there would only be a 0.03% and 0.7% chance of obtaining data as different as the observed data.

[0343] Exploratory p-value calculations performed through post hoc analyses are limited to the primary objective of the study. This approach reduces the risk of obtaining low p-values due to chance, which can occur when p-values are calculated based on multiple criteria, even by cross-referencing criteria against each other. These calculations allow for the observation of effects that persist longer than anticipated, while honoring the spirit of the protocol and its primary objective.

[0344] Limitations of the PRECESTO study

[0345] In the PRECESTTO study, the mCEQ was completed by smokers who did not want to quit and smoked freely (“as often as needed”). It is recognized that reducing smoking or quitting smoking can have a significant impact on perceptual and behavioral responses related to smoking. The cross-over design cannot include participants who plan to reduce smoking or try to quit smoking because this would result in an overly significant carry-over effect (continuation or continuation of the effect) from period 1 to period 2. In addition, recruiting participants who want to quit smoking would risk information loss if they quit smoking because participants who quit smoking can no longer assess their smoking satisfaction and therefore cannot complete the questionnaire. By recruiting smokers who do not want to quit smoking, it is more difficult to demonstrate the effect of NFL-101 on certain criteria (such as craving) because this will usually be reduced regularly due to smoking without any restrictions.

[0346] c.4. Conclusion: First placebo-controlled clinical trial with encouraging results

[0347] This exploratory study:

[0348] Demonstrated that a single administration of NFL-101 reduced smoking satisfaction in a clinically relevant manner, with effects that were longer-lasting than expected, confirming the therapeutic benefit of NFL-101, either alone or in combination with nicotine replacement therapy, for reducing withdrawal symptoms;

[0349] Represents the second clinical trial, following CESTO, to demonstrate that NFL-101 reduces smoking gratification, this time with a placebo control, strengthening the evidence base for NFL-101's effectiveness; and

[0350] Confirmed the clinical results mentioned previously (CESTO trial).

Claims

1. A method for preconditioning a subject to quit smoking, comprising the steps of: administering to the subject percutaneously or subcutaneously an effective dose of an aqueous solution of a tobacco leaf extract that is substantially free of nicotine and rich in protein, during which the subject continues to smoke freely.

2. The method of claim 1, wherein a second effective dose of an aqueous solution of a substantially nicotine-free and protein-enriched tobacco leaf extract is administered transdermally or subcutaneously to the subject, during which time the subject continues to smoke freely.

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

4. The method of treatment according to any one of claims 1 to 3, 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 dry extract thereof, and is substantially free of molecules with a molecular weight less than 10 kDa and RuBisCO protein; and in, The content of proteins with a molecular weight greater than 500 kDa in the tobacco leaf extract is less than 15 wt % based on the total protein weight of the dry extract.

5. The method of treatment according to claim 4, wherein the protein is selected from the group consisting of the following protein families: lignin-forming anionic peroxidases, glucan endo-1,3-β-glucosidases, endochitinases, pathogenesis-related proteins, osmomodulins, protease inhibitors and mixtures thereof.

6. The method of treatment according to any one of claims 1 to 5, wherein: The content of proteins with a molecular weight greater than 100 kDa in the tobacco leaf extract is less than 15% by weight based on the total protein weight of the dry extract.

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

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 endochitinase family selected from the group consisting of acid endochitinase P (P17513 according to the UniProt database), acid endochitinase Q (P17514 according to the UniProt database), endochitinase B (P24091 according to the UniProt database) and mixtures thereof.

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

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

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 protease inhibitor family 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.

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

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

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, in a form suitable for administration by an adhesive transdermal therapeutic system such as a patch, or in a form suitable for administration by spray or vaporization.

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. A method for enabling a subject to quit smoking, comprising the following two consecutive steps: i) a preconditioning step during which at least one effective dose of an aqueous solution of a substantially nicotine-free and protein-rich tobacco leaf extract is administered to the subject percutaneously or subcutaneously, during which the subject continues to smoke tobacco freely, and ii) a second step, which is after the pre-adaptation step i), and is carried out within 2 to 180 days after the subject is administered the product during the pre-adaptation step i), during which the subject completely quits smoking by optionally administering one or more supplementary administrations of at least one product capable of controlling nicotine withdrawal symptoms.

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 substantially nicotine-free and protein-rich tobacco leaf extracts, during which time the subject continues to smoke freely.

19. A method of treatment according to claim 18, wherein the effective doses of two or three aqueous solutions of substantially nicotine-free and protein-rich tobacco leaf extracts are administered transdermally two or three times, each of said administrations being separated from each other by at least 10 days, preferably at least 20 days, during which time the subject continues to smoke freely.

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

21. The method of any one of claims 17 to 20, wherein the second step after the preconditioning step i) is: within 2 to 30 days after the administration to the subject during the preconditioning step i), the subject completely quits smoking by one or more optional supplementary administrations of at least one product capable of controlling nicotine withdrawal symptoms.

22. The method of treatment according to any one of claims 17 to 21, wherein the one or more optional supplemental administrations of at least one product capable of controlling nicotine withdrawal symptoms are performed in a manner that gradually weans the subject from nicotine.

23. The method of treatment according to any one of claims 17 to 22, wherein when several products capable of controlling nicotine withdrawal symptoms are administered, they are administered at times independent of one another.

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

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

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

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

28. The method of any one of claims 17 to 27, 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 dry extract thereof, and is substantially free of molecules having a molecular weight less than 10 kDa and RuBisCO protein; and in, 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 dry extract.

29. The method 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-β-glucosidases, endochitinases, pathogenesis-related proteins, osmomodulins, protease inhibitors, and mixtures thereof.

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

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

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

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

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

36. The method of any one of claims 17 to 35, wherein the tobacco leaf extract comprises at least one protein belonging to the protease inhibitor family 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.

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

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

39. A method of treatment according to 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, in a form suitable for administration by an adhesive transdermal therapeutic system such as a patch, or in a form suitable for administration by spray or vaporization.

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. A kit comprising: - an aqueous solution of a protein-rich tobacco leaf extract; and independent - At least one product that can manage nicotine withdrawal symptoms.

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

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

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

45. The kit of any one of claims 41 to 44, 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 dry extract thereof, and is substantially free of molecules having a molecular weight less than 10 kDa and RuBisCO protein; and in, 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 dry extract.

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-β-glucosidases, endochitinases, pathogenesis-related proteins, osmomodulins, protease inhibitors, and mixtures thereof.

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

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

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

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

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

53. The kit according to any one of claims 41 to 52, wherein the tobacco leaf extract comprises at least one protein belonging to the protease inhibitor family 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.

54. A kit according to any one of claims 41 to 53, wherein the protein content of the dry extract of the tobacco leaf extract is at least 10% by weight, based on the total weight of the dry extract.

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

56. A kit according to 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, in a form suitable for administration by an adhesive transdermal therapeutic system such as a patch, or in a form suitable for administration by spray or vaporization.

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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