Nicotine bag reproductive development safety evaluation method

Abstract

The invention relates to a method for constructing a male non-human animal nicotine exposure model, which comprises the following steps: dissolving and extracting contents in a nicotine bag to obtain a nicotine bag content extracting solution; and selecting a male non-human animal, and performing intragastric administration with the nicotine bag content extracting solution to construct the male non-human animal nicotine exposure model. The invention also relates to a nicotine pocket reproductive development safety evaluation method based on the male non-human animal. The method comprises the following steps: taking the male non-human animal nicotine exposure model as a male parent to mate with a normal female non-human animal to generate a filial generation; detecting a parent reproduction index and a filial generation development index; and performing nicotine pocket reproductive development safety evaluation according to the male parent reproductive index and the offspring development index. The nicotine bag reproductive development safety evaluation comprises comparison with a nicotine bag safety standard substance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of experimental animal models and safety evaluation, and particularly relates to a method for constructing a male non-human animal nicotine exposure model and a nicotine pouch reproductive development safety evaluation method. BACKGROUND

[0002] Under the influence of international tobacco control movements, the consumption of cigarettes is showing a downward trend year by year, and the control of tobacco products and the demand for alternative products are showing an unprecedented urgency. Smokeless tobacco products have gradually become an important supplement to tobacco consumption in a smoke-free environment due to their obvious advantages such as non-combustion and no smoke generation, and have attracted widespread attention from major tobacco companies around the world. Among the many potential smokeless tobacco alternatives, nicotine pouches, as a new type of nicotine product, are gradually entering the field of view of consumers with their convenient and low-hazard characteristics, and are developing rapidly with a faster growth rate.

[0003] Nicotine pouches have a wide market base globally, particularly in Northern Europe and the United States, and their market share is gradually expanding with the rise of new tobacco products. Globally, in 2023, nicotine pouches (modern snus) as a rapidly rising tobacco product in recent years continued to grow at a high speed, with sales growing 43.5% year-on-year to 15.51 billion bags, and sales revenue growing 61.1% year-on-year to 7.86 billion US dollars. The sales revenue of nicotine pouches in the United States accounted for 75.8% of the world's total. With the continuous development of market trade and the continuous expansion of the market for new tobacco products, the production and sales of nicotine pouches are gradually showing a global trend. More and more international tobacco companies have begun to layout the nicotine pouch market worldwide, promoting the globalization of nicotine pouch production.

[0004] The use of nicotine pouches is different from the traditional cigarette inhalation method. Nicotine pouches do not need to be burned or inhaled, but are placed between the gums and lips to directly absorb nicotine through the oral mucosa, avoiding harmful substances such as tar and carbon monoxide produced by tobacco combustion; its use process is smokeless and ashless, and can be used secretly in smoke-free places without producing secondhand smoke.

[0005] Recently, the U.S. Food and Drug Administration (FDA) approved the first 20 flavors of nicotine pouch (oral tobacco) products for marketing, which is the first time in tobacco history that such products are allowed to be marketed through advertising. In terms of nicotine absorption mechanism, traditional cigarettes rely on the nicotine-smoke mixture generated by combustion for pulmonary absorption; electronic cigarettes form aerosols by heating nicotine glycerol solution for pulmonary circulation into blood; the natural tobacco leaf components in oral tobacco are absorbed into the blood circulation through the oral mucosa; and nicotine pouches directly absorb high-purity nicotine salt through the oral mucosa, resulting in a significantly higher blood nicotine concentration peak than other products, which is the key mechanism for heavy smokers to experience "smoking intoxication". Compared with more than 7000 chemical substances (69 of which are definitely carcinogenic) generated by cigarette combustion, the composition of nicotine pouches is relatively simple, but the increase in the bioavailability of nicotine per unit dose also poses the risk of excessive intake.

[0006] Due to the unique absorption mechanism of nicotine pouches, the evaluation methods for other tobacco products cannot be used for nicotine pouches, and there is a gap in the safety evaluation system for nicotine pouches. That is, existing research lacks a safety evaluation system for the characteristics of nicotine pouches, especially in the fields of reproductive toxicity and transgenerational effects. Therefore, it is urgent to develop a method for constructing a nicotine animal exposure model for nicotine pouches and a method for evaluating the safety of reproductive development, so as to be able to simulate a parent exposure model that simulates real use scenarios, such as intermittent dosing cycles (to simulate the use habits of pouches); to establish an evaluation index for transgenerational developmental toxicity; and to establish a control system based on standard safe products to realize the quantitative risk comparison between samples and standard safe products. The establishment of the above methods will fill the technical gap in the reproductive safety evaluation of nicotine pouch products. SUMMARY

[0007] In a first aspect, the present application provides a method for constructing a male non-human animal nicotine exposure model, comprising the following steps:

[0008] Step A: dissolving and extracting the contents of the nicotine pouch to obtain a nicotine pouch content extract;

[0009] Step B: selecting a male non-human animal and gavaging the nicotine pouch content extract to construct a male rat nicotine exposure model.

[0010] In some embodiments, the step A can comprise:

[0011] Step A1: immerse the nicotine bag in a solvent; the duration of immersion is 5-10 minutes, 10-20 minutes, 20-30 minutes, 30-60 minutes; the temperature of the solvent is 35.0-36.0℃, 36.0-37.5℃ or 37.5-39.0℃; the volume of the solvent used per bag of the nicotine bag is 5-15 mL / bag, 15-50 mL / bag, 50-100 mL / bag or 100-200 mL / bag;

[0012] Step A2: take out the nicotine bag, centrifuge the remaining liquid and take the supernatant to obtain the nicotine bag content extract; the nicotine bag content extract is stored in the dark.

[0013] In some embodiments, the solvent can be water or artificial saliva; the artificial saliva can contain water and one or more of the following components: sodium carboxymethyl cellulose, sorbitol, potassium chloride, sodium chloride, magnesium chloride, calcium phosphate, sodium phosphate, nipagin, distilled water, pH adjuster, alpha-amylase, lysozyme, lipase, peroxidase.

[0014] In some embodiments, the non-human animal is a rat or a mouse, the rat is 6-10 weeks old, 10-14 weeks old, 14-22 weeks old; the mouse is 4-6 weeks old, 6-10 weeks old, 10-16 weeks old.

[0015] In some embodiments, step B is specifically: gavage the male non-human animal in the manner of interval length T, gavage amount V each time, duration D;

[0016] The interval length T can be 0.5-1.0 hour, 1.0-2.0 hours or 2.0-4.0 hours;

[0017] The gavage amount V each time can be 0.5-1.0 mL, 1-1.5 mL or 1.5-2.0 mL;

[0018] The duration D can be 30-45 days, 45-60 days or 60-75 days.

[0019] In some embodiments, step B includes constructing a male non-human animal nicotine pre-pregnancy exposure model, wherein after the gavage duration D, gavage is stopped, and then the offspring are produced after mating with normal female non-human animals.

[0020] In some embodiments, step B includes constructing a male non-human animal nicotine pregnancy exposure model, wherein after the gavage duration D, gavage is continued until the female mice successfully become pregnant, and the offspring are produced after mating with normal female non-human animals.

[0021] In a second aspect, the present application also provides use of the male non-human animal nicotine exposure model obtained by the method of any of the embodiments of the first aspect for nicotine pouch reproductive development safety evaluation.

[0022] In a third aspect, the present application also provides a method for nicotine pouch reproductive development safety evaluation based on a male non-human animal, comprising the following steps:

[0023] Step C: mating the male non-human animal nicotine exposure model obtained by the method of any of the embodiments of the first aspect with a normal female non-human animal as a father to produce offspring;

[0024] Step D: detecting the father reproductive index and the offspring development index;

[0025] Step E: performing nicotine pouch reproductive development safety evaluation according to the father reproductive index and the offspring development index;

[0026] The nicotine pouch reproductive development safety evaluation comprises comparison with a nicotine pouch safety standard product.

[0027] In some embodiments, the father reproductive index comprises evaluation of testosterone synthesis function and / or evaluation of sperm quality; the evaluation of testosterone synthesis function comprises blood testosterone level, intratesticular testosterone level and / or expression of key enzymes for testosterone synthesis;

[0028] The evaluation of sperm quality comprises sperm density, sperm motility and / or sperm abnormality rate.

[0029] In some embodiments, the offspring development index comprises basic reproductive output, offspring growth, placental development, offspring blood biochemical index and / or organ development and morphology,

[0030] For example, the basic reproductive output comprises litter size and / or sex ratio;

[0031] For example, the offspring growth comprises body weight and / or body length;

[0032] For example, the placental development comprises placental weight and / or placental index;

[0033] For example, the offspring blood biochemical index comprises endocrine hormone, sex hormone, adrenal hormone and / or blood lipid metabolism. BRIEF DESCRIPTION OF DRAWINGS

[0034] The above content of the present application and the following detailed description will be better understood when read in conjunction with the accompanying drawings. It should be noted that the accompanying drawings are only examples of the claimed technical solutions.

[0035] Figure 1 is the nicotine release profile of sample 1 according to example 1 in constant temperature water at 37°C (abscissa: time, ordinate: concentration);

[0036] Figure 2 is the nicotine release profile of sample 2 according to example 1 in constant temperature water at 37°C (abscissa: time, ordinate: concentration);

[0037] Figure 3 is the nicotine release profile of sample 3 according to example 1 in constant temperature water at 37°C (abscissa: time, ordinate: concentration);

[0038] Figure 4 is the HE staining result chart of the male rats in exposure group 1 according to example 2;

[0039] Figure 5 is the testicular testosterone synthesis enzyme system immunofluorescence result chart of the male rats in exposure group 1 according to example 2;

[0040] Figure 6 is the light microscope chart of the sperm of the male rats in exposure group 1 according to example 2. DETAILED DESCRIPTION

[0041] "ranges" disclosed herein are defined, stated, or otherwise specified in the form of a lower limit and an upper limit that the range is defined by selecting a lower limit and an upper limit, the selected lower limit and upper limit defining the boundaries of the particular range. Ranges defined by such limits can be either inclusive or exclusive of the end values, and can be arbitrarily combined in any manner. For example, if ranges of 60-120 and 80-110 are listed, it is understood that ranges of 60-110 and 80-120 are also contemplated. Furthermore, if ranges of 1 and 2 are listed as minimum values, and if ranges of 3, 4, and 5 are listed as maximum values, then ranges of 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5 are all contemplated. In this application, the use of a numerical range "a-b" to indicate that any and all real combinations of the numbers between a and b are contemplated, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between 0 and 5 have been listed herein, and "0-5" is merely a shorthand for listing all of those numbers. In addition, when a parameter is stated to be an integer ≥ 2, it is equivalent to state that the parameter is, for example, an integer 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0042] In this text, all the embodiments and preferred embodiments mentioned herein can be combined with each other to form new technical solutions, unless otherwise specified.

[0043] In the present text, all technical and preferred features mentioned herein can be combined with each other to form new technical solutions, unless anything else is stated.

[0044] In the present text, all steps mentioned herein can be performed in the order mentioned, but also in a random order, unless anything else is stated. For example, the method comprises steps (a) and (b) means that the method can comprise steps (a) and (b) performed in the order mentioned, but also steps (b) and (a) performed in the order mentioned. For example, the method can further comprise step (c) means that step (c) can be added to the method in any order, for example, the method can comprise steps (a), (b) and (c), but also steps (a), (c) and (b), steps (c), (a) and (b), etc.

[0045] In the present text, the terms "comprising" and "including" mean open terms, but can also mean closed terms, unless anything else is stated. For example, the terms "comprising" and "including" can mean that further components not listed can also be included, but can also mean that only the listed components are included.

[0046] In the present description, it is to be understood that, unless otherwise specified, "above", "below", "one or more" means two or more.

[0047] In the present description, the term "or" is inclusive, unless otherwise stated. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following satisfy the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0048] In the present text, percentages (%) refer to mass percentages relative to the composition, unless anything else is stated.

[0049] In the present text, the sum of the amounts of the components in the composition is 100%, unless anything else is stated.

[0050] In the present text, "combinations thereof" means a multi-component mixture of the elements mentioned, for example, two, three, four, and up to the maximum possible, unless anything else is stated.

[0051] The term "one" as used in the present description means "at least one", unless anything else is stated.

[0052] The terms "first", "second", "third", etc. are used only for descriptive purposes and are not to be construed as indicating or implying relative importance or an ordered ranking of the indicated technical features. Thus, features defined with "first", "second" can include at least one of the features explicitly or implicitly. In the description of the application, the meaning of "a plurality" is at least two, for example two, three, etc., unless otherwise explicitly and specifically limited.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing specific embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0054] The detailed features and advantages of the present application are described in detail in the following detailed description of specific embodiments, which should be considered together with the drawings, the claims and the specification, and which are sufficient to understand the technical content of the present application and to implement it by any person skilled in the art. The skilled person in the art can easily understand the related purposes and advantages of the present application according to the description, claims and drawings disclosed in the specification.

[0055] Dissolution extraction of nicotine pouch contents

[0056] As used herein, "Nicotine Pouch" generally refers to a small fiber bag that absorbs nicotine through the inner wall of the mouth, and the main ingredient is nicotine powder or nicotine salt. They are usually placed between the gums and the upper lip, and nicotine is absorbed by the human body through the oral mucosa.

[0057] As used herein, "Nicotine" (also known as Nicotinamide), chemical formula C 10 H 14 N2, belongs to pyridine alkaloids. In this paper, the term "nicotine" and "nicotinamide" can be used interchangeably.

[0058] In some aspects, the method of the present application comprises dissolving and extracting the contents of the nicotine pouch to obtain a nicotine pouch content extract.

[0059] In some aspects, the dissolving and extracting comprises immersing the nicotine pouch in a solvent, then taking out the nicotine pouch, centrifuging the remaining liquid to take the supernatant, obtaining the nicotine pouch content extract, and storing it in the dark.

[0060] The duration of the immersion can range from 5-10 minutes, 10-20 minutes, 20-30 minutes, 30-60 minutes. For example, the duration of the immersion can be 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes, or any range therebetween.

[0061] The temperature of the solvent can range from 35.0-36.0°C, 36.0-37.5°C, or 37.5-39.0°C. For example, the temperature of the water can be 37°C.

[0062] The volume of the solvent used per bag of the nicotine pouches can range from 5-15 mL / bag, 15-50 mL / bag, 50-100 mL / bag, or 100-200 mL / bag. For example, the volume of the solvent can be 20 mL / bag, 30 mL / bag, 40 mL / bag, 50 mL / bag, 60 mL / bag, 70 mL / bag, 80 mL / bag, or 90 mL / bag, or any range therebetween.

[0063] In some aspects, the solvent is water or artificial saliva.

[0064] The artificial saliva can include water and one or more of the following components: sodium carboxymethylcellulose, sorbitol, potassium chloride, sodium chloride, magnesium chloride, calcium phosphate, sodium phosphate, nipagin, distilled water, pH adjuster, alpha-amylase, lysozyme, lipase, peroxidase.

[0065] In one aspect, the artificial saliva is prepared by adding sodium carboxymethylcellulose to 700 milliliters of distilled water, soaking overnight, and then adding sorbitol, sodium chloride, magnesium chloride, potassium chloride, nipagin after the sodium carboxymethylcellulose is swollen. After dissolving, a solution of calcium phosphate and sodium phosphate is added, and distilled water is added to the appropriate amount. After stirring, 0.01% hydrochloric acid or 0.05% sodium hydroxide is used to adjust the pH to 7. Finally, the relevant enzymes are added, and the enzyme concentrations are adjusted to alpha-amylase: 50-500 U / mL; lysozyme: 1-10 μg / mL; lipase: 0.5 U / mL; peroxidase: 0.5 U / mL. The bottle is sterilized with 100-degree flow steam for 30 minutes. The saliva thus prepared has the properties of a colorless, translucent liquid, salty taste, and certain viscosity.

[0066] Non-human animal selection

[0067] As used herein, “non-human animals” refers to all animals that are selected by laboratories for experiments, excluding humans, such as mice, rats, etc. These animals are selected because of certain similarities in genetic, physiological, or behavioral characteristics with humans.

[0068] In some exemplary embodiments, rats are selected as the "non-human animals" for experiments.

[0069] As used herein, "rat" refers to an animal belonging to the class Mammalia, order Rodentia, family Muridae, genus Rattus, and the scientific name Rattus norvegicus. Wistar rats were bred by the Wistar Institute of the United States in 1907, and later through standardized inbreeding screening became an important foundation strain for experimental animal models.

[0070] In the present application, male rats are selected as the model construction object. The sexual maturity time of male rats is usually 6 to 8 weeks (42-56 days) after birth, corresponding to a daily age range of 45-60 days. 30-35 days of age is too early and does not meet the physiological maturity standard.

[0071] In some aspects, the present application selects male rats of 6-10 weeks old, 10-14 weeks old, or 14-22 weeks old. For example, male rats of 6 weeks old, 7 weeks old, 8 weeks old, 9 weeks old, 10 weeks old, 11 weeks old, 12 weeks old, 13 weeks old, 14 weeks old, 15 weeks old, 16 weeks old, 17 weeks old, 18 weeks old, 19 weeks old, 20 weeks old, 21 weeks old, or 22 weeks old, or any range therebetween, are selected.

[0072] Gavage of nicotine pouch content extract

[0073] In some exemplary embodiments, the gavage administration of the nicotine pouch content extract is performed on rats.

[0074] As used herein, "gavage" generally refers to a technique for directly delivering liquid or semi-liquid substances into the stomach through a catheter, which can be used for drug administration in experimental animals.

[0075] Gavage of rats can be performed as follows: a gavage device is used, which is composed of a syringe and a special gavage needle. The gavage needle for rats is 6-8 cm long and about 1.2 mm in diameter. The tip of the gavage needle is welded with a small metal ball, and the metal ball is hollow. The purpose of welding the metal ball is to prevent the needle from piercing the trachea or damaging the digestive tract. The needle metal ball end can be bent to an angle of about 20 degrees to adapt to the physiological curvature of the oral cavity and esophagus. The rat is fixed with the left hand, and the right hand holds the gavage device. The gavage needle is inserted from the oral cavity of the rat, the head is pressed to make the oral cavity and esophagus in a straight line, and the gavage needle is slowly inserted along the posterior pharyngeal wall into the esophagus, and a slight resistance can be felt. At this time, the direction of the gavage needle can be slightly changed to stimulate the swallowing action, and then pushed in until the desired depth. A small amount of nicotine pouch content extract is pushed in and the rat's reaction is observed. If the rat appears calm and has normal breathing, the remaining nicotine pouch content extract is slowly pushed in, and then the needle is gently withdrawn. Generally, the gavage needle is inserted 4-6 cm into the esophagus of the rat.

[0076] In some aspects, the present application comprises gavage at an interval duration T, a gavage volume V, and a duration D.

[0077] The interval duration T can range from 0.5-1.0 hours, 1.0-2.0 hours, or 2.0-4.0 hours. For example, the interval duration T can be 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, or 4.0 hours, or any range therebetween.

[0078] The gavage volume V can range from 0.5-3.0 mL, or 0.7-2.0 mL, or 1-1.7 mL. For example, the gavage volume V can be 0.5 mL, 1.0 mL, 1.2.0 mL, 1.4 mL, 1.5 mL, 1.6 mL, or 2.0 mL.

[0079] The duration D can range from 30-45 days, 45-60 days, or 60-75 days. For example, the duration D can be 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, 60 days, 65 days, 70 days, or 75 days, or any range therebetween.

[0080] In some aspects, the present application comprises stopping gavage after the gavage duration D, and then mating with normal female rats to produce offspring. This constructs a male rat pre-pregnancy nicotine exposure model.

[0081] In some aspects, the present application comprises continuing gavage until the female rats successfully get pregnant when the gavage duration reaches two months, and then mating with normal female rats to produce offspring. This constructs a male rat pregnancy nicotine exposure model.

[0082] Paternal reproductive index testing

[0083] The paternal reproductive indicators are selected from one or more of the group consisting of testosterone synthesis function and sperm quality assessment.

[0084] For example, the testosterone synthesis function is selected from one or more of the group consisting of blood testosterone level, intratesticular testosterone level, and expression of key enzymes for testosterone synthesis.

[0085] As used herein, the term "blood testosterone level" refers to the concentration of testosterone (the primary male sex hormone) in the blood, typically detected by blood draw, which reflects the overall level of testosterone throughout the body.

[0086] As used herein, the term "intratesticular testosterone level" refers to the concentration of testosterone produced locally by testicular tissue, which is directly involved in spermatogenesis. The testis is the main site of testosterone synthesis, and the concentration inside is usually much higher than the blood level (about 50-100 times). Local deficiency can lead to spermatogenic dysfunction. Detection methods can be through testicular biopsy or microdialysis technology.

[0087] As used herein, the term "testosterone synthesis key enzyme expression" refers to the gene or protein expression level of key enzymes involved in testosterone synthesis. The amount of enzyme expression in testicular tissue is analyzed by molecular biology techniques such as RT-PCR, Western blot. A decrease in expression can indicate a disorder of testosterone synthesis.

[0088] For example, the sperm quality evaluation is selected from one or more of the following: sperm density, sperm motility, and sperm deformity rate.

[0089] As used herein, the term "sperm density" refers to the number of sperm in a milliliter of semen, also known as "sperm concentration". A value below the normal range can reduce the probability of natural conception.

[0090] As used herein, the term "sperm motility" refers to the sperm movement ability, which is divided into three categories: forward movement (PR), non-forward movement (NP), and immotility (IM). Low motility (asthenozoospermia) can cause sperm to be unable to penetrate the cervical mucus or reach the egg.

[0091] As used herein, the term "sperm deformity rate" refers to the proportion of morphologically abnormal sperm to total sperm. Head, neck, or tail deformities are observed under a microscope after staining. A high deformity rate can affect the sperm's ability to penetrate the egg, but fertility should be evaluated comprehensively in combination with other indicators.

[0092] Offspring development index testing

[0093] The offspring development indicators are selected from one or more of the following: basic reproductive output, offspring growth, placental development, offspring blood biochemical indicators, and organ development and morphology.

[0094] For example, the basic reproductive output is selected from one or more of the following: litter size and sex ratio.

[0095] For example, the offspring growth is selected from one or more of the following: body weight and body length.

[0096] For example, the placental development is selected from one or more of the following: placental weight and placental index.

[0097] For example, the offspring blood biochemical indicators are selected from one or more of the following: endocrine hormones, sex hormones, adrenal hormones, and blood lipid metabolism.

[0098] As used herein, the term "litter size" refers to the total number of offspring produced by a female in a single pregnancy or birth, and in the present application refers to the total number of offspring produced by a female in a single pregnancy or birth.

[0099] As used herein, the term "sex ratio" refers to the proportion of female to male individuals in the offspring.

[0100] As used herein, the term "weight" refers to the weight measurement of an offspring at a specific time point after birth.

[0101] As used herein, the term "body length" refers to the length measurement of an offspring from head to tail (or a specific anatomical landmark).

[0102] As used herein, the term "placental weight" refers to the total weight of placental tissue.

[0103] As used herein, the term "placental index" refers to the ratio of placental weight to fetal weight (placental index = placental weight / fetal weight x 100%).

[0104] As used herein, the term "endocrine hormone" refers to hormones secreted by endocrine glands (eg, pituitary gland, thyroid gland), such as growth hormone, thyroid hormone (T3 / T4).

[0105] As used herein, the term "sex hormone" refers to hormones related to sexual development.

[0106] As used herein, the term "adrenal hormones" refers to hormones secreted by the adrenal cortex, such as cortisol (stress hormone) and aldosterone (regulates water and salt balance). Elevated cortisol may reflect long-term stress in offspring, and abnormal aldosterone may be associated with electrolyte imbalance.

[0107] As used herein, the term "blood lipid metabolism" refers to the concentration and metabolic status of lipid substances (such as total cholesterol, triglycerides, high / low density lipoprotein) in the blood. Dyslipidemia (such as hypercholesterolemia) may indicate the risk of cardiovascular disease or metabolic syndrome in offspring in the future.

[0108] Comparison to safety standard samples

[0109] As used herein, "nicotine pouch safety standard products" refers to nicotine pouch products approved by regulatory authorities in China, the United States, Japan, South Korea, Europe and other countries.

[0110] The "nicotine pouch safety standard products" described in this article include nicotine pouch products approved for sale by the U.S. Food and Drug Administration (FDA) through the premarket tobacco product application (PMTA) pathway that meet the public health standards set forth in the Family Smoking Prevention and Tobacco Control Act of 2009.

[0111] The "nicotine pouch reference standard" described herein includes the following products released by FDA for marketing authorization, each with two nicotine concentrations (3 mg and 6 mg): ZYN Chill, ZYN Cinnamon, ZYN Citrus, ZYN Coffee, ZYN Cool Mint, ZYN Menthol, ZYN Peppermint, ZYN Smooth, ZYN Spearmint, and ZYN Wintergreen (Philip Morris USA, New York, USA).

[0112] The indicators compared herein include the indicators described above.

[0113] Examples

[0114] Example 1 - Nicotine pouch release profile determination

[0115] Table 1. Nicotine pouch sources

[0116] Sample number Company Model Nicotine content 1 Philip Morris International ZYN Chill 6 mg 2 British American Tobacco VELO SOUR LIME MINI 9.5 mg 3* Hubei Tobacco R&D product 20 mg

[0117] *Hubei YCRI R&D product ingredients: nicotine 20 mg, microcrystalline cellulose, mannitol, menthol, propylene glycol, glycerin, and potassium sorbate.

[0118] Experimental materials and testing instruments

[0119] 1) Analytical balance (precision class), Sartorius, Germany;

[0120] 2) Shaker, Silek, China;

[0121] 3) Palm centrifuge, Thermo Fisher Scientific, USA;

[0122] 4) BBX24B-CE homogenizer, Bullet Blender Blue, USA;

[0123] 5) RNAfree pipette tips, various sizes of EP tubes, Takara, Japan;

[0124] 6) General EP tubes, Takara, Japan;

[0125] 7) Dry thermostat, Itslib Instrument Manufacturing Co., Ltd., Haimen, Jiangsu, China;

[0126] 8) Micropipettes of different sizes, Eppendorf, Germany;

[0127] 9) Ultra-low temperature refrigerator, Panasonic, Japan;

[0128] 10)QB-208 shaker, Haimen Kelinbel Co. Ltd, China;

[0129] 11)Constant temperature shaker, Changzhou Zhiborui Instrument Manufacturing Co. Ltd, China;

[0130] 12)Pressure steam sterilization pot, Hirayama, Japan;

[0131] 13)Carboxymethylcellulose sodium, sorbitol, potassium chloride, sodium chloride, magnesium chloride, calcium phosphate, sodium phosphate, nipagin, pH adjuster, alpha-amylase, lysozyme, lipase, peroxidase, Wuhan Seville Biological Technology Co. Ltd, China.

[0132] Experimental procedure

[0133] Using the above experimental materials and detection instruments, according to the conventional experimental method, artificial saliva was prepared: carboxymethylcellulose sodium was taken into 700 ml of distilled water, soaked overnight, after swelling, sorbitol, sodium chloride, magnesium chloride, potassium chloride, nipagin was added, after stirring, calcium phosphate and sodium phosphate solution was added, distilled water was added to the full amount, after stirring, 0.01% hydrochloric acid or 0.05% sodium hydroxide was added to adjust the pH to 7, finally the related enzymes were added, and the enzyme concentration was adjusted to alpha-amylase: 50-500 U / mL; Lysozyme: 1-10 μg / mL; Lipase: 0.5 U / mL; Peroxidase: 0.5 U / mL was bottled, and sterilized with 100 degree flow steam for 30 minutes. The saliva prepared in this way is colorless and translucent liquid, salty, with certain viscosity.

[0134] Take a complete nicotine bag sample and place it in 50 mL of 37°C constant temperature ultrapure water, interval time with 1 mL of pipette gun to take 0.5 mL solution in EP tube, after sampling, centrifuge at 3000 rpm for 1 minute, take the supernatant as the dissolved sample. The results of samples 1-3 are shown in Figures 1-3 .

[0135] Take a complete nicotine bag sample and place it in 50 mL of 37°C constant temperature artificial saliva, interval time with 1 mL of pipette gun to take 0.5 mL solution in EP tube, after sampling, centrifuge at 3000 rpm for 1 minute, take the supernatant as the dissolved sample.

[0136] The nicotine concentration was detected by national standard (YC / T 160-2002 and YC / T 468-2013)

[0137] Result analysis

[0138] For the use of ultrapure water as solvent, the nicotine dissolution curve of each sample was detected (take a complete nicotine bag sample and place it in 50 mL aqueous solution, take 0.5 mL solution in a non-enzyme EP tube with a 1 mL pipette every certain time, take sample every 10 s within the first 1 min, take sample every 30 s within 2-3 min, take sample every 1 min within 4-8 min, select 10 min, 12 min, 15 min, 20 min, 25 min, 30 min as sampling points after 8 min, gently shake the beaker before sampling, and all samples are used for rapid detection of nicotine dissolution). The comparison results of three samples of Philip Morris International-ZYN, British American Tobacco-Velo, and Hubei China Tobacco-research and development products were compared. The detection results of Philip Morris International-ZYN (nicotine content of 6 mg) showed that within the first 120 s, the nicotine release rate of ZYN was faster, and the concentration could reach 0.074144595 mg / mL at 120 s, and the peak concentration was 0.1 mg / mL. In summary, the nicotine release rate of ZYN was faster; the detection results of British American Tobacco-Velo (nicotine content of 10 mg) showed that within the first 120 s, the nicotine release rate of Velo was faster, and the concentration could reach 0.085028463 mg / mL at 120 s, and the peak concentration was 0.1328481 mg / mL. Given that the nicotine content of Velo is higher than that of ZYN, the peak concentration of Velo is also higher than that of ZYN; the detection results of Hubei China Tobacco-research and development products (nicotine content of 20 mg) showed that within the first 120 s, considering that the nicotine content of Hubei China Tobacco-research and development products was 20 mg, the concentration reached 0.118433615 mg / mL at 120 s, and the peak concentration was 0.196032423 mg / mL. Using artificial saliva as a solvent to extract the contents of nicotine bags, similar experimental results were obtained.

[0139] Example 2 - Reproductive safety evaluation of nicotine

[0140] Sources of experimental materials and detection instruments

[0141] 1) Test kit for testosterone ELISA, North Bio, China;

[0142] 2) PBS buffer, Thermo Fisher Scientific, USA;

[0143] 3) DAB staining solution, Gene Technology, China;

[0144] 4) 4% paraformaldehyde fixing solution, Servicebio, China;

[0145] 5) Triton, Servicebio, China;

[0146] 6) Tris, Servicebio, China;

[0147] 7) Glycerol, Servicebio, China;

[0148] 8) Primary antibody diluent, TBS buffer, Servicebio, China;

[0149] 9) StAR antibody (A16432), Abclonal Technology, China;

[0150] 10) 3β-HSD antibody (A8035), Abclonal Technology, China;

[0151] 11) RNA RNA Stabilization Reagent, Qiagen, Germany;

[0152] 12) reagent, Invitrogen, USA;

[0153] 12) Reverse transcription kit (DRR047A), Takara, Japan;

[0154] 13) RT-qPCR kit, Takara, Japan;

[0155] 14) Select Master Mix, Takara, Japan;

[0156] 15) Primers, Shanghai Tianyi Huayu Company, China;

[0157] 16) Isoflurane, Baxter Healthcare, USA;

[0158] 17) Analytical balance (precision class), Sartorius, Germany;

[0159] 18) General microscope, Nikon CKX41, Japan;

[0160] 19) Fluorescence inverted microscope, OLYMPUS, Japan;

[0161] 20) Oscillator, Silek, China;

[0162] 21) Small animal anesthesia machine, Hubei Ruwood Technology Co., Ltd., China;

[0163] 22) Microplate reader, BIO-TEK, USA;

[0164] 23) Microtome, LEICA, USA;

[0165] 24) Paraffin embedding machine, Hubei Fengyuan Medical Instrument Co., Ltd., China;

[0166] 25) Dehydration machine, Hubei Fengyuan Medical Instrument Co., Ltd., China;

[0167] 26) 384 fluorescence quantitative PCR instrument, Applied Biosystems, USA;

[0168] 27) Low-temperature high-speed refrigerated centrifuge, Thermo Fisher Scientific Inc., USA;

[0169] 28) Applied Biosystems StepOne Plus RT-qPCR instrument, Applied Biosystems, USA;

[0170] 29) NanoDrop 3000C UV spectrophotometer, Thermo Fisher Scientific, USA;

[0171] 30) Conventional PCR instrument, Applied Biosystems, USA;

[0172] 31) Palmtop centrifuge, Thermo Fisher Scientific Inc., USA;

[0173] 32) BBX24B-CE homogenizer, Bullet Blender Blue, USA;

[0174] 33) RNAfree pipette tips, EP tubes of various specifications, Takara, Japan;

[0175] 34) Ordinary EP tubes, cell culture dishes, and cell culture flasks, Takara, Japan;

[0176] 35) Dry incubator, Jiangsu Haimen Qilin Bell Instrument Manufacturing Co., Ltd., China;

[0177] 36) Micropipettes of different specifications, Eppendorf, Germany;

[0178] 37) Ultra-low temperature refrigerator, Panasonic Corporation, Japan;

[0179] 38) QB-208 shaking table, Qilin Bell Company, Haimen, China;

[0180] 39) Constant temperature shaker, Changzhou Zhiborui Instrument Manufacturing Co., Ltd., China;

[0181] 40) Pressure steam autoclave, Hirayama, Japan.

[0182] Whole experimental procedure

[0183] Table 2. Experimental groups

[0184]

[0185]

[0186] Using the above experimental materials and detection instruments, according to the conventional experimental method: select adult about 12 weeks old male Wistar rats, 10 in each group, according to the above table according to example 1, respectively, the contents of nicotine bag extraction liquid is gavaged, the gavage dose is 1 ml, the interval time is about 1 hour, and the duration is two months. After two months, each group of Wistar male mice is mated with 12-week-old Wistar female mice.

[0187] According to the above content, after the completion of administration and mating, the next morning, the female mouse vagina is examined, the vaginal secretion of the female mouse is taken with a cotton swab dipped in PBS to make a smear, and whether sperm is observed under a microscope is observed immediately. If a large number of sperm are observed, the animal is considered to be pregnant (if only a few sperm are observed, it is not considered to be pregnant), and the day is recorded as GD0. On GD20, a part of the pregnant mice in each group is randomly selected, anesthetized and killed, the fetal mice are taken out by laparotomy, one fetal mouse is taken from each litter, fetal blood is taken, and is stored at-80℃. The blood indexes of endocrine hormones, sex hormones, adrenal hormones and / or blood lipid metabolism are measured. The placental weight, placental index, fetal mouse weight, fetal mouse length, litter size, sex ratio, live fetus rate, dead fetus rate, resorption fetus rate are recorded. The development changes caused by the use of nicotine are judged by the above indexes.

[0188] After the completion of mating of male mice, 5 male Wistar rats are selected after exposure and mating, anesthetized with 2% isoflurane and killed. Blood is taken from the carotid artery for subsequent blood index detection. In addition, the testis and adrenal gland are frozen in liquid nitrogen and stored at-80℃ for subsequent detection. The right testis of another 5 male rats is taken, the exposure group is fixed with 4% paraformaldehyde for morphological index detection. At the same time, the left epididymis is taken, the epididymal tail is washed with 37℃ PBS preheated medium, and then 4 ml of PBS is added to a six-well plate. The epididymal tail is cut into pieces and incubated at 37℃ for 2-3 min. After the sperm suspension is absorbed, mixed and diluted, the diluted liquid is dropped into a counting plate. Sperm counting and motion video detection are performed under a light microscope. In addition, diluent is added to 4% paraformaldehyde and fixed for sperm morphology detection. The changes of reproductive function after the use of nicotine bag are judged by the above indexes.

[0189] Result analysis

[0190] The morphology and function of testis after mating (including the function of testosterone synthesis and spermatogenesis): HE staining results showed that the testis interstitial width of exposure group 1 was normal, the number of interstitial cells was normal, the arrangement of Sertoli cells was neat (see Figure 4 the red arrow mark), and the spermatogenic epithelium and seminiferous tubules did not have obvious pathological changes. Further through the immunofluorescence results, it was found that the testicular testosterone synthesis enzyme system had high expression abundance in the testicular interstitial cells, indicating that the testosterone synthesis function was at a normal level, as shown in Figure 5 Further detection of the spermatogenesis function of rats found that the sperm movement was fast, the movement distance was long, the movement frequency was high, there was no obvious abnormal morphological sperm (such as no head, no tail, etc.), and the number and activity of sperm were normal, as shown in Figure 6 In addition, all the offspring development indicators were normal.

[0191] The results of exposure groups 2, 3 and the control group were equivalent to exposure group 1 (safety standard), indicating the safety of the samples in the experiment in terms of reproductive development.

[0192] The terms and expressions based on the present document are used only to describe the present application. The present application should not be limited to these terms and expressions. The use of these terms and expressions does not mean that any illustration and description (or part thereof) is excluded. It should be recognized that various modifications can exist and should be included in the scope of the claims. Other modifications, changes and substitutions can also exist. Accordingly, the claims should be considered to cover all such equivalents.

[0193] Similarly, it should be noted that although the present application has been described with reference to the current specific embodiments, those skilled in the art should recognize that the above embodiments are only used to illustrate the present application, and various equivalent changes or substitutions can be made without departing from the spirit of the present application, therefore, any changes, modifications of the above embodiments within the scope of the spirit of the present application will fall within the scope of the claims of the present application.

Claims

1. A method for constructing a nicotine exposure model in male non-human animals, characterized in that: The following steps are involved: Step A: dissolving and extracting the contents of the nicotine bag to obtain a nicotine bag content extract; Step B: Select a male non-human animal and gavage the nicotine bag content extract to establish a nicotine exposure model for the male non-human animal.

2. The method for constructing a male non-human animal nicotine exposure model according to claim 1, wherein: The step A comprises: Step A1: immersing the nicotine pouch in a solvent; the immersion duration is 5-10 minutes, 10-20 minutes, 20-30 minutes, or 30-60 minutes; the temperature of the solvent is 35.0-36.0° C., 36.0-37.5° C., or 37.5-39.0° C.; the volume of the solvent used in each nicotine pouch is 5-15 mL / bag, 15-50 mL / bag, 50-100 mL / bag, or 100-200 mL / bag; Step A2: taking out the nicotine bag, centrifuging the remaining liquid and taking the supernatant to obtain the nicotine bag content extract; the nicotine bag content extract is stored in the dark.

3. The method for constructing a male non-human animal nicotine exposure model according to claim 2, wherein: The solvent is water or artificial saliva; The artificial saliva comprises water and one or more of the following components: sodium carboxymethylcellulose, sorbitol, potassium chloride, sodium chloride, magnesium chloride, calcium phosphate, sodium phosphate, paraben, distilled water, pH regulator, α-amylase, lysozyme, lipase, and peroxidase.

4. The method for constructing a male non-human animal nicotine exposure model according to claim 1, wherein: The non-human animal is a rat or a mouse, wherein the rat is 6-10 weeks old, 10-14 weeks old, or 14-22 weeks old; the mouse is 4-6 weeks old, 6-10 weeks old, or 10-16 weeks old.

5. The method for constructing a male non-human animal nicotine exposure model according to claim 1, wherein: The step B specifically comprises: gavage the male non-human animal with an interval length T, a gavage volume V per gavage, and a duration D; The interval duration T is 0.5-1.0 hours, 1.0-2.0 hours or 2.0-4.0 hours; The gavage volume V for each time is 0.5-1.0 mL, 1-1.5 mL or 1.5-2.0 mL; The duration D is 30-45 days, 45-60 days or 60-75 days.

6. The method for constructing a male non-human animal nicotine exposure model according to claim 1, wherein: The step B comprises constructing a nicotine pre-pregnancy exposure model for male non-human animals, wherein after oral administration for a duration of D, oral administration is stopped, and then the male non-human animals are mated with normal female non-human animals to produce offspring; and / or The step B includes constructing a nicotine exposure model for male non-human animals during pregnancy, wherein after the gavage duration D, gavage is continued until the female mouse successfully becomes pregnant, and then mates with a normal female non-human animal to produce offspring.

7. Use of the male non-human animal nicotine exposure model obtained by the method for constructing a male non-human animal nicotine exposure model according to any one of claims 1 to 6 for evaluating the reproductive developmental safety of nicotine pouches.

8. A method for evaluating the reproductive developmental safety of nicotine pouches in male non-human animals, characterized in that: The following steps are involved: Step C: using the male non-human animal nicotine exposure model obtained by the method for constructing a male non-human animal nicotine exposure model according to any one of claims 1 to 6 as a father and mating with a normal female non-human animal to produce offspring; Step D: Testing of paternal reproductive indicators and offspring development indicators; Step E: conducting a reproductive development safety evaluation of the nicotine pouch based on the paternal reproductive indicators and the offspring development indicators; The reproductive developmental safety evaluation of the nicotine pouch includes comparison with nicotine pouch safety standards.

9. The method for evaluating reproductive developmental safety of nicotine pouches in male non-human animals according to claim 8, wherein: The paternal reproductive indicators include: testosterone synthesis function assessment and / or sperm quality assessment; the testosterone synthesis function assessment includes: blood testosterone level, testosterone level in testicles and / or expression of key enzymes for testosterone synthesis; The sperm quality assessment includes: sperm density, sperm motility and / or sperm deformity rate.

10. The method for evaluating reproductive developmental safety of nicotine pouches in male non-human animals according to claim 8, wherein: The offspring development indicators include: basic reproductive output, offspring growth, placental development, offspring blood biochemical indicators and / or organ development and morphology, The basic information on reproductive output includes: litter size and / or sex ratio; The offspring growth conditions include: body weight and / or body length; The placenta development condition includes: placenta weight and / or placenta index; The offspring blood biochemical indicators include: endocrine hormones, sex hormones, adrenal hormones and / or blood lipid metabolism.

Images