Low-dose nicotine sustained-release administration preparation and use thereof

Abstract

The application provides a low-dose nicotine sustained-release drug preparation and an application thereof, and relates to the technical field of medicaments.The effective component of the preparation is (-)-di-L-(+)-nicotine tartrate dihydrate (purity is greater than or equal to 98.0 %), the drug is administered by subcutaneous implantation of an osmotic pressure driven sustained-release pump, and the administration dose is 0.1-0.3 mg / kg / d.The administration preparation of the application can significantly improve depressive and anxious behaviors, restore intestinal barrier function and microbial homeostasis, reshape a central 5-hydroxytryptamine (5-HT) system and is related to intestinal flora metabolism, and has good safety.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a low-dose nicotine sustained-release formulation and its application. Background Technology

[0002] Depression is a common mental disorder, clinically characterized by persistent low mood, loss of interest, anhedonia, cognitive impairment, and hopelessness, severely impacting patients' social functioning and quality of life. In recent years, the prevalence and disability rates of depression have been rising globally, becoming one of the major public health problems second only to cardiovascular disease. The World Health Organization (WHO) reports that the number of people suffering from depression worldwide has exceeded 380 million and is increasing annually, seriously threatening human health. Currently, clinical treatment mainly involves medication, including selective serotonin reuptake inhibitors (SSRIs), serotonin-norepinephrine reuptake inhibitors (SNRIs), and monoamine oxidase inhibitors (MAOIs). However, these traditional antidepressants generally suffer from slow onset of action (usually 4-6 weeks), unstable efficacy, significant individual variability, and frequent adverse reactions, leading to poor patient compliance and high relapse rates. Therefore, developing novel antidepressant treatment strategies with novel mechanisms of action, rapid onset of action, and high safety is an urgent scientific problem to be solved.

[0003] Studies have shown that the occurrence of depression is closely related to the dysfunction of monoamine neurotransmitters in the brain, especially the serotonin (5-HT) system. 5-HT is an important neurotransmitter regulating behaviors such as mood, motivation, and cognition, and the dorsalraphe nucleus (DRN) is one of the main areas in the brain where 5-HT is synthesized and released. In recent years, multiple clinical and animal studies have found that nicotine, as the main bioactive alkaloid in tobacco, can activate 5-HTergic neurons in the DRN and enhance 5-HT release, showing certain antidepressant potential. In addition, nicotine also has certain neuroprotective, anti-inflammatory, and cognitive-improving effects, which can provide new ideas for the treatment of mood disorders. However, traditional nicotine administration methods (such as injection, patch, and oral administration) have significant shortcomings: first, the blood drug concentration fluctuates greatly after administration, making it difficult to maintain a stable efficacy level; second, peak concentrations can easily induce activation of the central reward system, thereby increasing the risk of addiction; and third, frequent injections can increase stress responses in animals, interfering with experimental results. These problems seriously limit the development and application of nicotine in the treatment of depression. Therefore, there is an urgent need to develop a low-dose, sustained-release nicotinic drug administration strategy that can effectively avoid the addiction risk caused by peak concentrations while maintaining a relatively stable blood drug concentration, thereby fully exerting its regulatory effect on the central 5-HT system and achieving safe and effective antidepressant treatment.

[0004] The gut-brain axis refers to the bidirectional regulatory network between the gut and the central nervous system through multiple pathways, including neural, immune, and endocrine pathways. Recent studies have shown that, in addition to maintaining gastrointestinal homeostasis, the gut microbiota can influence mood and neurological function through mechanisms such as short-chain fatty acid metabolism, inflammation regulation, and tryptophan-5-hydroxytryptamine (5-HT) metabolism. Dysbiosis is believed to be associated with impaired 5-HT synthesis, elevated inflammatory factors, and changes in neural plasticity. Microecological interventions such as probiotics, prebiotics, and fecal microbiota transplantation have been observed in some studies to improve depression-related behaviors, suggesting the potential interventional value of the gut microbiota. However, existing research has largely focused on traditional probiotics such as *Lactobacillus* and *Bifidobacterium*, while studies on the dominant mucus-layer bacteria *Akkermansia muciniphila* (…) have been less explored. A. muciniphila The specific role of nicotine in mood disorders and its involvement in multiple pathways such as intestinal barrier function, inflammatory response, metabolic regulation, and neurotransmitter regulation remain unclear, and the relevant mechanisms are still under investigation. Currently, research on whether low-dose nicotine can participate in mood regulation through the peripheral system, particularly by regulating the gut microbiota, is relatively limited, and verifiable causal evidence is lacking. In particular, it is unclear whether nicotine regulates specific bacterial genera, and whether such changes are related to gut homeostasis and 5-HT metabolism.

[0005] In summary, the existing technology has the following shortcomings: (1) A. The specific mechanism of action of muciniphila in depressive disorders is not yet clear, and its functional validation is insufficient; (2) There is a lack of direct experimental support as to whether low-dose nicotine can exert behavioral regulatory effects by modulating gut microbiota; (3) There are few reports on the transferability verification of nicotine-related bacterial community changes and the functional verification of specific bacterial genera in existing studies; (4) Existing studies are mostly observations at the single-mechanism level, lacking a multi-link integrated mechanism model based on the gut-brain axis; The aforementioned technological gaps provide the necessary background for proposing new technical solutions in this invention. Summary of the Invention

[0006] This invention addresses the current technical problems of nicotine in existing technologies, such as slow onset of action, unstable efficacy, large individual variability, frequent adverse reactions, and limited application scope. It provides a low-dose, sustained-release nicotine formulation and its application. By employing a sustained-release strategy, this invention can maintain stable blood drug concentrations under low-dose conditions, improve efficacy consistency, and reduce the risk of addiction. It reveals that nicotine works by upregulating... A. muciniphilaA novel mechanism by which abundance improves depression and a new use for nicotine to synergistically improve depression are proposed, expanding the application of microecological therapy in the field of mental illness.

[0007] One objective of this invention is to provide a low-dose sustained-release nicotine formulation, wherein the active ingredient of the formulation is (-)-di-L-(+)-nicotine dihydrate (TCI), with a purity ≥98.0%, and is administered subcutaneously via an osmotic pressure-driven sustained-release pump at a dose of 0.1-0.3 mg / kg / day.

[0008] Preferably, the dosage is 0.2 mg / kg / day.

[0009] Preferably, the slow-release pump is selected from Alzet®2006 osmotic pump, Alzet®1002 osmotic pump or Alzet®1004 osmotic pump, with an effective volume of 200 μL and a release rate of 0.11-0.25 μL / h.

[0010] Preferably, the slow-release pump is implanted in the subcutaneous tissue of the back of the scapula of the experimental animal, with the outlet fixed towards the tail to avoid the animal biting and reduce mechanical damage.

[0011] Preferably, the solvent of the preparation is selected from 0.9% sodium chloride injection, PBS buffer, or glycerol isotonic buffer.

[0012] Preferably, the formulation is administered subcutaneously for a period of 4 weeks.

[0013] A second aspect of the invention relates to the use of the above-described drug formulation in the preparation of a medicament for treating depression or depressive-like behavior.

[0014] Preferably, the drug induces an increase in the abundance of Akkermansia myxophilus.

[0015] Compared with the prior art, the present invention has the following beneficial effects: the drug delivery formulation of the present invention can significantly improve depressive-like and anxiety-like behaviors, restore intestinal barrier function and microbial homeostasis, remodel the central 5-hydroxytryptamine (5-HT) system and is related to intestinal flora metabolism, and has good safety. Attached Figure Description

[0016] Figure 1 : Schematic diagram of the nicotine sustained-release drug delivery device and implantation method of the present invention; wherein: 1-osmotic pump body, 2-drug storage chamber, 3-semi-permeable membrane shell, 4-release catheter, 5-subcutaneous tissue on the back of a mouse; Figure 2 : Effect of subcutaneous sustained-release nicotine intervention on depressive-like and anxiety-like behaviors in CUMS mice; Figure 3 The weakening effect of antibiotic pretreatment on the antidepressant-like effects of nicotine; Figure 4 Nicotine reverses CUMS-induced gut microbiota dysbiosis and A. muciniphila Abundance decreased; Figure 5 Nicotine restores intestinal barrier integrity and alleviates colonic inflammation in CUMS mice; Figure 6 Nicotine regulates the tryptophan-serotonin metabolic pathway in CUMS mice through gut microbiota. Figure 7 Transplantation of nicotine-derived microbiota and A. muciniphila Supplementing the ameliorative effect on behavior and 5-HT metabolism in CUMS mice; Figure 8 Transplantation of nicotine-derived microbiota and A. muciniphila It supplements the restorative effect on the gut microbiota structure; Figure 9 Transplantation of nicotine-derived microbiota and A. muciniphila It supplements the repair of the intestinal barrier and alleviates inflammation. Detailed Implementation

[0017] The following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the invention. Those skilled in the art can make any alterations or modifications to the invention, and these equivalent changes and modifications also fall within the scope defined by the claims.

[0018] Experimental Example 1: An experimental study on the improvement of depressive-like behavior in CUMS mice by different doses of sustained-release nicotine administration 1. Experimental Grouping and Methods SPF-grade male C57BL / 6J mice aged 6-8 weeks were selected and randomly divided into 5 groups (n=8-10 per group) after 1 week of acclimatization. All mice were housed in a stable environment and, except for the control group, received continuous chronic unpredictable mild stress (CUMS) treatment for 4 weeks. This included diurnal variation, restraint, cold / heat stimulation, wet bedding, noise, tail clips, and fasting / water deprivation. One stressor was randomly applied daily, with no consecutive repetitions. The treatments for each group are shown in Table 1 below.

[0019] Table 1: Grouping List

[0020] Nicotine was continuously released via subcutaneous implantation using an osmotically driven micro-release pump (Alzet® 2006). The active ingredient, (-)-2-L-(+)-tartrate nicotine dihydrate, was dissolved in 0.9% sodium chloride injection solution, sterilized through a 0.22 μm filter, and then loaded into the osmotically driven release pump (Alzet® 2006, 200 μL capacity, release rate 0.15 μL / h, DURECT, US). After pre-equilibration in PBS (Gibco, 10010023) at 37°C for 4 hours, the pump was implanted subcutaneously in the dorsal scapula of C57BL / 6J mice under isoflurane anesthesia (RWD Life Science), with the release catheter fixed towards the tail. A schematic diagram of the nicotine sustained-release delivery device and implantation method is shown below. Figure 1 As shown in Table 2. Weight was continuously monitored during the 4-week experiment. Behavioral tests were performed at the end of the fourth week of the experiment.

[0021] Table 2: Behavioral Tests

[0022] Statistical analysis method: One-way ANOVA + post-hoc test; data are expressed as mean ± SEM.

[0023] 2. Experimental Results The results are as follows Figure 2 As shown in the figure. A represents the randomization of mice into 5 groups (n=8-10 per group) after a 1-week acclimatization period: control group (no stress), CUMS group, CUMS + low-dose nicotine group (Nic-L, 0.05 mg / kg / day, 4 weeks), CUMS + medium-dose nicotine group (Nic-M, 0.2 mg / kg / day, 4 weeks), and CUMS + high-dose nicotine group (Nic-H, 0.5 mg / kg / day, 4 weeks); B represents daily weight monitoring during the 4-week experiment; C represents the sucrose preference test (SPT); D represents the forced swimming test (FST); E represents the tail suspension test (TST); F represents the dwell time in the central region of the open field test (OFT); G represents the distance moved in the central region of the open field test (OFT); H represents the dwell time on the open arm of the elevated cross maze test (EPM); I represents the distance moved on the open arm of the elevated cross maze test (EPM); and J represents the representative movement trajectories in the OFT and EPM experiments. Statistical significance was determined using one-way ANOVA with post-hoc tests: ns (no significant difference, P ≥ 0.05); compared with the control group, # P<0.05, ## P<0.01, ### P<0.001; compared with the CUMS group, P<0.05, P<0.01, P<0.001.

[0024] (1) Weight changes ( Figure 2 (B) The CUMS group showed slow weight gain, while the Nic-M and Nic-H groups showed a better trend in weight loss compared to the CUMS group.

[0025] (2) Sucrose Preference Experiment (SPT) Figure 2 (C) CUMS significantly reduced sucrose preference (P<0.001). The Nic-M group significantly restored sucrose preference (P<0.01), and the Nic-H group also showed improvement. The Nic-L group showed no significant restoration.

[0026] (3) Forced swimming test (FST) Figure 2 (D) and the suspended tail test (TST) Figure 2 (E): Immobility time was significantly increased in the CUMS group (P<0.001). Immobility time was significantly decreased in the Nic-M group (P<0.001). High-dose Nic-H also decreased immobility time, but showed a tendency towards overactivity. Nic-L showed no improvement.

[0027] (4) Open Field Experiment (OFT) Figure 2 Central region exploration was reduced in F and G CUMS mice. Nic-M treatment significantly increased the time spent in the central region and the distance traveled in the central region (P<0.01). Nic-H treatment showed some improvement, but it was not as stable as Nic-M treatment.

[0028] (5) Elevated Cross Maze Experiment (EPM) Figure 2 In H and I CUMS mice, the time spent in the open arm and the distance traveled decreased. Nic-M mice significantly recovered their open arm exploration behavior (P<0.01). Nic-H mice showed some improvement. Nic-L mice showed no significant change.

[0029] (6) Representative motion trajectory ( Figure 2 In the J)CUMS group, the trajectory density was concentrated in the edge region; the exploration pattern of the Nic-M group tended to be normal, similar to the control group.

[0030] 3. Summary This embodiment demonstrates that a medium-dose sustained-release administration of nicotine (0.2 mg / kg / day) significantly improved depressive-like and anxiety-like behaviors in CUMS mice, including: recovery of anhedonia (SPT), reduced behavioral hopelessness (FST / TST), improved anxiety-like behavior (OFT / EPM), and normalization of activity trajectories. Low doses (0.05 mg / kg / day) did not significantly improve depressive-like behaviors, while high doses (0.5 mg / kg / day), although effective, showed a tendency towards hyperactivity in some indicators.

[0031] Therefore, in the technical system of this invention, 0.2 mg / kg / d is the optimal therapeutic dose, which combines effectiveness and safety, and provides a clear basic model for subsequent microbiota analysis, intestinal barrier detection and FMT experiments.

[0032] Example 2: Antibiotic-mediated bacterial depletion (ABX) significantly attenuated the antidepressant-like effect of sustained-release nicotine. 1. Experimental Grouping and Methods This embodiment aims to verify whether the antidepressant effect of nicotine depends on the gut microbiota. SPF-grade male C57BL / 6J mice (6-8 weeks old) were selected and randomly divided into the following five groups (n=10 in each group) after 1 week of acclimatization. The experimental groups and treatment methods are shown in Table 3.

[0033] Table 3: List of Experimental Groups

[0034] (1) Construction of pseudo-sterile model: Vancomycin 100 mg / kg, Neomycin sulfate 200 mg / kg, Metronidazole 200 mg / kg, and Ampicillin 200 mg / kg were administered by gavage once daily for 7 days. Except for the ABX treatment group, the other groups received an equal volume of physiological saline. Feces were collected before antibiotic treatment (week 0) and after treatment (week 1), and the fecal DNA concentration was measured to reflect the degree of bacterial clearance.

[0035] (2) The methods for establishing the CUMS model and conducting behavioral tests (SPT, FST, TST, OFT, EPM) are the same as in Example 1.

[0036] 2. Experimental Results The results are as follows Figure 3As shown in the figure, A represents the body weight of mice at week 0; B represents the results of the sugar preference test (SPT) at week 0; and C represents the experimental design for the antibiotic intervention on the antidepressant effect of nicotine. After one week of acclimatization, mice were randomly divided into five groups: control group, chronic unpredictable stress group (CUMS), CUMS+Nic-M group, antibiotic-treated CUMS group (ABX-CUMS), and antibiotic-treated CUMS+Nic-M group (ABX-CUMS+Nic-M). Before the formal experiment began, the ABX-CUMS and ABX-CUMS+Nic-M groups received antibiotic treatment for one week, while the other groups received an equal volume of saline. D represents the gut microbiota content (fecal DNA concentration) of mice in the ABX-CUMS group and ABX-CUMS+Nic-M group after 1 week of antibiotic treatment, compared with the CUMS group and CUMS+Nic-M group; E represents the body weight of mice at week 5; F represents the results of the sugar water preference test (SPT) at week 5; G represents the results of the forced swimming test (FST) at week 5; H represents the results of the tail suspension test (TST) at week 5; I represents the movement trajectory of mice in the open field test (OFT) and elevated cross maze test (EPM) at week 5; J represents the distance of movement in the central area of ​​the open field test (OFT); K represents the distance of movement in the open arm of the elevated cross maze test (EPM). Data are expressed as mean ± standard error (SEM) (n=10 per group); statistical significance was determined by one-way ANOVA with post-hoc test: ns (no significant difference, P≥0.05); compared with the control group, # P<0.05, ## P<0.01, ### P<0.001; compared with the CUMS group, P<0.05, P<0.01, P<0.001.

[0037] (1) Antibiotics significantly reduce the content of intestinal flora ( Figure 3 After one week of ABX treatment, the fecal DNA concentration in mice decreased by approximately 80%, significantly lower than that in the CUMS group and the CUMS+Nic-M group (P<0.001). These results confirm that ABX treatment successfully disrupted the gut microbiota, achieving a pseudo-sterile (PGF) state.

[0038] (2) Weight changes Figure 3 The weight of CUMS mice decreased significantly. The Nic-M group partially recovered its weight (P<0.05). The weight improvement effect of ABX treatment disappeared, and there was no significant difference between the ABX+CUMS+Nic-M group and the CUMS group.

[0039] (3) Sucrose Preference Experiment (SPT) Figure 3 In the CUMS group (F), sucrose preference decreased significantly (P<0.001). In the Nic-M group, sucrose preference recovered (P<0.01). After ABX cleared the bacterial community, the effect of nicotine on restoring sucrose preference completely disappeared (P>0.05).

[0040] (4) FST and TST ( Figure 3 The immobility time was significantly increased in the G and H CUMS groups (P<0.001). The immobility time was significantly decreased in the Nic-M group (P<0.001). The immobility time in the ABX+Nic-M group was similar to that in the CUMS group, indicating a significant weakening of the nicotinic antidepressant-like effect.

[0041] (5) OFT and EPM behavioral indicators Figure 3 In the I–K)CUMS mice, central area activity was reduced and open-arm exploration was decreased. The Nic-M group showed a significant recovery in exploration behavior (P<0.01). The ABX+Nic-M group showed no improvement in exploration level, similar to the CUMS group.

[0042] 3. Summary The results of this embodiment indicate that antibiotic cocktails effectively clear the gut microbiota of mice (fecal DNA decreased by approximately 80%). The antidepressant-like effect of nicotine on CUMS mice was significantly weakened or completely eliminated under conditions of microbiota clearance. Behavioral recovery was as follows: loss of pleasure (SPT) was not restored, immobility time (FST / TST) did not decrease, and anxiety-like behavior (OFT / EPM) was not improved. There were no significant differences between the ABX+CUMS+Nic-M group and the CUMS group in all core behavioral indicators. In summary, this embodiment clearly demonstrates that the antidepressant effect of nicotine is gut microbiota-dependent. Gut microbiota integrity is a necessary condition for nicotine to improve depressive-like behavior. This embodiment thus establishes the causal basis of the "nicotine-microbiota-behavior" chain and lays the foundation for subsequent studies on... A. muciniphila This provides key theoretical support for the verification.

[0043] Example 3: Low-dose nicotine sustained-release administration by adjusting A. muciniphila Experimental study on restoring gut microbiota homeostasis, repairing the intestinal barrier, and improving 5-HT metabolism. 1. Experimental Grouping and Methods (1) SPF-grade male C57BL / 6J mice (6–8 weeks old) were selected and randomly divided into three groups after 1 week of acclimatization (n=7-8 in each group). The specific groups and treatment methods are shown in Table 4.

[0044] Table 4: Specific Groups and Treatment Methods

[0045] In the Nic-M group, an Alzet® 2006 osmotic pump was implanted, releasing nicotine at a rate of 0.2 mg / kg / day for four consecutive weeks, consistent with Example 1.

[0046] During the experiment, feces were collected regularly for metagenomic sequencing and short-chain fatty acid analysis; colon tissue was collected for HE staining, immunofluorescence, and inflammatory factor determination; hippocampus, serum, and feces were collected to detect 5-HTP, 5-HT, and 5-HIAA, and to detect the expression of related genes.

[0047] (1) Metagenomic sequencing Mouse fecal samples were collected (avoiding stress days, all samples were collected at 9:00 AM), immediately flash-frozen in liquid nitrogen, and stored at -80°C for later use. Total DNA was extracted using the EZNA® Stool DNA Kit (Omega Bio-tek), metagenomic sequencing was performed using the MGISEQ-2000 platform (BGI, China), and species annotation was performed based on Kraken2 + Bracken. A non-redundant gene set was constructed using MetaGeneMark + CD-HIT, gene abundance was quantified using Salmon, and functional pathways were annotated using the KEGG database. Microbial α-diversity was assessed using the Shannon and Simpson indices, and β-diversity was analyzed using principal coordinate analysis (PCoA) based on the Bray–Curtis distance. Microbial community composition and differential abundance were analyzed using LEfSe, with a threshold set at LDA score > 2.0 and p < 0.05.

[0048] (2) Detection of short-chain fatty acids (SCFAs) The content of major short-chain fatty acids, including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid, in the colon contents of mice in the control group (CON), CUMS group, and nicotinic acid sustained-release group (Nic-M) was detected. Sample pretreatment: Approximately 50 mg of mouse colon contents was added to 400 μL of 50% methanol (HPLC grade, Merck) and magnetic beads, and homogenized thoroughly. The mixture was centrifuged at 20,000 × g for 10 min at 4 °C. 200 μL of the supernatant was collected, and 10 μL of internal standard solution (isooctanoic acid, Sigma-Aldrich) was added, followed by 5 μL of 0.8 M NaOH. After thorough mixing, the mixture was freeze-dried under vacuum. The residue was redissolved in 180 μL of ethanol and 20 μL of 0.4 M succinic acid solution, precipitated at -20 °C for 2 h, and then centrifuged at 4 °C for 20,000 × g for 10 min. The supernatant was then injected for analysis.

[0049] GC-MS detection: An Agilent 7890B gas chromatograph with a 5977B mass spectrometer detector (Agilent Technologies, USA) was used, equipped with a DB-FFAP capillary column (30m × 0.25mm × 0.25μm). Injection volume: 1μL, split ratio 20:1; carrier gas: high-purity helium, flow rate 1mL / min; injection port temperature: 250℃, transfer line temperature: 240℃; temperature program: initial 80℃ held for 1min → increased to 180℃ at 10℃ / min → increased to 240℃ at 20℃ / min held for 2min, run time 16min; detection mode: electron impact (EI, 70eV), selected ion monitoring (SIM) mode, solvent delay 4min; the standard curve was prepared using a series of gradient dilutions of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid standards (Sigma-Aldrich), with a linear correlation coefficient R² > 0.999.

[0050] (3) Hematoxylin-eosin (H&E) staining of colon tissue Mice euthanized after experimental treatment had their colon segments (approximately 0.5 cm in length) harvested immediately after opening the abdominal cavity. The tissue was rapidly placed in 10% paraformaldehyde fixative and fixed at room temperature for 24 hours. The fixed tissue was then subjected to a gradient dehydration process using 50%, 70%, 90%, and 100% (anhydrous ethanol), followed by xylene clearing and paraffin embedding. The embedded tissue was sectioned using a Leica RM2135 rotary microtome (Germany) at a thickness of 5 μm. The sections were dewaxed in xylene for 10 min, then rehydrated in 100%, 90%, 70%, and 50% ethanol, respectively, and rinsed with distilled water for 5 min. Hematoxylin and eosin (H&E) staining was performed according to standard procedures: first, hematoxylin staining, followed by differentiation with acidic ethanol, blue resizing with alkaline blue solution, and finally eosin staining. After dehydration, clearing, and mounting, the stained sections were observed and photographed under an Olympus microscope (Japan).

[0051] (4) Immunofluorescence (IF) staining of colon tissue Approximately 0.5 cm of distal colon was harvested, rinsed with PBS, and fixed in 4% paraformaldehyde (Servicebio, G1101) for 24 h. After graded alcohol dehydration, paraffin embedding, and sectioning (4 μm thickness), intestinal barrier-related proteins were detected using immunofluorescence staining: primary antibodies: rabbit anti-ZO-1 (1:1000, GB115686, Servicebio), rabbit anti-Occludin (1:1000, GB111401, Servicebio), rabbit anti-MUC2 (1:1000, GB11344, Servicebio); secondary antibody: Cy3-labeled goat anti-rabbit IgG (1:400, GB21303, Servicebio); nuclear staining: DAPI (G1012, Servicebio). Images were acquired using a laser confocal microscope (Nikon Digital Sight DS-FI2, Japan), and the average density of protein fluorescence signals was calculated using the Aipathwell image analysis system (Servicebio).

[0052] (5) Enzyme-linked immunosorbent assay (ELISA) of inflammatory factors in colon and hippocampus tissues Colon tissue blocks were homogenized in pH 7.4 PBS solution. The homogenate was centrifuged at 11,000×g for 20 min at 4℃, and the supernatant was collected for analysis. The total protein concentration of the samples was determined using a BCA protein quantification kit (PC0020, Solarbio, Beijing). Subsequently, the following indicators were detected using an ELISA kit (Enzyme-Linked Biotechnology, Shanghai): IL-6 (JL20268), IL-1β (JL18442), TNF-α (JL10484), TPH1 (JL43839), ZO-1 (JL20409), and Occludin (JL20408) in colon and hippocampal tissues.

[0053] For serum samples containing IL-6, IL-1β, and TNF-α (using the same kit as above), the absorbance (OD value) was measured according to the standard curve provided in the kit, and the actual concentration was calculated.

[0054] (6) Targeted detection of hippocampal neurotransmitters Hippocampus tissue from mice was collected, and the levels of 5-HT, 5-hydroxytryptophan (5-HTP) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were detected by UHPLC-QTOF-MS / MS (Waters I-Class+ABSciex 6500, USA).

[0055] (7) Real-time quantitative PCR (RT-qPCR) detection of gene expression in colon tissue Total RNA was extracted from colon tissue using the SteadyPure Universal RNA Extraction Kit (AG11732, Agbio, Hunan). An equal volume of RNA was used for reverse transcription according to the Evo M-MLV Reverse Transcription and gDNA Removal Kit (AG11705, Agbio) instructions to obtain cDNA. The obtained cDNA was diluted 1:10 and used as a template for subsequent PCR. SYBR Green Pro Taq HS premixed reagent (AG11701, Agbio) and a QuantStudio™ 6 Flex real-time PCR system (Applied Biosystems, USA) were used. PCR reaction conditions included 95°C for 30 s (pre-denaturation); 40 cycles: 95°C for 5 s; 60°C for 30 s; 72°C for 2 min (extension). Genes detected included IL-6, IL-1β, TNF-α, ZO-1, Occludin, Tph1, and Ido1.

[0056] The internal reference gene is GAPDH. Relative expression levels are calculated at 2... ΔΔCt Method calculation.

[0057] (8) Multi-omics association analysis To explore the association mechanism between gut microbiota, metabolites, and the nervous system in brain regions, a multi-omics joint analysis was conducted based on gut metagenomics and hippocampal targeted neurotransmitter metabolomics data. Spearman correlation analysis showed that the top 20 bacterial genera were associated with acetic acid, propionic acid, and... Akkermansia Relative abundance analysis 2. Experimental Results See results Figures 4-6 .

[0058] Figure 4In the diagram, A represents the assessment of gut microbiota richness and evenness using α-diversity indices (Shannon, Simpson); BC represents β-diversity analysis using principal coordinate analysis (PCoA) and box plots based on Bray-Curtis distance to assess differences in gut microbiota composition among different groups; D represents a comparison of changes in gut microbiota composition among different experimental groups at the phylum level; E represents a phylogenetic descent diagram based on LEfSe analysis showing taxa with significant differences in abundance among different groups at the family level; F represents visualization of genus-level changes using STAMP, highlighting genera that showed significant alterations under CUMS and nicotine treatments; G represents a quantitative analysis of relative abundance at the species level, with particular attention to changes in *A. muciniphila* after nicotine treatment; H represents a violin plot showing the distribution and differences in *A. muciniphila* abundance among groups; IJ represents PCoA and Bray-Curtis box plots based on microbial gene profiles revealing trends in metabolic potential; and K represents KEGG pathway enrichment analysis identifying microbial functional pathways affected by treatment. Data are expressed as mean ± standard error (SEM) (n=7-8 per group); statistical significance was determined by Kruskal–Wallis or LEfSe test (as appropriate), with significance indicated as follows: ns (no significant difference, P≥0.05); P<0.05; P<0.01; P<0.001.

[0059] Figure 5 In the table, A represents representative H&E-stained sections of colon tissue from the control group, CUMS group, Nic-M group, and ABX+Nic-M group; BC represents the immunofluorescence staining results of tight junction proteins ZO-1 and Occludin; DE represents the quantitative analysis of the fluorescence intensity of ZO-1 and Occludin; F, H, and M represent the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in colon tissue as measured by ELISA; G and L represent the longitudinal changes in the concentrations of acetic acid and propionic acid in colon tissue; data are expressed as mean ± standard error (SEM) (n=6-10 per group). Statistical significance is indicated as follows: # P<0.05, ## P<0.01, ### P<0.001 compared with the control group; *P<0.05, **P<0.01, ***P<0.001 compared with the CUMS group.

[0060] Figure 6In this context, AC represents the content of 5-HTP (A) and 5-HT (B) in the hippocampus and the 5-HIAA / 5-HT ratio (C); DF represents the content of 5-HTP (D) and 5-HT (E) in serum and the 5-HIAA / 5-HT ratio (F); GI represents the content of 5-HTP (G) and 5-HT (H) in feces and the 5-HIAA / 5-HT ratio (I); JL represents the gene expression of Tph2 (J) in the hippocampus and Tph1 (K) and Ido1 (L) in the colon. The data were presented as mean ± standard error (SEM) (n=6–8 per group). M represents the Spearman correlation heatmap, showing the correlation between representative gut microbiota and various host parameters, including behavioral indicators, short-chain fatty acid (SCFA) levels, inflammatory factors (IL-1β, IL-6, TNF-α), serotonin metabolites (Trp, 5-HTP, 5-HT, 5-HIAA), and rate-limiting enzymes (Tph1, Tph2, Ido1). Data were expressed as mean ± standard error (SEM) (n=6–8 per group). Statistical significance was assessed using one-way ANOVA with post-hoc tests: ns (no significant difference, P≥0.05); #P<0.05, ##P<0.01, ###P<0.001 compared to the control group; P<0.05, P<0.01, P<0.001 compared with the CUMS group.

[0061] (1) Nicotine significantly restored the diversity and structural diversity of the gut microbiota in CUMS mice (Shannon and Simpson indices), indicating a significant decrease in richness and evenness in the CUMS group (P<0.01). After Nic-M treatment, α-diversity significantly increased, approaching that of the control group (P<0.05). β-diversity (PCoA, Bray–Curtis) showed that the microbiota structure in the Nic-M group deviated significantly from that in the CUMS group, clustering towards the control group. At the phylum level: Firmicutes, Bacteroidota, and Verrucomicrobiota showed significant changes in the CUMS group. The Nic-M group restored the CUMS-induced disorder, with a significant increase in A. muciniphila, a key member of Verrucomicrobiota (P<0.01). LEfSe further showed that the CUMS group was enriched with inflammation-related microbiota (e.g., certain Enterobacteriaceae). The Nic-M group was significantly enriched with A. muciniphila and metabolically health-related genera. STAMP showed that multiple probiotic genera involved in mucus layer maintenance and SCFA synthesis were significantly restored (P<0.05).

[0062] (2) Nicotinic acid restores intestinal barrier integrity. HE staining showed that in the CUMS group, the colonic villus structure was damaged, the crypts were irregular, and the infiltration of inflammatory cells was increased. In the Nic-M group, the colonic tissue structure was significantly restored and the inflammation was reduced. Immunofluorescence and quantification showed that the expression of ZO-1 and Occludin was significantly decreased in CUMS (P<0.001). In the Nic-M group, the expression of both was significantly restored (P<0.01), indicating that the barrier function was restored.

[0063] (3) Nicotine significantly reduced intestinal inflammation levels. ELISA showed that the levels of IL-1β, IL-6, and TNF-α were significantly increased in the CUMS group (P<0.01). Nic-M treatment significantly reduced the levels of the three inflammatory factors (P<0.05–0.01).

[0064] (4) Short-chain fatty acids (SCFAs) showed significant recovery. Acetic acid and propionic acid content decreased significantly in the CUMS group (P<0.05). In the Nic-M group, SCFAs increased significantly (P<0.05), indicating the recovery of microbial metabolic function.

[0065] (5) Nicotine improves 5-HT (5-HT) metabolic disorders. Results from hippocampal, serum, and fecal tests consistently showed that in the CUMS group, 5-HTP and 5-HT were significantly decreased, and the 5-HIAA / 5-HT ratio was increased (P<0.001). In the Nic-M group, 5-HTP and 5-HT were significantly restored, and the 5-HIAA / 5-HT ratio was reduced (P<0.05–0.01). In terms of gene expression, Nic-M significantly upregulated the rate-limiting enzymes Tph1 / Tph2 and inhibited Ido1 (P<0.05), causing tryptophan metabolism to shift back from the kynurenine pathway to 5-HT synthesis.

[0066] (6) Correlation analysis (Spearman) Akkermansia Significantly positively correlated with ZO-1, Occludin, SCFAs, and 5-HT (P<0.05). Significantly negatively correlated with IL-1β, IL-6, and TNF-α.

[0067] 3. Summary This embodiment illustrates that low-dose sustained-release nicotine can significantly restore CUMS-induced gut microbiota imbalance, especially significantly improve... A.muciniphila Relative abundance. Intestinal barrier structure and function are repaired, tight junction protein expression is restored, and inflammation levels are reduced. Microbial metabolites SCFAs are significantly increased, and the serotonin metabolic pathway is fully restored. A complete gut-brain pathway mechanism chain is formed between microbes, the barrier, metabolism, and neurotransmitters. A.muciniphila It is an important effector bacterium for nicotine to exert its antidepressant effect.

[0068] Example 4: Nicotine-derived microbiota transplantation (Nic-FMT) and A.muciniphila Supplements can improve depressive-like behaviors and restore 5-HT metabolism through the gut-brain axis. 1. Experimental Grouping and Methods Using PGF mice as receptors, this study aimed to verify whether nicotine exerts an antidepressant effect through gut microbiota and to validate... A.muciniphila Its key role.

[0069] After one week of adaptation, the mice were divided into five groups. The specific grouping and treatment methods are shown in Table 5.

[0070] Table 5: Specific Grouping and Processing Methods

[0071] During the experiment, feces were collected periodically for metagenomic sequencing and short-chain fatty acid analysis; colon tissue was collected for HE staining, immunofluorescence, and inflammatory factor assay; hippocampus, serum, and feces were collected to detect 5-HTP, 5-HT, and 5-HIAA, and to detect the expression of related genes, consistent with Example 3. The PGF construction method was consistent with Example 2.

[0072] 2. Experimental Results See results Figures 7-8 .

[0073] Figure 7 In the diagram, A: Experimental flowchart; Mice were divided into: Control group, Con+CUMS-FMT group (CUMS donor fecal microbiota transplantation into uncompressed PGF recipients), CUMS+PBS group (buffer-treated), CUMS+Nic-FMT group (nicotine donor fecal microbiota transplantation), CUMS+AKK-FMT group (… A.muciniphila Intervention); B: Body weight change in mice at 5-9 weeks; CD: Immobility time in FST and TST; EF: Central region dwell time and movement distance in OFT experiment; GH: Open arm dwell time and movement distance in EPM; I: OFT and EPM trajectory plots; JL: 5-HTP, 5-HT, and 5-HIAA / 5-HT ratios in the hippocampus; M: Tph2 gene expression level in the hippocampus. Data are expressed as mean ± SEM (BI: n=10-14; JM: n=10). P<0.05, P<0.01, P<0.001; ns indicates no statistically significant difference (P>0.05).

[0074] Figure 8 In the diagram, A: α-diversity analysis (Shannon and Simpson indices); BC: β-diversity box plots and principal coordinate analysis (PCoA); D: A.muciniphila Relative abundance; E: Phylum-level microbial community structure (bar chart); F: Species-level microbial community structure (bar chart); GH: LEfSe analysis of control group vs CUMS-FMT group (LDA>3.0); IJ: LEfSe analysis of CUMS+PBS vs Nic-FMT group; K: LDA distribution of CUMS+PBS vs AKK-FMT; L: STAMP analysis showing changes in the abundance of key species; Data are expressed as mean ± SEM (n=6 per group); ns indicates no statistically significant difference (P>0.05); P<0.05, P<0.01, P<0.001.

[0075] Figure 9 In the middle, A: HE staining of the colon (magnification: ×200μm, ×50μm) (n=6 per group); BC: Immunofluorescence staining of ZO-1 and Occludin in the colon; DE: Quantification of fluorescence intensity of ZO-1 and Occludin in the colon (pixels / mm) 2 (×50 μm, n=7-8 per group); FG: ZO-1 and Occludin protein expression in the colon (n=6 per group); HJ: IL-6, IL-1β and TNF-α expression in the colon (n=6 per group); KL: Acetic acid and propionic acid levels in the colon (n=6 per group). Data are expressed as mean ± standard error (mean ± SEM). ns indicates no statistically significant difference (P>0.05); P<0.05, P<0.01, P<0.001.

[0076] (1) Both Nic-FMT and AKK-FMT can significantly improve depressive-like behaviors. Figure 6 Behavioral results (B–I): Mice in the Con+CUMS-FMT group exhibited significant depressive-like behavior, demonstrating that the CUMS microbiota itself can induce abnormal behavior. Both the Nic-FMT and AKK-FMT groups showed significant improvements in the following indicators (P<0.05–0.001): FST / TST: reduced immobility time; OFT: increased time spent in the central region and distance traveled; EPM: increased open-arm exploration time. This indicates that the nicotine-regulated microbiota has an antidepressant effect. Akkermansia This effect can be replicated independently by a single bacterium.

[0077] (2) Nic-FMT and AKK-FMT restore neurotransmitter metabolism ( Figure 6 Hippocampal analysis (J–M) showed that in the CUMS+PBS group, 5-HTP and 5-HT were significantly decreased, while 5-HIAA / 5-HT was increased. Both the Nic-FMT and AKK-FMT groups showed significant recovery of 5-HTP and 5-HT (P<0.01). Tph2 Upward adjustment Maoa Downregulation indicates enhanced synthesis and weakened decomposition. The results suggest that nicotine-regulated gut microbiota can restore 5-HT metabolism, while... A.muciniphila These are key effector bacteria.

[0078] (3) Restoration of gut microbiota structure ( Figure 7 α-Diversity: The Nic-FMT and AKK-FMT groups showed significantly higher diversity than the CUMS+PBS group. β-Diversity: The microbial community structure of both groups converged towards that of the healthy group. A. muciniphila The relative abundance was significantly increased (P<0.001). LEfSe analysis showed that: CUMS-FMT was enriched with a variety of inflammation-related bacterial communities; Nic-FMT and AKK-FMT were enriched with beneficial bacteria, including mucus layer maintaining bacteria and SCFA-producing bacteria; STAMP showed a significant recovery of a variety of key bacterial genera.

[0079] (4) Nic-FMT and AKK-FMT repair the intestinal barrier and reduce inflammation. Figure 8 HE staining: Colonic villus structure and crypt integrity restored; ZO-1 / Occludin expression level significantly increased (P<0.01); inflammatory factors (IL-1β, IL-6, TNF-α) significantly decreased; SCFAs (acetic acid, propionic acid) significantly increased. 3. Summary This embodiment demonstrates that nicotine can transfer its antidepressant effect to recipient mice by regulating the gut microbiota. Nic-FMT can replicate the antidepressant effect of nicotine, proving that its effect is microbially transferable. A. muciniphila Single-strain supplementation is sufficient to restore behavioral patterns, barrier function, inflammation levels, and 5-HT metabolism, indicating that... A. muciniphila It is a key effector bacteria of nicotine, and has constructed a complete mechanism chain of "nicotine → microbial community → barrier / inflammation / 5-HT → behavior".

[0080] In summary, the four embodiments systematically support the technical solution of the present invention from multiple dimensions, including behavioral science, microbiome, immunology, metabolomics, and transferability verification: the low-dose nicotine sustained-release formulation significantly upregulates... A. muciniphila This invention aims to improve the abundance of nicotine, thereby restoring the intestinal barrier, reducing inflammation, and rebuilding the metabolic homeostasis of short-chain fatty acids and 5-HT, ultimately achieving the technical effect of improving depressive symptoms. The invention thus constructs a clear and reproducible pathway of "nicotine →..." A. muciniphila The causal chain of "→gut–brain axis→depression improvement" provides a new application basis for microbial-targeted intervention for depression.

[0081] The above embodiments demonstrate that the low-dose nicotine sustained-release formulation of the present invention can stabilize blood drug concentration, improve depressive-like behavior, restore intestinal barrier and flora homeostasis, increase SCFAs levels and remodel the central 5-HT system, without enhancing motor ability or interfering with addiction risk, and has significant technical effects and innovation.

[0082] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A low-dose nicotine sustained-release formulation, characterized in that, The active ingredient of the formulation is (-)-di-L-(+)-tartrate nicotine dihydrate with a purity ≥98.0%. It is administered subcutaneously via an osmotic pressure-driven sustained-release pump at a dose of 0.1-0.3 mg / kg / day.

2. The drug delivery formulation according to claim 1, characterized in that, The dosage is 0.2 mg / kg / day.

3. The drug delivery formulation according to claim 1, characterized in that, The slow-release pump is selected from Alzet®2006, Alzet®1002, or Alzet®1004 osmotic pumps, with an effective volume of 200 μL and a release rate of 0.11-0.25 μL / h.

4. The drug delivery formulation according to any one of claims 1-3, characterized in that, The solvent for the formulation is selected from 0.9% sodium chloride injection, PBS buffer, or glycerol isotonic buffer.

5. The drug delivery formulation according to any one of claims 1-3, characterized in that, The formulation is administered subcutaneously for a period of 4 weeks.

6. Use of a drug delivery formulation according to any one of claims 1-5 in the preparation of a medicament for treating depression or depressive-like behavior.

7. The use according to claim 6, characterized in that, The drug induces an increase in the abundance of Akkermansia myxophilus.

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