Application of nicotine in preparation of medicine for relieving, adjunctively treating or treating Gaucher disease

Abstract

The invention relates to the technical field of Gaucher disease treatment, in particular to application of nicotine in preparation of drugs for relieving, adjuvant therapy or treatment of Gaucher diseases. The invention reveals for the first time that nicotine can enhance mitochondrial autophagy mediated by a PGC1 alpha / Parkin pathway by reducing accumulation of glucosylceramide and up-regulating protein expression of PGC1 alpha and Synaptophysin in a hippocampal region, also improves synaptic function and various neurotransmitter levels, enhances learning memory and application ability, and further improves neurodegenerative diseases caused by Gaucher disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of Gaucher disease treatment, and particularly relates to the use of nicotine in the preparation of a drug for relieving, adjuvant treatment or treating Gaucher disease. BACKGROUND

[0002] Gaucher disease is an autosomal recessive lysosomal storage disease caused by mutations in the gene of glucocerebrosidase (GBA1). The loss of GBA1 enzyme activity leads to the abnormal accumulation of its substrate, glucosylceramide (GluCer) and its derivative, glucosphingosine, in macrophages, liver and spleen, and central nervous system, and further causes multi-organ lesions. According to the degree of nervous system involvement, Gaucher disease can be divided into three subtypes: type I (non-neurological disease), type II (acute neurological disease) and type III (chronic neurological disease). Among them, type II and type III patients have severe neurodegenerative symptoms due to intracerebral lipid deposition, including cognitive impairment, motor disorder, epilepsy, etc., and there is currently no effective treatment, with a very high mortality rate.

[0003] Currently, the clinical treatment of Gaucher disease mainly relies on the following two strategies: (1) Enzyme replacement therapy (ERT). By intravenous injection of recombinant glucocerebrosidase (such as imiglucerase), the degradation of GluCer in peripheral organs is accelerated. This therapy has a significant improvement on hepatosplenomegaly and bone disease in type I patients, but the recombinant enzyme cannot penetrate the blood-brain barrier and has no effect on neurological disease. In addition, ERT needs to be administered every week for a lifetime, with high treatment costs (annual cost of more than 300,000 US dollars), and may cause immunogenic reactions.

[0004] (2) Substrate reduction therapy (SRT). Using glucosylceramide synthase inhibitors (such as Eliglustat) to reduce the generation of GluCer. Although SRT can partially relieve the symptoms of mild patients, it has no effect on the accumulated lipids, and has side effects such as cardiotoxicity.

[0005] For neurological Gaucher disease, existing therapies can only provide palliative support (such as antiepileptic drugs), and cannot delay disease progression. Therefore, it is urgent to develop a new treatment strategy that can target the central nervous system and repair neuronal damage.

[0006] Nicotine is a natural alkaloid that can regulate dopamine release, inhibit neuroinflammation and oxidative stress by activating neuronal nicotinic acetylcholine receptors (nAChRs). However, whether the mechanism of action of nicotine is related to the key pathways of neurodegeneration in Gaucher disease, such as PGC1α / Parkin-mediated mitochondrial autophagy and synapse protein SYNA expression, has not been reported in the literature. In addition, the potential application of nicotine in the treatment of Gaucher disease has not been explored at all, and there are no related patents or clinical studies in this field. SUMMARY

[0007] The present application provides the use of nicotine in the preparation of a medicament for alleviating, adjuvant treating or treating Gaucher disease, and provides a new idea for alleviating, adjuvant treating or treating Gaucher disease and preparing a medicament for alleviating, adjuvant treating or treating Gaucher disease.

[0008] In a first aspect, the present application provides the use of nicotine in the preparation of a medicament for alleviating, adjuvant treating or treating a disease related to abnormal accumulation of ceramide or its derivative, abnormal accumulation of glucosylceramide or its derivative, reduced mitochondrial autophagy function, reduced synapse function and / or reduced neurotransmitter level.

[0009] In an alternative embodiment, the nicotine is used to prepare a medicament for alleviating, adjuvant treating or treating a disease related to abnormal accumulation of ceramide or its derivative, abnormal accumulation of glucosylceramide or its derivative, reduced mitochondrial autophagy function, reduced synapse function and / or reduced neurotransmitter level by reducing abnormal accumulation of ceramide or its derivative, reducing abnormal accumulation of glucosylceramide or its derivative, improving mitochondrial autophagy function, improving synapse function and / or improving neurotransmitter level.

[0010] In an alternative embodiment, the derivative of glucosylceramide includes glucosylsphingosine; And / or, the neurotransmitter includes at least one of gamma-aminobutyric acid, 5-hydroxytryptamine, acetylcholine and dopamine; And / or, the nicotine improves mitochondrial autophagy function by regulating the PGC1α / Parkin pathway; And / or, the nicotine improves synapse function by regulating Synaptophysin protein.

[0011] In a second aspect, the present application provides the use of nicotine in the preparation of a medicament for alleviating, adjuvant treating or treating Gaucher disease.

[0012] In an alternative embodiment, the Gaucher disease includes non-neuropathic type (type I), acute neuropathic type (type II) and chronic neuropathic type (type III).

[0013] In an alternative embodiment, the Gaucher disease comprises symptoms of cognitive impairment, motor incoordination and / or epilepsy.

[0014] In an alternative embodiment, the Gaucher disease is mediated by abnormal accumulation of ceramide or derivatives thereof, abnormal accumulation of glucosylceramide or derivatives thereof, decreased mitochondrial autophagy function, decreased synaptic function and / or decreased neurotransmitter levels.

[0015] In a third aspect, the present application provides a medicament for treating Gaucher disease, wherein the medicament comprises nicotine as an active substance.

[0016] In an alternative embodiment, the medicament further comprises a pharmaceutically acceptable excipient or carrier; optionally, the medicament is an oral preparation or a non-oral preparation.

[0017] In an alternative embodiment, the oral preparation is any one of a capsule, a tablet, an oral liquid, a granule, a pill, a powder, a bolus or a paste; Alternatively, the non-oral preparation is any one of an injection, a cream, a patch, an ointment or a spray.

[0018] The technical solution of the present application has the following advantages: 1. The present application provides the use of nicotine in the preparation of a medicament for alleviating, adjuvant treating or treating a disease mediated by abnormal accumulation of ceramide or derivatives thereof, abnormal accumulation of glucosylceramide or derivatives thereof, decreased mitochondrial autophagy function, decreased synaptic function and / or decreased neurotransmitter levels. The present application researches find that nicotine has the effects of reducing abnormal accumulation of ceramide or derivatives thereof, reducing abnormal accumulation of glucosylceramide or derivatives thereof, improving mitochondrial autophagy function, improving synaptic function and / or improving neurotransmitter levels, and thus can be used in the preparation of a medicament for alleviating, adjuvant treating or treating a disease mediated by abnormal accumulation of ceramide or derivatives thereof, abnormal accumulation of glucosylceramide or derivatives thereof, decreased mitochondrial autophagy function, decreased synaptic function and / or decreased neurotransmitter levels.

[0019] 2. The present application provides the use of nicotine in the preparation of a medicament for alleviating, adjuvant treating or treating Gaucher disease. The present application researches find that nicotine has the effect of regulating the key pathway of Gaucher disease, i.e. the effect of reducing abnormal accumulation of ceramide or derivatives thereof, reducing abnormal accumulation of glucosylceramide or derivatives thereof, improving mitochondrial autophagy function, improving synaptic function and / or improving neurotransmitter levels, and thus nicotine can be used in the preparation of a medicament for alleviating, adjuvant treating or treating Gaucher disease. BRIEF DESCRIPTION OF DRAWINGS

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 These are the cognitive function and motor ability assessment results of each group of mice in Example 1 of the present invention; where A is the spontaneous alternation rate, B is the total movement distance, and C is the rotarod test time; Figure 2 This is the result of Western blot analysis of the relative expression levels of PGC1α and Synaptophysin proteins in the hippocampus in Example 1 of this invention; where SYNA represents Synaptophysin protein. Figure 3 yes Figure 2 Quantitative results; Figure 4 These are the neurotransmitter detection results for each group in Embodiment 1 of the present invention; Figure 5 These are the detection results of the content of ceramide and glucosamine in the hippocampus of each group in Example 1 of the present invention; wherein, the left figure is ceramide and the right figure is glucosamine. In the above figures, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.001. Detailed Implementation

[0022] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0023] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0024] Nicotine was sourced from Zhengzhou Tobacco Research Institute; Conduritol Be epoxide was purchased from MedChemexpress (MCE) Biotechnology Company in the United States, product number HY-100944.

[0025] RIPA lysis buffer was purchased from Beyotime (Catalog No. P0013B); protease inhibitor mixture was purchased from Beyotime (Catalog No. P1045); BCA protein quantification kit was purchased from Beyotime (Catalog No. P0010S); HRP-goat anti-mouse IgG was purchased from Abcam (Catalog No. ab205719); HRP-goat anti-rabbit IgG was purchased from Abcam (Catalog No. ab205718); ECL chemiluminescence kit was purchased from Beyotime (Catalog No. P0018AS); stripping was purchased from Vazyme (Catalog No. E701-02).

[0026] The γ-aminobutyric acid (GABA) colorimetric assay kit was purchased from Elabscience, catalog number E-BC-K852-M; the 5-hydroxytryptamine (ST / 5-HT) enzyme-linked immunosorbent assay kit was purchased from Elabscience, catalog number E-EL-0033; the acetylcholine (ACH) enzyme-linked immunosorbent assay kit was purchased from Elabscience, catalog number E-EL-0081; and the dopamine (DA) enzyme-linked immunosorbent assay kit was purchased from Elabscience, catalog number E-EL-0046.

[0027] Example 1: Use of nicotine in the preparation of drugs for the relief, adjunctive treatment, or treatment of Gaucher disease. 1. Animal model construction and dosing regimen Thirty 3-month-old male C57BL / 6J mice were randomly divided into three groups: a control group (n=10), a Gaucher disease model group (CBE group) (n=10), and a nicotinic acid treatment group (CBE+Nic group) (n=10). The Gaucher disease model group and the nicotinic acid treatment group received intraperitoneal injections of 100 mg / kg Conduritol Be epoxide (an irreversible, covalent β-glucosidase inhibitor, CBE) daily for 4 weeks to establish the Gaucher disease model. Mice in the nicotinic acid treatment group received intraperitoneal injections of 0.25 mg / kg nicotinic acid daily until the model was established. The control group received physiological saline. Each injection volume was 500 μL.

[0028] 2. Assessment of cognitive function and motor ability (1) Y-shaped maze experiment The Y-maze consists of three identical arms connected at 120-degree angles. Mice are naturally inclined to explore new environments. When they move freely in the maze, they can remember the arm they just visited (short-term spatial memory), thus tending to enter a "new" arm they haven't recently visited. This behavior of "entering three different arms in sequence" is called "spontaneous alternation behavior." The accuracy of spontaneous alternation is an important indicator for assessing working memory in mice. The specific experiment includes the following steps: A. Place the mouse: Gently place the mouse at the end of a designated arm (usually called the “starting arm”), ensuring that the mouse’s head is facing the end of the arm.

[0029] B. Free exploration: Immediately activate the camera system and timer, allowing the mouse to freely explore the maze for 8 minutes.

[0030] C. End of Experiment: After 8 minutes, gently remove the mouse from the maze. Immediately clean the maze thoroughly as described above, and wait for it to dry completely before proceeding with the next mouse.

[0031] D. Data Statistics: Calculate the total number of arm entries. The total number of times the mouse enters each arm (denoted as T) is calculated, along with the total number of correct alternations (denoted as A). The formula is shown below: Spontaneous alternation accuracy (%) = [A / (T - 2)] × 100%.

[0032] Total movement distance reflects the mouse's ability to move independently.

[0033] (2) Rotating bar experiment A. The rotator experiment includes the following three stages, as detailed below: Day 1 is the training phase. Placing the mouse: Gently place the mouse facing the ramp (avoiding direct view of the drop area) on the roller in its path. Before starting the device, ensure the mouse can stand steadily on the roller. Training: Start the device and allow the mouse to walk according to the set training parameters. Recording and Assistance: The device automatically records the latency when the mouse falls. Each training session for each mouse is typically 2-5 minutes, repeated 2-3 times, with at least 15-30 minutes between sessions to prevent over-fatigue. Ending Training: After training, return the mouse to its cage, thoroughly clean the roller, and then begin training the next mouse.

[0034] The second day was for intensive training. The training process from the first day was repeated to reinforce the mice's learning.

[0035] Day 3: Formal Testing. Preparation: Same as the training phase, ensure consistent environment and equipment settings. Testing: Place the mouse in the instrument and start the pre-set acceleration program. Testing Rules: During the formal test, if the mouse falls, it will not be placed back on the roller. One test completes the test. Data Recording: The instrument will automatically record key indicators. Latency: The time (in seconds) from the start of the test to the mouse falling. Drop Speed: The speed (rpm) of the roller when the mouse falls. Cleaning: Clean the roller after testing one mouse before testing the next.

[0036] B. Rotation experiment data: Calculate the mean ± standard error of the rotation time for each group of mice.

[0037] (3) Data processing methods When comparing differences between different groups using appropriate statistical methods (such as t-tests or one-way ANOVA), p < 0.05 is considered statistically significant.

[0038] (4) Experimental results The results of the Y-shaped maze experiment are as follows: Figure 1 As shown in Figures A and B, the Y-maze experiment is used to test spatial working memory. Figure 1 As shown in Figures A and B, compared to the control group, the model group showed a significant decrease in spatial working memory, with a statistically significant difference (p < 0.001), indicating that the model group mice had impaired spatial working memory, and the model was successfully constructed. Compared to the model group, in the nicotine treatment group, nicotine treatment significantly increased the turnover rate in Gaucher disease mice. Figure 1 The results showed a statistically significant difference (p < 0.05), indicating that nicotine can improve the reduction in spatial working memory caused by Gaucher disease and treat the cognitive decline caused by Gaucher disease.

[0039] The results of the rotator experiment are as follows Figure 1 As shown in Figure C, the rotarod test was used to assess the motor function of mice. The figure shows that compared to the control group, the model group exhibited a significant decrease in motor function, with a statistically significant difference (p < 0.0001), indicating that the Gaucher disease model mice had a significantly reduced time spent on the rotarod, i.e., reduced motor function. Compared to the model group, the nicotinic acid treatment group increased the time mice spent on the rotarod. Figure 1 (C) This study demonstrated that nicotine can improve the motor function of Gaucher disease mice.

[0040] 3. Western blot analysis was used to detect the relative expression levels of PGC1α and Synaptophysin proteins in the hippocampus. Hippocampal tissues from mice in each group were homogenized in a mixture of RIPA lysis buffer and protease inhibitors. After centrifugation, the supernatant was collected, and protein concentration was determined according to the BCA protein quantification kit instructions. After quantification, 15 µg of protein was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% (w / v) skim milk powder (dissolved in TBST containing 0.1% Tween-20) at room temperature for 1 h, followed by overnight incubation with primary antibody at 4°C. The next day, the membrane was washed three times with TBST (10 min each time) and incubated with HRP-labeled secondary antibodies for 1 h at room temperature: HRP-goat anti-mouse IgG (1:10,000) and HRP-goat anti-rabbit IgG (1:10,000). After washing again, the membrane was developed using an ECL chemiluminescence kit, and images were acquired using a Tanon-5200 gel imaging system. For cases where the target protein and internal control had overlapping molecular weights, sequential incubation with mild stripping buffer was used, and complete removal of the primary antibody was confirmed by incubating with only the secondary antibody. ImageJ software was used to analyze the integrated grayscale values ​​and calculate the relative protein expression levels.

[0041] The detection results of Western blot method are as follows Figure 2 and Figure 3 As shown, compared with the control group, synaptophysin (SYNA) was significantly reduced in the Gaucher disease model group (p < 0.05), while the nicotine treatment group completely recovered to normal levels, showing a statistically significant difference compared with the model group (p < 0.05). At the molecular level, compared with the control group, PGC1α protein, which is involved in mitochondrial synthesis in the hippocampus of Gaucher disease mice, was significantly reduced (p < 0.01), while the nicotine treatment group restored it to normal levels, showing a statistically significant difference compared with the model group (p < 0.05).

[0042] 4. Determination of neurotransmitter, ceramide, and glucosamine content in the hippocampus Neurotransmitters were measured using the γ-aminobutyric acid (GABA) colorimetric assay kit, the 5-hydroxytryptamine (ST / 5-HT) enzyme-linked immunosorbent assay kit, the acetylcholine (ACH) enzyme-linked immunosorbent assay kit, and the dopamine (DA) enzyme-linked immunosorbent assay kit. The experimental procedures were performed in accordance with the instruction manual.

[0043] The results are as follows Figure 4As shown, compared with the control group, the levels of the major neurotransmitters γ-aminobutyric acid (GABA), serotonin (5-HT), acetylcholine (Ach), and dopamine (DA) in the hippocampus of mice in the Gaucher disease model group were significantly decreased (p < 0.001, p < 0.05, p < 0.05, p < 0.0001). In contrast, compared with the model group, nicotine treatment increased the levels of GABA, 5-HT, Ach, and DA, with statistically significant differences (p < 0.05, p < 0.05, p < 0.05, p < 0.001). These results indicate that nicotine can enhance spatial working memory in aged rats by improving dopaminergic signaling and synaptic integrity.

[0044] The main characteristic of Gaucher disease is the accumulation of glucosamine. Lipomics analysis was used to analyze the levels of ceramide and glucosamine in the hippocampus. Figure 5 The results showed that the Gaucher disease model group had significant accumulation of ceramides and glucosamines of different chain lengths, while the nicotinic acid treatment group could reduce the accumulation of both ceramides and glucosamines.

[0045] The above results demonstrate that nicotine can enhance memory and motor abilities in Gaucher disease model mice by reducing the accumulation of ceramides and glucosamine, increasing mitochondrial production, and increasing synaptic plasticity.

[0046] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Use of nicotine in the preparation of medicaments for the relief, adjunctive treatment or treatment of diseases mediated by abnormal accumulation of ceramides or their derivatives, abnormal accumulation of glucosamine or its derivatives, decreased mitophagy, decreased synaptic function and / or decreased neurotransmitter levels.

2. The use according to claim 1, characterized in that, The nicotine is prepared as a drug to alleviate, adjuvantly treat, or treat diseases mediated by abnormal accumulation of ceramides or their derivatives, abnormal accumulation of glucosamine or its derivatives, reduced mitophagy function, reduced synaptic function, and / or reduced neurotransmitter levels by reducing abnormal accumulation of ceramides or their derivatives, abnormal accumulation of glucosamine, reduced mitophagy function, reduced synaptic function, and / or reduced neurotransmitter levels.

3. The use according to claim 1 or 2, characterized in that, The derivatives of the glucose ceramide include glucosinolate; And / or, the neurotransmitter includes at least one of γ-aminobutyric acid, serotonin, acetylcholine and dopamine; And / or, the nicotine enhances mitophagy by regulating the PGC1α / Parkin pathway; And / or, the nicotine enhances synaptic function by regulating the Synaptophysin protein.

4. Use of nicotine in the preparation of drugs for the relief, adjunctive treatment or treatment of Gaucher disease.

5. The use according to claim 4, characterized in that, Gaucher disease includes non-neuropathy, acute neuropathic, and chronic neuropathic forms.

6. The use according to any one of claims 4 to 5, characterized in that, Gaucher disease includes symptoms of cognitive impairment, motor incoordination, and / or epilepsy.

7. The use according to any one of claims 4 to 6, characterized in that, Gaucher disease is mediated by abnormal accumulation of ceramides or their derivatives, abnormal accumulation of glucosamine or its derivatives, decreased mitophagy, decreased synaptic function, and / or decreased neurotransmitter levels.

8. A drug for treating Gaucher disease, characterized in that, The drug uses nicotine as its active ingredient.

9. The use according to any one of claims 1 to 7 and the medicament according to claim 8, characterized in that, The drug may also include pharmaceutically acceptable excipients or carriers; optionally, the drug may be an oral or non-oral formulation.

10. The use according to claim 9 and the drug according to claim 9, characterized in that, The oral preparation is any one of capsules, tablets, oral liquids, granules, pills, powders, elixirs, or ointments; Alternatively, the non-oral preparation may be any one of an injection, cream, patch, ointment, or spray.

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