Use of l-nrb in the preparation of a medicament for treating parkinson's disease

Abstract

The application provides application of a hydroxy pentylbenzoic acid double ester compound L-NRB in treatment of Parkinson's disease. Animal model test proves that L-NRB can effectively improve the MPTP-induced Parkinson's disease mouse behavior index, increase the content of DA and its metabolites DOPAC and HVA in the brain, inhibit the loss of dopamine neurons in the substantia nigra and striatum, inhibit the expression of inflammatory factors and the activation of astrocytes and microglia in the substantia nigra; meanwhile, L-NRB can also inhibit the activity of MAO-B in the striatum. L-NRB has no toxic side effects in vivo and in vitro; has the advantages of high safety, small dosage, long-term stability and the like, and is suitable for further development for actual clinical treatment.

Description

Invention Field

[0001] This application pertains to the fields of neurodegenerative diseases and biomedicine. Specifically, this application provides the use of L-NRB in the preparation of a drug for treating Parkinson's disease. Background Technology

[0002] Parkinson's disease (PD), also known as paralysis agitans, is a common neurodegenerative disease affecting middle-aged and elderly people. Clinically, it is mainly characterized by resting tremor, bradykinesia, rigidity, and postural instability. The prevalence in my country among people aged 65 and above is 1700 per 100,000. According to the "Chinese Guidelines for the Treatment of Parkinson's Disease (Fourth Edition)," current treatments for Parkinson's disease, whether medication or surgery, cannot stop the progression of the disease, let alone cure it. Therefore, finding new drugs to treat Parkinson's disease is currently a top priority in its treatment.

[0003] The main pathological changes in Parkinson's disease (PD) are progressive degeneration of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies. The main biochemical changes are a decrease in dopamine neurotransmitters in the striatum and an imbalance between dopamine and acetylcholine. Therefore, detecting the levels of dopamine and its metabolites in the brain and immunostaining tyrosine hydroxylase (TH)-positive neurons in the substantia nigra pars compacta and striatum are the gold standard for evaluating the efficacy of Parkinson's disease treatment in animal experiments.

[0004] Neuroinflammation is involved in the occurrence and development of Parkinson's disease (PD). Microglial activation is closely associated with the accumulation of misfolded proteins in various neurodegenerative diseases, including PD, AD, and ALS. The release of aggregated α-synuclein, mSOD1, or Aβ / tau oligomers from neurons into the extracellular space can directly induce microglia to develop the M1 phenotype. During the pathogenesis phase, M2 microglia may primarily phagocytose cellular debris, enhance tissue remodeling, and produce anti-inflammatory factors to maintain tissue homeostasis. However, endogenous stimuli, including aggregated α-synuclein, mSOD1, Aβ plaques, and tau oligomers, as well as the persistent presence of environmental toxins, induce microglia to adopt the M1 phenotype and impair immune degradation processes in the later stages of disease progression, ultimately leading to irreversible neuronal loss. Therefore, inhibiting neuroinflammation is an important direction for drug development in neurodegenerative diseases.

[0005] Monoamine oxidase (MAO) is widely distributed in the central and peripheral nervous systems. MAO-A is abundant in noradrenergic neurons projecting from the locus coeruleus, while MAO-B is abundant in raphe nuclei and serotonergic and histaminergic neurons in the posterior hypothalamus. In the substantia nigra, MAO-A is primarily expressed in dopaminergic neurons, while MAO-B is mainly expressed in glial cells of the substantia nigra. Quantitative analysis revealed that MAO-B is three times more abundant than MAO-A in the substantia nigra. Due to the tissue-specific distribution of the two MAO subtypes, MAO also exhibits substrate specificity. In the human brain, MAO-B activity accounts for more than 80%, suggesting that striatal dopaminergic oxidase (DA) is primarily inactivated by MAO-B deamination. Therefore, developing highly potent, selective, and reversibly inhibitory MAO-B inhibitors remains an important task in the field of psychogenic disorders (PD).

[0006] Butylphthalide (NBP) was approved by the China Food and Drug Administration (CFDA) in 2002 for the treatment of acute ischemic stroke, and is China's third innovative drug with independent intellectual property rights. The "Guidelines for the Diagnosis and Treatment of Acute Ischemic Stroke in China (2021 Edition)" describes butylphthalide as a drug that improves microcirculation, promotes angiogenesis, and increases cerebral blood flow. However, the limited efficacy and extremely poor water solubility of NBP significantly restrict its application. L-NRB is a levorotatory product formed by combining the ring-opening product of butylphthalide with dextromethorphanol in a 1:2 molar ratio. According to previous studies by our research group, L-NRB has good activity in treating cerebral ischemia, and its effect is superior to that of butylphthalide. L-NRB also has therapeutic effects on amyotrophic lateral sclerosis (ALS) and Alzheimer's disease due to its neuroprotective and anti-inflammatory effects, but its therapeutic effect on Parkinson's disease has not yet been reported. Summary of the Invention

[0007] On the one hand, this application provides the use of L-NRB in the prevention and treatment of Parkinson's disease, wherein the chemical formula of L-NRB is shown in Formula I:

[0008]

[0009] Furthermore, the Parkinson's disease mentioned is Parkinson's disease caused by MPTP.

[0010] Furthermore, the drug increases the levels of DA, DOPAC, and HVA in the brain.

[0011] Furthermore, the drug inhibits the expression of inflammatory factors and the activation of astrocytes and microglia in the substantia nigra.

[0012] Furthermore, the drug inhibits the activity of MAO-B.

[0013] Furthermore, the drug is in oral, injectable, or transdermal form.

[0014] Furthermore, the drug is an oral dosage form.

[0015] Furthermore, the drug also includes pharmaceutically acceptable excipients or excipients.

[0016] On the other hand, this application provides a medicament for treating Parkinson's disease, the medicament comprising L-NRB, the chemical formula of which is shown in Formula I:

[0017]

[0018] Furthermore, the drug is an oral dosage form.

[0019] The available drug dosage forms for this application include various injectable, oral, and topical dosage forms, including but not limited to tablets, capsules, oral solutions, aqueous injections, powder injections, eye drops, and transdermal drug delivery preparations.

[0020] The medicament described in this application may also include various pharmaceutically acceptable excipients or excipients, including but not limited to coating materials, solvents, solubilizers, binders, stabilizers, antioxidants, pH adjusters, flavoring agents, etc.

[0021] The "L-NRB" mentioned in this application refers to a compound with the chemical formula I below, which is disclosed in the applicant's earlier application CN202210205661.2. Its chemical name, chemical formula, and various abbreviations all have the same meaning and can be used interchangeably.

[0022]

[0023] The MPTP described in this application has the chemical name 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, CAS NO. 23007-85-4. It is a compound that inhibits the destruction of nerve cells in the substantia nigra. It is present in the environment and in certain drugs, and is one of the known causes of Parkinson's disease. It has been used to model Parkinson's disease.

[0024] This application clarifies that L-NRB treats Parkinson's disease by inhibiting neuroinflammation and reducing MAO-B activity, and therefore can be used for the further development of drugs for the prevention / treatment of Parkinson's disease, increasing the available drugs for the clinical treatment of Parkinson's disease. Attached Figure Description

[0025] Figure 1A Representative trajectories for each group of mice in the open field experiment;

[0026] Figure 1B The total distance traveled in the open field experiment, the change in the central area visit time, and the number of times the equipment was erected;

[0027] Figure 2A Determination of the content of DA in the striatum;

[0028] Figure 2B Determination of the content of DOPAC in the striatum;

[0029] Figure 2C Determination of the content of HVA in the striatum;

[0030] Figure 3 TH immunohistochemical staining in the substantia nigra (Part A) and TH immunohistochemical staining in the striatum (Part B);

[0031] Figure 4A Determination of the content of IL-1β in the serum;

[0032] Figure 4B Determination of the content of TNF-α in the serum;

[0033] Figure 4C Determination of the content of IL-6 in the serum;

[0034] Figure 5 Iba1 immunohistochemical staining in the substantia nigra (Part A) and GFAP immunohistochemical staining in the substantia nigra (Part B);

[0035] Figure 6 Determination of the activity of MAO-B in the striatum. Detailed implementation mode

[0036] Example 1 Construction of a Parkinson's animal model and behavioral experiments

[0037] Experimental materials:

[0038] L-NRB was synthesized by the Chemical Laboratory of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, and the structure was synthesized and confirmed according to Example 2 in CN202210205661.2.

[0039] MPTP·HCl was purchased from Aladdin Biochemical Technology Co., Ltd. (product number: M132847-100mg). Seventy-two CBA / J mice were purchased from Beijing SPF Biotechnology Co., Ltd., license number: SYXK (Beijing) 2024-0010. The spontaneous activity behavior experiment box was purchased from Shanghai Xinruan Information Technology Co., Ltd. (product number: XR-XZ301). All animals were raised in the animal center of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences. All mice were raised in an environment with a temperature of 22-26°C, a humidity of 40-70%, and a 12-hour alternating dark and light cycle, with free access to water and food. The use of experimental animals followed the Guidelines for the Care and Use of Laboratory Animals promulgated by the National Institutes of Health of the United States and the regulations of the Animal Care and Use Committee of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences.

[0040] Experimental methods:

[0041] Grouping and Drug Administration: Mice were randomly divided into a control group, a model group, an L-NRB 38.125 mg / kg group, an L-NRB 76.25 mg / kg group, an L-NRB 152.5 mg / kg group, and a selegiline positive control group, with 12 mice in each group. The drug administration groups were administered the drug via gavage once daily for 20 consecutive days, while the control and model groups were administered an equal volume of the drug via gavage. Modeling began on day 8 of drug administration and lasted for 6 days. Two hours after the end of drug administration, mice in the model and drug administration groups were intraperitoneally injected with MPTP·HCl at a dose of 35.1 mg / kg, while the control group was injected with an equal volume of physiological saline. One day after modeling, an open field test was performed.

[0042] Processing and sample collection methods: 20 days after drug administration, mice were sacrificed and serum was collected. The brains of 5 mice from each group were fixed, and the striatum of the other 7 mice was cryopreserved.

[0043] Experimental results:

[0044] One day after MPTP modeling, the central region access time, central region distance ratio, and number of standing times in the model group mice were significantly lower than those in the control group (Figure 1, p < 0.05). All drug administration groups could improve the decrease in central region access time, central region distance ratio, and number of standing times in the model mice (Figure 1, p < 0.05). This demonstrates that L-NRB can improve the motor ability of PD mice.

[0045] Example 2: Determination of DA, DOPAC, and HVA content in mouse striatum

[0046] Experimental methods:

[0047] After homogenizing the mouse striatum with physiological saline and centrifuging, the supernatant was used for BCA protein quantification. The protein concentration of the homogenate of all samples was then adjusted to 1.178 mg / ml, and the relevant experiments were performed according to the kit instructions.

[0048] DA detection: The mouse dopamine detection kit (catalog number: HY-K0027) purchased from Beijing Huaying Biotechnology Research Institute was used for detection.

[0049] DOPAC detection: The mouse dihydroxyphenylacetic acid detection kit (catalog number: HY-NE202) purchased from Beijing Huaying Biotechnology Research Institute was used for detection.

[0050] HVA detection: The mouse homovanillic acid detection kit (product number: HY-NE201) purchased from Beijing Huaying Biotechnology Research Institute was used for detection.

[0051] Experimental results:

[0052] After MPTP modeling, the levels of DA and its metabolites in the striatum of mice in the model group were significantly reduced. Figures 2A-2C (p < 0.001), all drug administration groups improved the decrease in DA, DOPAC, and HVA levels in model mice ( Figures 2A-2C (p < 0.001).

[0053] Example 3: Immunohistochemical detection of the number of dopaminergic neurons in the substantia nigra and striatum

[0054] Experimental methods:

[0055] 1. Fixation of frozen sections: Air-dry frozen sections at room temperature, bake in an oven at 37°C for 10-20 minutes, fix in methanol for 20 minutes, and wash three times in PBS (pH 7.4) on a decolorizing shaker for 5 minutes each time.

[0056] 2. Antigen retrieval: See the table above for retrieval instructions. During this process, excessive evaporation of the buffer solution should be prevented, and the slides should not be dried out. After natural cooling, place the slides in PBS (pH 7.4) and wash them three times on a decolorizing shaker for 5 minutes each time.

[0057] 3. Blocking endogenous peroxidase: Place the slides in a 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 minutes. Then, place the slides in PBS (pH 7.4) and wash them three times on a decolorizing shaker for 5 minutes each time.

[0058] 4. Serum blocking: After the sections are dried, draw a circle around the tissue with a histochemical pen, and add 3% BSA evenly to the inside of the circle to block the tissue at room temperature for 30 minutes.

[0059] 5. Add primary antibody: Discard the blocking solution, add the primary antibody (recombinant anti-tyrosine hydroxylase antibody, Seville Biotechnology, catalog number: GB15182-100, dilution ratio 1:500) prepared in PBS to the slide, and incubate the slide flat in a humidified chamber at 4°C overnight.

[0060] 6. Add secondary antibody: Place the slide in PBS (pH 7.4) and wash three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, add the secondary antibody (HRP-labeled) of the corresponding species from the histochemistry kit to the inside of the slide and incubate at room temperature for 50 minutes.

[0061] 7. DAB staining: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, add freshly prepared DAB staining solution to the circle. Control the staining time under a microscope. A positive result is brownish-yellow. Rinse the slide with tap water to stop the staining process.

[0062] 8. Counterstaining cell nuclei: Counterstain with hematoxylin for about 3 minutes, wash with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, re-blue with hematoxylin blue solution, and rinse with running water.

[0063] 9. Dehydration and mounting: Place the sections in the following solutions in sequence: 75% alcohol for 5 min, 85% alcohol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, n-butanol for 5 min, and xylene I for 5 min to dehydrate and clear the sections. Remove the sections from the xylene and let them dry slightly before mounting with mounting adhesive.

[0064] 10. Microscopic examination: The results were interpreted under a white light microscope (Nikon Instruments Co., Ltd., model: E100).

[0065] Experimental results:

[0066] MPTP modeling resulted in a decrease in the number of dopamine neurons in the substantia nigra of model mice. All drug administration groups could improve the reduction in the number of dopamine neurons in model mice. Figure 3 Part A of the drug, while MPTP modeling led to the loss of striatal fibers in the model mice, and each drug administration group could improve the loss of striatal fibers in the model mice ( Figure 3 Part B)

[0067] Example 4: Determination of serum inflammatory factors in mice

[0068] Experimental methods:

[0069] Mouse serum was collected, and the levels of pro-inflammatory factors IL-1β, TNF-α, and IL-6 in the mouse serum were detected using the ELISA method according to the kit instructions.

[0070] IL-1β: The mouse IL-1β detection kit (catalog number: HY-H0001) purchased from Beijing Huaying Biotechnology Research Institute was used for detection.

[0071] TNF-α: The mouse TNF-α detection kit (catalog number: HY-H0019) purchased from Beijing Huaying Biotechnology Research Institute was used for detection.

[0072] IL-6: The mouse IL-6 detection kit (catalog number: HY-H0007) purchased from Beijing Huaying Biotechnology Research Institute was used for detection.

[0073] Experimental results:

[0074] After MPTP modeling, the levels of pro-inflammatory factors in the serum of the model group increased significantly. Figures 4A-4C (p < 0.001), all treatment groups improved the increase of IL-1β, TNF-α, and IL-6 in the serum of model mice. Figures 4A-4C (p < 0.05).

[0075] Example 5: Immunohistochemical detection of the number of astrocytes and microglia in the substantia nigra.

[0076] Experimental methods:

[0077] The immunohistochemical staining method was the same as in Example 3. The primary antibodies used were recombinant anti-GFAP antibody (Seville Biotech, catalog number: GB15100-100, dilution ratio 1:1000) and recombinant anti-Iba1 antibody (Seville Biotech, catalog number: GB153502-100, dilution ratio 1:1000).

[0078] Experimental results

[0079] L-NRB administration significantly inhibited MPTP-induced microglia in mouse substantia nigra. Figure 5 Part A) and astrocytes ( Figure 5 Activation of Part B)

[0080] Example 6: Determination of MAO-B activity in mouse striatum

[0081] Experimental methods:

[0082] The striatum was lysed using a lysis buffer containing protease inhibitors and phosphatase inhibitors, and proteins were extracted. After standing at 4°C for 30 min, the mixture was centrifuged at 12000 rpm for 30 min at 4°C to remove insoluble impurities. The protein concentration was then determined using a BCA protein quantification kit (Kangwei Century, catalog number: CW0014S). Amplex was used according to the kit instructions. TM The Red Monoamine Oxidase Assay Kit (Thermo Fisher Scientific, catalog number: A12214) detects the activity of MAO-B in samples.

[0083] Experimental results:

[0084] In the model group, the activity of MAO-B was significantly increased compared with that in the control group. Figure 6 (p < 0.01), medium and high doses of L-NRB significantly reduced the activity of MAO-B in the striatum. Figure 6 (p < 0.05).

[0085] 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 L-NRB, whose chemical formula is shown as formula I, in the preparation of a drug for preventing and treating Parkinson's disease: Formula I.

2. The use according to claim 1, wherein the Parkinson's disease is MPTP-induced Parkinson's disease.

3. The use according to claim 1 or 2, wherein the drug is in the form of oral, injection or transdermal.

4. The use according to claim 3, wherein the drug is in the form of oral.

5. The use according to claim 3, wherein the drug further comprises pharmaceutically acceptable adjuvants or excipients.

Images