Use of bimatoprost for the preparation of a medicament for the prevention and / or treatment of parkinson's disease

Abstract

This invention discloses the application of bimatoprost in the preparation of drugs for the prevention and / or treatment of Parkinson's disease, within the field of drug reuse. Bimatoprost is a prostaglandin F2α analog, currently mainly used for glaucoma and eyelash growth. This invention has found that bimatoprost can improve Parkinson's disease-related behavioral disorders, increase the expression of tyrosine hydroxylase, a protein associated with dopaminergic neurons in the substantia nigra, and reduce the abnormal accumulation of α-synuclein, thus exhibiting good ameliorative and protective effects against Parkinson's disease. This invention provides a new drug option for the treatment of Parkinson's disease.

Description

Technical Field

[0001] This invention relates to novel uses of pharmaceuticals in the field of medicine, specifically to the use of bimatoprost in the preparation of drugs for the prevention and / or treatment of Parkinson's disease. Background Technology

[0002] Parkinson's disease (PD) is a common neurodegenerative disorder of the central nervous system, characterized by the gradual loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain and the abnormal aggregation of α-synuclein (α-syn) to form Lewy bodies. Numerous studies have shown that the accumulation of misfolded α-syn within neurons, forming protein aggregates, is a core neuropathological feature of various PD subtypes. To date, α-syn pathological testing remains the gold standard for definitive diagnosis. Furthermore, the abnormal aggregation of α-syn is not only a pathological marker but is also considered a key driver of the early onset and continued progression of the disease. As the disease progresses, patients develop typical motor symptoms such as bradykinesia, resting tremor, rigidity, and postural instability, and may also experience non-motor symptoms such as sleep disturbances, mood disorders, and autonomic dysfunction. Due to its complex etiology, slow progression, and involvement of multiple pathological processes, there are currently no effective treatments to alter the course of Parkinson's disease. Recent studies have shown that the glycoprotein nonmetastatic melanoma protein B (GPNMB) is closely related to neurodegenerative diseases, and it has potential research value in the pathological processes of dopaminergic neuron damage and Parkinson's disease. Furthermore, research has shown that GPNMB can interact with α-Syn, and its expression changes are associated with abnormal aggregation and intercellular transmission of α-Syn protein. Currently, there is no research on the application of drug intervention to regulate GPNMB-related changes for the prevention and treatment of Parkinson's disease.

[0003] Current clinical treatments primarily focus on symptomatic relief with medications, including levodopa, dopamine receptor agonists, MAO-B inhibitors, and COMT inhibitors. While these drugs can improve early symptoms, long-term use often leads to diminished efficacy, dose-dependent side effects, and motor complications (such as dyskinesia and fluctuating treatment response). Furthermore, none of these drugs effectively prevents further degeneration of dopaminergic neurons, failing to fundamentally intervene in disease progression. Therefore, developing novel treatment strategies that can intervene in disease progression and possess neuroprotective effects is urgently needed.

[0004] In recent years, the strategy of drug repurposing has received widespread attention. By conducting new indication studies on already marketed drugs, the repeated verification of pharmacokinetics and safety in the early stages of drug development can be bypassed, significantly shortening the translational cycle. A growing body of research demonstrates that some drugs initially used in ophthalmology, cardiovascular diseases, or the immune system may possess novel biological activities in neurological diseases. However, research on drug repurposing for Parkinson's disease remains limited, particularly in interventions targeting core pathological aspects such as α-synuclein aggregation, decreased TH expression, and neurodegenerative damage.

[0005] Bimatoprost is a prostaglandin F2α analog currently used primarily as an ophthalmic drug to lower intraocular pressure, and has been shown to have good pharmacokinetic properties and clinical safety. Despite its widespread use in ophthalmology, no studies have been reported on the effects of bimatoprost on Parkinson's disease. Summary of the Invention

[0006] The purpose of this invention is to provide a novel pharmaceutical use for bimatoprost, opening up new areas for the clinical application of this drug and providing a novel and promising candidate drug for the treatment of Parkinson's disease.

[0007] In a first aspect, to achieve the above objectives, the present invention adopts the following technical solution: the use of bimatoprost in the preparation of drugs for the prevention and / or treatment of Parkinson's disease.

[0008] Furthermore, the drug is used to improve motor and behavioral disorders associated with Parkinson's disease.

[0009] Furthermore, the motor behavior disorder includes one or more of the following: motor coordination disorder, bradykinesia, and balance disorder.

[0010] Furthermore, the bimatoprost is used to increase the expression level of tyrosine hydroxylase, a marker of dopaminergic neurons.

[0011] Furthermore, the bimatoprost is used to reduce the expression level or abnormal aggregation level of α-synuclein.

[0012] Preferably, as an improvement, the dosage of bimatoprost is 10–30 mg / kg. More preferably, it is 10 mg / kg or 30 mg / kg.

[0013] Preferably, as an improvement, the method of administration of the drug is selected from at least one of nasal administration, intraperitoneal injection, subcutaneous administration, or oral administration.

[0014] In a second aspect, the present invention provides a pharmaceutical composition for the prevention and / or treatment of Parkinson's disease, comprising bimatoprost as an active ingredient and a pharmaceutically acceptable carrier.

[0015] Preferably, as an improvement, the pharmaceutical composition is formulated for use by at least one of nasal administration, intraperitoneal injection, subcutaneous administration, or oral administration.

[0016] The beneficial technical effects of this invention are as follows: This study found that bimatoprost can effectively improve motor behavioral disorders in a Parkinson's disease model, increase TH expression, and reduce abnormal expression or aggregation of α-syn. Furthermore, after treatment with bimatoprost, disease-related molecular markers (including GPNMB) in the Parkinson's disease model showed favorable changes, suggesting the potential application value of bimatoprost in the treatment of Parkinson's disease. As a marketed drug, bimatoprost has advantages such as well-established safety, good stability, and clear pharmacokinetic characteristics, making it suitable for further development and widespread application in the treatment of Parkinson's disease. Attached Figure Description

[0017] Figure 1 This is a statistical graph showing the time spent on the rotarod in each group of mice during the rotarod experiment; Figure 2 This is a statistical graph showing the time each group of mice spent in the grid during the grid experiment; Figure 3 This is a statistical graph showing the time it took for each group of mice to reach the bottom in the pole climbing experiment; Figure 4 This is a statistical graph showing the time taken for each group of mice to cross the balance beam in the balance beam experiment; Figure 5 These are representative trajectory diagrams and total distance statistics of each group of mice in the open field experiment; Figure 6 These are representative immunohistochemical staining images of the substantia nigra pars compacta in the brain of mice from each group, along with their statistical diagrams. Figure 7 This is a schematic diagram of the interaction network between bimatoprost and GPNMB protein; Figure 8 This is a schematic diagram of the molecular docking between bimatoprost and the GPNMB protein binding site; Figure 9 This is a Western blot diagram and statistical graph of the expression of GPNMB, TH, and α-syn proteins in the brain tissue of mice in different groups of MPTP-induced Parkinson's disease model. Figure 10 This is a Western blot diagram and statistical graph showing the expression of GPNMB, TH, and α-syn proteins in the brain tissue of mice in different groups of a 6-OHDA-induced Parkinson's disease model. Detailed Implementation

[0018] This invention has discovered that bimatoprost can be used to prepare drugs for improving and / or preventing and / or treating Parkinson's disease. After treatment with bimatoprost, motor dysfunction in Parkinson's disease model mice was improved, dopaminergic neuron-related indicators showed favorable changes, abnormal expression or aggregation of α-synuclein decreased, and molecular indicators related to Parkinson's disease (including GPNMB) changed, thus demonstrating good preventive and therapeutic effects against Parkinson's disease.

[0019] The drug uses bimatoprost as its active ingredient, which can be administered via pharmaceutically acceptable routes of administration. These routes include intraperitoneal injection, subcutaneous administration, nasal administration, or oral administration.

[0020] The present invention does not impose any particular limitation on the administration method or the specific preparation method, and any method known to those skilled in the art can be used.

[0021] In this invention, the dosage of bimatoprost is 10–30 mg / kg. Bimatoprost within this dosage range is more effective in preventing and treating Parkinson's disease.

[0022] In a specific embodiment of the present invention, taking a 6-hydroxydopamine (6-OHDA) or MPTP-induced Parkinson's disease mouse model as an example, the experimental animals were randomly divided into a control group, a model group, and a bimatoprost treatment group. The bimatoprost treatment group was administered the drug according to the above-mentioned dosage and administration method. After a certain period of administration, behavioral experiments were performed on the mice in each group, and brain tissue was collected for relevant indicator detection, thereby further illustrating the technical effects of bimatoprost in improving and / or treating Parkinson's disease.

[0023] experiment: 1.1 Laboratory Animals C57 male mice, weighing approximately 20 g, were purchased from the Experimental Animal Center of Zunyi Medical University. The animals were housed in the SPF animal facility of the Key Laboratory of Basic Pharmacology of the Ministry of Education at Zunyi Medical University. Each group of experimental animals was kept under constant temperature and humidity conditions, with free access to clean drinking water and basic feed. The circadian rhythm was 12 h.

[0024] 1.2 Main Instruments and Equipment Mouse rotarod test apparatus, mouse open field test apparatus, mouse balance beam apparatus, mouse pole climbing apparatus, mouse grid apparatus, stereotaxic apparatus, Bio-Rad chemiluminescence imaging system, etc.

[0025] 1.3 Main Experimental Reagents Bimatoprost (BIM, CAS: 35121-78-9); 6-hydroxydopamine (6-OHDA); MPTP; DMSO; 0.9% physiological saline; 4% paraformaldehyde; RIPA lysis buffer, PMSF; BCA protein quantification kit; ECL luminescent reagent; immunohistochemistry and immunoblotting related reagents; primary antibodies: TH, GPNMB, α-Syn, GAPDH, etc.; secondary antibodies: HRP-labeled secondary antibodies, etc.

[0026] 2. Experimental Methods 2.1 Grouping and Administration SPF-grade male C57BL / 6J mice, weighing 20–25 g, were randomly divided into 8 groups (n=10). BIM below refers to bimatoprost.

[0027] Control group; Control + BIM (30 mg / kg) group; 6-OHDA group; 6-OHDA + BIM (10 mg / kg) group; 6-OHDA + BIM (30 mg / kg) group; MPTP group; MPTP + BIM (10 mg / kg) group; MPTP + BIM (30 mg / kg) group.

[0028] 1) Control group: No modeling treatment was given, but an equal volume of physiological saline was injected into the peritoneum.

[0029] 2) Control + BIM group: No modeling treatment was given, and bimatoprost 30 mg / kg was injected intraperitoneally.

[0030] 3) 6-OHDA group: Parkinson's disease model was established by stereotactic injection of 6-OHDA, followed by intraperitoneal injection of an equal volume of physiological saline.

[0031] 4) 6-OHDA + BIM group: After establishing the 6-OHDA Parkinson's disease model, bimatoprost was administered intraperitoneally at doses of 10 mg / kg or 30 mg / kg, respectively.

[0032] 5) MPTP group: Parkinson's disease model was established by intraperitoneal injection of MPTP, followed by intraperitoneal injection of an equal volume of physiological saline.

[0033] 6) MPTP + BIM group: After establishing the MPTP Parkinson's disease model, bimatoprost was administered intraperitoneally at doses of 10 mg / kg or 30 mg / kg, respectively.

[0034] 2.2 Preparation of PD animal models This study used two classic PD animal models for joint validation: the 6-OHDA stereotactic injection model and the MPTP intraperitoneal injection model.

[0035] (1) 6-OHDA modeling: C57 male mice were anesthetized and fixed in a stereotaxic apparatus, with the anterior fontanelle exposed. The anterior fontanelle was used as the origin of the coordinate system, and the positioning points were: 2.2 mm posterior to the anterior fontanelle, 1.5 mm lateral to the midline, and 4.8 mm subdural depth. 6-OHDA solution (5 μg / μL, prepared with physiological saline containing 0.2% ascorbic acid) was slowly injected at a rate of 1 μL / min, with a total volume of 2 μL. The needle was stopped for 3 min after injection, and then the needle was slowly withdrawn and the wound was sutured.

[0036] (2) MPTP modeling: The subacute MPTP modeling method was used, with MPTP (30 mg / kg, dissolved in physiological saline) injected intraperitoneally daily for 7 consecutive days. The control group was injected with an equal volume of physiological saline. Body weight and general condition were monitored during the modeling period.

[0037] Example 1: The ameliorative effect of bimatoprost on the behavior of a Parkinson's disease model mouse 2.3 Behavioral Testing After the intervention, the following behavioral tests will be conducted: 2.3.1 Rotating bar experiment The rotarod test is used to evaluate the motor coordination and balance abilities of laboratory animals. Before the experiment, the animals undergo acclimatization training to reduce the impact of environmental changes on the experimental results. During the formal experiment, mice are placed on the rotarod apparatus, and the time it takes for them to fall off the rotarod is recorded to reflect changes in the animals' motor coordination function.

[0038] 2.3.2 Grid Experiment The grid test was used to assess neuromuscular strength and coordination in mice. Mice were placed on a horizontal 12 cm × 12 cm metal grid. Once the mice had gripped the grid firmly with all four limbs, the device was rotated 180° and timing began. Timing stopped when the mouse completely released the grid (i.e., its paws left the grid). A suspension time exceeding 3 minutes was recorded as 180 seconds. Each mouse was tested three times, with 30-minute intervals between tests, and the average suspension time was taken as the result.

[0039] 2.3.3 Pole Climbing Experiment The pole-climbing test is used to assess the motor coordination and balance abilities of mice. During the test, mice are placed head-up at the top of a vertical pole, and their ability to automatically turn and descend to the bottom is recorded. Typically, the mice will descend naturally to the ground and return to their cage. The latency from the start of the turn to completion and the total time to descend to the bottom of the pole are recorded. Each mouse is tested three times, with 30-minute intervals between tests, and the average value is taken as the final result.

[0040] 2.3.4 Balance Beam Experiment The balance beam test is used to assess the motor coordination and balance ability of mice. In the experiment, mice are asked to walk upright on a narrow, elevated beam to reach a finish platform. The experiment lasts for 3 days, with the first 2 days for acclimatization training and the third day for the formal test. During the test, the mice are placed at the starting point of the balance beam, and the time required to cross it is recorded. Each mouse is tested 3 times, and the average value is taken as the final result.

[0041] 2.3.5 Open Field Experiment The open field test was conducted in a quiet environment to assess the spontaneous activity and exploratory behavior of mice. C57BL / 6J mice were gently placed in the center of the open field chamber, and their movement was recorded using a video recording system for 5 minutes. After each test, the bottom and inner walls of the chamber were wiped with 75% ethanol to remove any odor residue and avoid affecting the behavior of the next animal. Mice were rotated sequentially to complete the test. After the experiment, the movement trajectory was recorded, and behavioral indicators such as the total distance traveled and the activity time were statistically analyzed.

[0042] Example 2: Protective effect of bimatoprost on dopaminergic neurons in a Parkinson's disease model mouse 2.4 Sampling and Specimen Preparation After behavioral testing, mice were anesthetized with 2% sodium pentobarbital (40 mg / kg) and fixed in a supine position. After opening the thoracic cavity, approximately 200 mL of PBS was perfused into the left ventricle using a peristaltic pump until the liver turned white. Protein analysis samples were taken from the substantia nigra region of the midbrain, placed in EP tubes, and flash-frozen in liquid nitrogen. Store at 80 °C for Western blot analysis. Histological samples: continue perfusion with 4% paraformaldehyde solution until limbs are rigid. Whole brain samples were harvested, fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and then used for immunohistochemical analysis.

[0043] 2.5 Immunohistochemical staining Paraffin sections (5 μm) were dewaxed and rehydrated for antigen retrieval, endogenous peroxidase was inhibited, serum blocked, and incubated with TH primary antibody at 4°C. The next day, they were incubated with HRP secondary antibody and DAB staining was performed, followed by dehydration, clearing, and mounting. TH-positive signals in the substantia nigra region were observed under a microscope and statistically analyzed.

[0044] Example 3: Effects of bimatoprost on the expression of Parkinson's disease-related proteins and exploration of the mechanism of interaction between bimatoprost and GPNMB protein. 2.6 Western blot assay 1) Weigh an appropriate amount of midbrain tissue, grind it on ice, add RIPA lysis buffer (containing PMSF inhibitor), mix well and let it stand for lysis; 2) Centrifuge the lysis buffer and collect the supernatant for subsequent protein detection; 3) The protein sample was quantified using the BCA method, and a loading system was prepared, mixed, and then denatured. 4) Add the processed protein sample to an SDS-PAGE gel for electrophoretic separation; 5) The protein was transferred onto a polyvinylidene fluoride (PVDF) membrane using a wet transfer method; 6) After the transfer is complete, seal the membrane with skim milk powder and then clean it; 7) Incubate with primary antibody and detect TH, GPNMB, α-Syn and GAPDH proteins; 8) After cleaning, add the appropriate secondary antibody for incubation, develop using chemiluminescence, and analyze the band signals.

[0045] 2.7 Statistical Analysis of Data Experimental data were analyzed using Graphpad Prism statistical software. All data are expressed as mean ± standard deviation. One-way ANOVA was used to compare differences in means between groups. The LSD test was used to test homogeneity of variance, and Dunnett's T3 was used to compare differences in means between groups. P <0.05 is statistically significant.

[0046] 3. Experimental Results 3.1 Effects of bimatoprost on motor behavior in a mouse model of Parkinson's disease After establishing a Parkinson's disease model and administering appropriate treatments, various behavioral tests were performed on mice in each group. For example... Figure 1 – Figure 5 As shown, compared with the Control group, mice in the 6-OHDA and MPTP groups exhibited reduced rotarod time in the rotarod test, time spent in the grid test in the grid test, and distance traveled in the open field test, while increasing the time required to cross the balance beam in the balance beam test and the time to reach the bottom of the pole in the pole climbing test. This suggests that the Parkinson's disease model was successfully established, and the differences were statistically significant. P < 0.05); Compared with the model group, the mice in the model drug group (30 mg / kg) showed beneficial changes in the relevant indicators in the above behavioral experiments and the differences were statistically significant (#P < 0.05), indicating that BIM can improve motor dysfunction in Parkinson's disease model mice.

[0047] 3.2 Effects of bimatoprost on dopaminergic neurons in a Parkinson's disease model mouse Immunohistochemistry was used to examine the substantia nigra pars compacta in the midbrain of mice in each group. Figure 6 As shown, compared with the Control group, the number of TH-positive neurons in the substantia nigra of mice in both the 6-OHDA group and the MPTP group was significantly reduced. P<0.05); Compared with the corresponding model group, the number of TH positive neurons in the model group was significantly increased (#P<0.05), suggesting that BIM can effectively reduce the damage of dopaminergic neurons in the two PD models.

[0048] 3.3 Schematic analysis of the interaction between bimatoprost and GPNMB protein The interaction relationship between equine prostrin and GPNMB protein was analyzed using protein interaction network analysis and molecular docking methods. Figure 7 and Figure 8 As shown, bimatoprost is associated with GPNMB in the protein action network and exhibits potential binding sites at the molecular level, providing a reference for its application in Parkinson's disease.

[0049] 3.4 Effects of bimatoprost on the expression of related proteins in different Parkinson's disease models The expression of relevant proteins in the brain tissue of different Parkinson's disease model mice was detected using Western blot. For example... Figure 9 and Figure 10 As shown, in MPTP and 6-OHDA-induced Parkinson's disease models, compared with the Control group, the expression of TH protein in the brain tissue of mice in the model group was decreased, while the expression of α-syn protein and GPNMB protein was increased. Compared with the model group, the expression level of TH protein was improved and the abnormal increase trend of α-syn protein and GPNMB protein was alleviated after treatment in the model drug group.

[0050] Summary of Experimental Results In summary, this invention systematically evaluated the effects of bimatoprost in an animal model of Parkinson's disease from an overall experimental perspective, using various methods such as rotarod experiments, grid experiments, pole climbing experiments, balance beam experiments, open field experiments, and protein expression detection. This verified that bimatoprost can improve motor dysfunction and regulate related pathological indicators in Parkinson's disease models, demonstrating its potential for application in the prevention and treatment of Parkinson's disease.

[0051] The advantage of this invention lies in its application of bimatoprost, specifically its use in the preparation of drugs for the prevention and / or treatment of Parkinson's disease. Animal experimental results demonstrate that bimatoprost can have a positive interventional effect on Parkinson's disease models.

[0052] (1) Bimatoprost can improve motor dysfunction in Parkinson's disease model mice to a certain extent and enhance their motor coordination and activity. (2) Bimatoprost can improve the abnormal changes of dopaminergic neurons in the substantia nigra region of the brain in Parkinson's disease model mice and has a certain protective effect on nerve function. (3) Bimatoprost can regulate the expression levels of disease-related proteins in the brain tissue of Parkinson's disease model mice, including TH, α-syn and GPNMB, making their expression status different from that of the model group.

[0053] In conclusion, bimatoprost has shown the effect of improving motor function and regulating related pathological indicators in the Parkinson's disease model, and has good application prospects. It can provide experimental evidence for its further development and application in the field of Parkinson's disease prevention and treatment.

[0054] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. Use of bimatoprost in the preparation of drugs for the prevention and / or treatment of Parkinson's disease.

2. The use according to claim 1, characterized in that: The drug is used to improve motor and behavioral disorders associated with Parkinson's disease.

3. The use according to claim 2, characterized in that: The motor behavior disorder includes one or more of the following: motor coordination disorder, bradykinesia, and balance disorder.

4. The use according to claim 3, characterized in that: The bimatoprost is used to increase the expression level of tyrosine hydroxylase, a marker of dopaminergic neurons.

5. The use according to claim 4, characterized in that: The bimatoprost is used to reduce the expression level or abnormal aggregation level of α-synuclein.

6. The use according to claim 5, characterized in that: The dosage of bimatoprost is 10–30 mg / kg.

7. The use according to claim 6, characterized in that: The method of administration of the drug is selected from at least one of nasal administration, intraperitoneal injection, subcutaneous administration, or oral administration.

8. A pharmaceutical composition for the prevention and / or treatment of Parkinson's disease, characterized in that, It contains bimatoprost as the active ingredient, and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition is formulated for use by at least one of the following methods: intranasal administration, intraperitoneal injection, subcutaneous administration, or oral administration.

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