Application of rotigotine microspheres in preparation of medicine for preventing and treating multiple sclerosis

Abstract

The invention discloses an application of rotigotine microspheres in preparation of a medicine for preventing or treating multiple sclerosis. The rotigotine microspheres can improve spinal cord demyelination and infiltration of spinal cord inflammatory cells, can also improve the weight problem, can be administrated once every 14 days as a microsphere preparation, greatly improves the use compliance of patients, and provides a new thought for treating multiple sclerosis.

Description

Technical Field

[0001] The present invention relates to the field of medicines, in particular to the application of rotigotine microspheres in the preparation of medicines for preventing and treating multiple sclerosis, and belongs to the field of medical technology. Background Art

[0002] Rotigotine is a non-ergotamine dopamine receptor agonist with a chemical formula of C 19 H 25 NOS, molecular weight is 315.47. Rotigotine can act on dopamine, among which DRD3 has the highest affinity and the strongest stimulatory effect, DRD2, DRD4 and DRD5 have relatively weak affinity, and DRD1 has the weakest affinity. Rotigotine is mainly used to treat idiopathic Parkinson's disease and restless legs syndrome. Its anti-Parkinson's effect is achieved by stimulating DRD1, DRD2 and DRD3.

[0003] Rotigotine microspheres are injected intramuscularly, which allows the drug to be released continuously over a long period of time, continuously and stably stimulating dopamine receptors, thereby maintaining compliance of patients.

[0004] Multiple sclerosis (MS) is a chronic autoimmune disease that often occurs in the central nervous system and is characterized by temporal and spatial multiplicity. Its pathology is multiple. According to the "Chinese Guidelines for the Diagnosis and Treatment of Multiple Sclerosis" (2023 edition), the cause of MS is still unclear. Epstein-Barr virus infection, low serum vitamin D levels, insufficient sun exposure, smoking, and obesity in adolescence may be risk factors for the onset of MS. MS lesions can involve CNS white matter and gray matter; the acute phase is manifested by T lymphocyte-mediated inflammation around venules, followed by demyelination, accompanied by varying degrees of axonal damage; progressive MS pathology shows more severe white and gray matter demyelination, axonal loss, meningeal inflammatory changes, and gray matter involvement.

[0005] The current prevention method for multiple sclerosis is regular examination, and the diagnostic criteria are determined by the number of attacks and the number of lesions. The main drugs currently used are: acute treatment period: ① glucocorticoids, with methylprednisolone as the first-line treatment, or oral prednisone acetate or prednisolone; ② plasma exchange: as the second-line treatment; remission treatment period: teriflunomide, fingolimod hydrochloride, siponimod, ozanimod, dimethyl fumarate, ofatumumab, glatiramer acetate, etc.

[0006] Multiple sclerosis can present a variety of symptoms, such as chronic pain, paresthesia, fatigue (a more obvious symptom in patients), impaired walking ability, ataxia and tremors, bladder dysfunction, sexual dysfunction, bowel dysfunction, depression and mood instability, and cognitive impairment. Therefore, symptom management and symptomatic treatment for multiple sclerosis are very important.

[0007] Currently, no studies or reports on the prevention or treatment of multiple sclerosis with rotigotine microspheres have been found. Summary of the Invention

[0008] The present invention has for the first time discovered the use of rotigotine microspheres in the preparation of a drug for preventing or treating multiple sclerosis, providing a new treatment plan for the treatment of multiple sclerosis.

[0009] On the one hand, the present invention provides the use of rotigotine microspheres in the preparation of a drug for preventing or treating multiple sclerosis.

[0010] Rotigotine microspheres can both prevent the onset of multiple sclerosis and treat multiple sclerosis.

[0011] Further, the use of the rotigotine microspheres lies in preventing or treating: spinal cord myelin loss, and / or infiltration of spinal cord inflammatory cells, and / or myelin regeneration.

[0012] Further, while preventing or treating multiple sclerosis, it is possible to combine and maintain body weight.

[0013] Further, the multiple sclerosis is caused by immune deficiency.

[0014] Further, rotigotine microspheres mainly complete the treatment of multiple sclerosis by increasing the level of IL-10 and / or decreasing the level of TNF-α.

[0015] Further, the pharmaceutical preparation is an oral preparation or an injection.

[0016] Further, the rotigotine microspheres are administered by intramuscular injection, once every 2 weeks.

[0017] Further, the dosage of the rotigotine microspheres is 20 mg / kg to 40 mg / kg, and the effect is better with the increase of the dosage.

[0018] Further, when preparing a drug for preventing or treating multiple sclerosis, the rotigotine microspheres can be used alone, or compounded with other drugs, compounds, carriers into a pharmaceutical composition.

[0019] Further, a drug for preventing or treating multiple sclerosis contains rotigotine microspheres.

[0020] Advantages of the present invention:

[0021] 1. The present invention first discovers the application of rotigotine microspheres in the preparation of drugs for preventing or treating multiple sclerosis. The rotigotine microspheres can directly act on spinal cord myelin loss and improve the infiltration of spinal cord inflammatory cells.

[0022] 2. The present invention can effectively improve the related symptoms of multiple sclerosis and combined physical problems.

[0023] 3. The rotigotine microspheres can be used alone or in combination with other known drugs, compounds or carriers to prepare various forms of medicaments or pharmaceutical compositions such as oral preparations and injections for prevention or treatment.

[0024] 4. The microsphere preparation has a long medication cycle, which greatly improves the compliance of use. The present invention provides a new idea for preventing or treating multiple sclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Effect of LARM on the body weight of mice.

[0026] Figure 2 Effect of LARM on the disease state of mice.

[0027] Figure 3 Effect of LARM on IL-10 in the spinal cord of mice.

[0028] Figure 4 Effect of LARM on TNF-α in the spinal cord of mice.

[0029] Figure 5 Effect of LARM on the histopathology of the spinal cord of mice (HE staining, 400×).

[0030] Figure 6 Effect of LARM on the histopathology of the spinal cord of mice (LFB staining, 400×). DETAILED DESCRIPTION OF THE INVENTION

[0031] The methods and techniques of the present invention are generally carried out according to the conventional methods known in the art, unless otherwise specified. The nomenclatures related to biology, pharmacology, and medical and pharmaceutical chemistry described herein, as well as the experimental methods and techniques, are known and commonly used in the art.

[0032] Unless otherwise specified, the scientific and technical terms used herein shall have the meanings commonly understood by those of ordinary skill in the art. However, the following terms have the following definitions:

[0033] TERM

[0034] The term "therapeutically effective dose" refers to any amount of a drug that, when used alone or in combination with another therapeutic agent, promotes the regression of a disease, which is manifested as a decrease in the severity of the disease symptoms, an increase in the frequency and duration of the disease-free period, or the prevention of a disorder or disability caused by the disease. The "therapeutically effective dose" of the drug of the present invention also includes a "prophylactically effective dose", which is any amount of a drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or suffering from a recurrence of the disease, inhibits the occurrence or recurrence of the disease.

[0035] As will be apparent to those skilled in the art, the effective in vivo dosage and the specific mode of administration will vary depending on the species, weight and age of the mammal being treated, the specific compound used, and the specific purpose for which these compounds are used. Those skilled in the art can determine the effective dose level (i.e., the dose level necessary to achieve the desired effect) according to conventional pharmacological methods. Generally, the human clinical application of the product starts at a lower dose level and the dose level is then continuously increased until the desired effect is achieved. Alternatively, the useful dosage and route of administration of the compositions identified by this method can be established by acceptable in vitro studies using existing pharmacological methods.

[0036] To better understand the content of the present invention, the content of the present invention will be further described below in conjunction with specific implementation methods, but the protection content of the present invention is not limited to the following examples.

[0037] Experimental materials and reagents

[0038] Experimental animals: C57BL / 6J mice, female, weighing 16 - 20 g, purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., certificate number SCXK(Lu)20190003.

[0039] Experimental drugs: Rotigotine microspheres, from Shandong Luye Pharmaceutical Co., Ltd.

[0040] Experimental reagents and consumables: 0.9% sodium chloride solution, mouse TNF-α kit, mouse IL-10 kit, absolute ethanol, paraformaldehyde, PBS, MOG35-55 peptide, 40μm purple cell strainer, Complete Freund's Adjuvant, pertussis toxin; medical dispensing three-way valve, screw syringe, straight tip surgical scissors, sterile centrifuge tube, disposable sampling trough.

[0041] Experimental instruments: AL 204 electronic balance, vortex mixer, centrifuge, electronic balance, incubator, ice maker, Forma902 upright ultra-low temperature refrigerator, pipette.

[0042] All experimental materials and reagents are commercially available.

[0043] Abbreviations:

[0044] Rotigotine microspheres: LARM

[0045] Sodium Carboxymethylcellulose: Sodium Carboxymethylcellulose, CMC-Na

[0046] Pertussis toxin: PTx

[0047] Phosphate Buffered Saline: PBS

[0048] Complete Freund's Adjuvant: CFA

[0049] Experimental autoimmune encephalomyelitis model: EAE

[0050] Preparation of the solution in Example 1

[0051] 1. Preparation of MOG35-55 solution:

[0052] Dilute the lyophilized MOG35-55 with normal saline and store it in a refrigerator at -20°C to obtain the MOG35-55 stock solution; before immunization, dilute the MOG35-55 stock solution with normal saline to a concentration of 1 mg / mL.

[0053] 2. Preparation of MOG35-55 / CFA emulsion:

[0054] With a syringe, draw out the CFA stock solution and the MOG35-55 solution respectively, and quickly push the MOG35-55 solution into the CFA stock solution. Transfer the solution between the two syringes about 10 minutes. After the syringe becomes easy to rotate, drop a drop into water. If the droplet does not spread on the water surface, it is obtained.

[0055] 3. Preparation of pertussis toxin (PTx):

[0056] Dilute the PTx freeze-dried powder with PBS to a concentration of 1 μg / mL.

[0057] 4. Preparation of LARM suspension:

[0058] Preparation of the stock solution: Take 1 bottle of LARM with a specification of 112 mg, add 2 mL of 1% CMC-Na to LARM to prepare a 56 mg / mL LARM suspension stock solution.

[0059] Preparation of LARM suspension: Take 0.6 mL of the stock solution and dilute it with 1% CMC-Na to make a 4 mg / mL LARM suspension.

[0060] Preparation of 5.4% paraformaldehyde solution:

[0061] Take 80 g of paraformaldehyde powder, add 1800 mL of PBS solution, make up the volume to 2 L, and dissolve it in an incubator.

[0062] Establishment and administration of experimental autoimmune encephalomyelitis (EAE) model in Example 2

[0063] 2.2.1 Experimental autoimmune encephalomyelitis model

[0064] Induce the EAE model by subcutaneous injection of MOG35-55 / CFA emulsion (100 μL) into the neck and lower back of mice respectively.

[0065] 2.2.2 Grouping and administration of experimental animals

[0066] Healthy 6-8-week-old female C57BL / 6J mice were randomly divided into 4 groups, namely Control group, EAE group, EAE + LARM 20 mg / kg group, and EAE + LARM 40 mg / kg group.

[0067] The mice in each group were weighed and recorded daily, and administered according to their body weight. The intramuscular injection volume was 0.05 mL / 10 g. The mice were anesthetized with isoflurane; except for the Control group, the other 4 groups were injected subcutaneously with MOG35-55 / CFA emulsion into the neck and lower back of the mice; on the day of immunization and the 2nd day, PTx 10 mL / kg (10 μg / kg PTx) was injected intraperitoneally; on the 2nd day and the 15th day after immunization, LARM was injected intramuscularly into the EAE + LARM 20 mg / kg group and the EAE + LARM 40 mg / kg group.

[0068] The specific administration methods are as follows in the table:

[0069] Summary table of drug administration and treatment for experimental mice

[0070]

[0071]

[0072] 2.2.3 Experimental content

[0073] 2.2.3.1 EAE monitoring

[0074] For the monitoring of EAE, on the 0th day (the day of drug administration), the mice were first evaluated after the anesthesia recovery, and on the 2nd day (the day of only injecting PTx), the evaluation was carried out immediately after drug administration.

[0075] Mouse disease status scoring table

[0076] Score Disease characteristics 0 points No defect 1 point Partial caudal paralysis* 2 points Complete caudal paralysis* 3 points Partial hindlimb paralysis 4 points Complete hindlimb paralysis 5 points Forelimb paralysis 6 points Death

[0077] *: Pinch the middle of the mouse's tail to determine if there are characteristics of paralysis.

[0078] 2.2.3.2 Mouse sample collection

[0079] After the mice reached the peak of the disease (Day 22), the mice were euthanized, whole blood was taken from the medial canthal vein of the eye into an EDTA anticoagulant tube, and the mice were dissected to isolate the spinal cord for later detection of pathology and the degree of damage to inflammatory cytokines.

[0080] 2.2.3.3 Preparation of mouse pathological sections

[0081] 1) Preparation of HE-stained pathological sections of mouse spinal cord tissue: The mouse spinal cord tissue was embedded, sectioned, then HE-stained, and then dehydrated and sealed. The pathological state of the tissue was observed under a microscope.

[0082] 2) Preparation of LFB-stained pathological sections of mouse spinal cord tissue: The mouse spinal cord tissue was embedded, sectioned, then LFB-stained, differentiated, counterstained with eosin, and then sealed. The pathological state of the tissue was observed under a microscope.

[0083] 2.2.3.4 Statistical analysis

[0084] GraphPad Prism 9 software was used for statistical analysis of the data. All data were expressed as mean ± standard deviation The means and differences between groups were compared using one-way ANOVA and T-Test. P < 0.05 indicated significant differences in the experimental data.

[0085] Example 3 Mouse body weight analysis

[0086] The body weights of the mice in each group in Example 2 were monitored. The body weights of the mice in each group were recorded daily, and the percentage change in body weight was calculated. Specifically, as Figure 1 shown, compared with the Control group, the body weight of the mice in the EAE group decreased significantly (P < 0.05); compared with the EAE group, the body weight of the mice in the EAE + LARM 40 mg / kg group increased significantly (P < 0.05), indicating that LARM can improve the decrease in body weight of EAE mice.

[0087] Example 4 Analysis of mouse disease status

[0088] The disease status of the mice in each group in Example 2 was monitored. The disease status was monitored and scored daily. The higher the score, the more severe the disease status of the mice. Specifically, as Figure 2As shown, compared with the Control group, the disease state score of mice in the EAE group increased significantly (P<0.05). Compared with the EAE group, the disease state scores of mice in the EAE+LARM 20mg / kg and EAE+LARM 40mg / kg groups decreased significantly (P<0.05), indicating that LARM can significantly alleviate the disease state of EAE mice.

[0089] Detection and analysis of IL-10 in spinal cord tissue of mice in Example 5

[0090] The detection results of the expression of cytokine IL-10 showed that, as Figure 3 , compared with the Control group, the level of IL-10 in the spinal cord of mice in the EAE group decreased significantly (P<0.05). Compared with the EAE group, the levels of IL-10 in the spinal cord of mice in the EAE+LARM 20mg / kg group and the EAE+LARM 40mg / kg group increased significantly (P<0.05).

[0091] Detection and analysis of TNF-α in spinal cord tissue of mice in Example 6

[0092] The detection results of the expression of cytokine TNF-α showed that, as Figure 4 , compared with the Control group, the level of TNF-α in the spinal cord of mice in the EAE group increased significantly (P<0.01). Compared with the EAE group, the levels of TNF-α in the spinal cord of mice in the EAE+LARM 20mg / kg group and the EAE+LARM 40mg / kg group decreased significantly (P<0.05).

[0093] Histopathological analysis of mice in Example 7

[0094] As Figure 5 and Figure 6 showed, no obvious histological structure changes were seen in the Control group; local gliosis in the white matter, swelling and disintegration of some nerve fibers were observed in the EAE group; local gliosis in the white matter and disintegration of some nerve fibers were found in the EAE+LARM 20mg / kg group; no obvious histological structure abnormalities were detected in the EAE+LARM 40mg / kg group; the results suggested that early administration of high-dose LARM has a certain inhibitory effect on the occurrence and development of EAE, and the pathological results showed that the infiltration of inflammatory cells and demyelination in the spinal cord improved significantly.

[0095] The above data were statistically analyzed using GraphPad Prism 9 software, and all data were expressed as mean ± standard deviation The means and differences between groups were compared using one-way ANOVA and T-Test. P<0.05 indicates significant differences in the experimental data.

[0096] In summary, rotigotine microspheres can be used in the preparation of drugs for preventing or treating multiple sclerosis. Rotigotine microspheres can be used alone or formulated into a pharmaceutical composition in combination with other drugs, compounds, or carriers. During use, the drug contains an effective therapeutic amount of rotigotine microspheres. The rotigotine microspheres can be prepared into various forms of medicaments such as oral preparations and injections for use.

[0097] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the embodiments, for those of ordinary skill in the art, it is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions required to be protected by the present invention.

Claims

1. Use of rotigotine microspheres in the preparation of a medicament for preventing or treating multiple sclerosis.

2. The use according to claim 1, wherein The use of the rotigotine microspheres is for preventing or treating: 1) Spinal cord myelin loss, and / or, 2) Infiltration of spinal cord inflammatory cells, and / or 3) Myelin regeneration.

3. The use according to claim 1, wherein The use is for preventing or treating a medicament for multiple sclerosis complicated with weight loss.

4. The use according to claim 1, wherein The multiple sclerosis is caused by immune deficiency.

5. According to any one of the uses described in claims 1 to 4, characterized in that, The use lies in that the rotigotine microspheres increase the level of IL-10 and / or decrease the level of TNF-α.

6. The use according to claim 1, characterized in that, The medicament for multiple sclerosis is an oral preparation or an injection.

7. The use according to claim 1, characterized in that, The rotigotine microspheres are administered once every 14 days by intramuscular injection.

8. The use according to claim 7, characterized in that, The dosage of the rotigotine microspheres is 20 mg / kg to 40 mg / kg.

9. The use according to claim 1, wherein The rotigotine microspheres can be used alone or compounded with other drugs, compounds, carriers into a pharmaceutical composition.

10. The use according to claim 1, wherein, The medicament contains an effective therapeutic amount of rotigotine microspheres.

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