CN116173217A

CN116173217A - Use of vitamin D receptor agonists for the treatment of motor neuron diseases

Info

Publication number: CN116173217A
Application number: CN202310199503.5A
Authority: CN
Inventors: 白戈; 刘华清; 唐明敏
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2023-02-24
Filing date: 2023-02-24
Publication date: 2023-05-30

Abstract

The invention provides application of a vitamin D receptor agonist in preparing a product for treating motor neuron diseases, and relates to the technical field of medicines. The inventor researches show that the vitamin D receptor agonist can obviously up-regulate the transcription level of VEGF and improve peripheral nerve and muscle lesions of fibula muscular atrophy and motor nerve dysfunction. Thus, vitamin D receptor agonists can be used in the preparation of products for the treatment of motor neuron diseases.

Description

Use of vitamin D receptor agonists for the treatment of motor neuron diseases

Technical Field

The invention relates to the technical field of medicines, in particular to application of a vitamin D receptor agonist in preparing a product for treating motor neuron diseases.

Background

In the past few decades, the social burden caused by global nervous system diseases has shown a significant upward trend. Taking 2015 as an example, various nervous system diseases all over the world lead to death of 940 ten thousand patients, accounting for 16.8% of the total population of deaths, which is the lethal factor of rank 2. Among these neurological diseases, the most causative causes remain unclear until now. Fibular muscular dystrophy (CMT), one of the most common hereditary peripheral neuropathy, has a prevalence of about 1:2500 worldwide. The clinical symptoms of patients often manifest as loss of strength and muscular atrophy of the distal muscles of the extremities, and impaired motor and sensory functions. The common onset of fibular muscular atrophy is early, and many patients begin to develop clinical symptoms from teenagers or young age stages, and the disease becomes so severe as to lose labor and life self-care capacity with the increasing age. CMT has high incidence and serious illness, and brings heavy burden to families and society of patients, but effective therapeutic drugs are still lacking clinically up to the present time.

Aminoacyl tRNA synthetases are the largest gene family associated with CMT (Patzko and Shy, 2011). Among them, the GARS encoding glycyl tRNA synthetases (GlyRS) are the first members to be discovered, whose mutations lead to a dominant axonal form of CMT (CMT 2D subtype). Although GlyRS function is essential in all cells, mutation of this gene results in selective degenerative changes of peripheral axons, resulting in loss of distal motor/sensory function. We have found that CMT2D mutation alters the conformation of GlyRS, causing mutation of GlyRS (GlyRS ^CMT2D ) Being able to bind to the Nrp1 receptor and disrupt the Vascular Endothelial Growth Factor (VEGF)/Nrp 1 pathway, down-regulating Nrp1 gene expression exacerbates CMT mouse phenotype, while increasing VEGF expression can improve motor function. Many evidences indicate that VEGF has neuroprotective effects and can reduce the effects of factors such as oxidative stress, nutrient deficiency, chemical toxicity, etc. on neuronal survival and function. Motor neurons express the Nrp1 receptor, which appears to depend on muscle-derived VEGF to sustain survival, and promoter mutations reduce VEGF levels, resulting in delayed motor neuron degeneration. These experiments confirm GlyRS ^CMT2D Proteins interfere with the VEGF/Nrp1 signaling pathway by interacting with Nrp1 abnormalities, thereby causing motor neurodegeneration.

However, there is currently a lack of effective therapeutic agents against peripheral nerve diseases such as CMT.

In view of this, the present invention has been made.

Disclosure of Invention

It is a first object of the present invention to provide the use of a vitamin D receptor agonist in the manufacture of a product for the treatment of motor neuron diseases, to address at least one of the above problems.

A second object of the present invention is to provide a medicament for treating motor neuron diseases.

In a first aspect, the present invention provides the use of a vitamin D receptor agonist in the manufacture of a product for the treatment of motor neuron diseases.

As a further aspect, the vitamin D receptor agonist comprises calcipotriol.

As a further aspect, the motor neuron disease comprises fibular muscular dystrophy.

As a further aspect, the product comprises a medicament.

In a second aspect, the present invention provides a medicament for the treatment of motor neuron diseases, the medicament comprising a vitamin D receptor agonist.

As a further aspect, the vitamin D receptor agonist comprises calcipotriol.

As a further technical scheme, the raw materials of the medicine also comprise pharmaceutically acceptable auxiliary materials.

As a further technical scheme, the auxiliary materials comprise at least one of diluents, fillers, excipients, binders, wetting agents, disintegrating agents, absorption promoters, surfactants, adsorption carriers, lubricants and flavoring agents.

As a further technical scheme, the medicament is an oral preparation or an injection.

Compared with the prior art, the invention has the following beneficial effects:

the inventor researches show that the vitamin D receptor agonist can obviously up-regulate the transcription level of VEGF and improve peripheral nerve and muscle lesions of fibula muscular atrophy and motor nerve dysfunction. Thus, vitamin D receptor agonists can be used in the preparation of products for the treatment of motor neuron diseases.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic representation of the mechanism by which vitamin D receptor agonists (calcipotriol) effect treatment of fibular muscular dystrophy;

FIG. 2 is a graph showing that vitamin D receptor agonists (calcipotriol) can up-regulate VEGF transcription levels in C2C12 cells;

FIG. 3 is a graph showing that vitamin D receptor agonists (calcipotriol) up-regulate VEGF transcription levels in fibular muscle tissue of CMT model mice;

FIG. 4 is a graph showing that calcipotriol significantly increases hind limb extension scores in mice with models of fibular muscular dystrophy;

FIG. 5 is a graph showing that vitamin D receptor agonist (calcipotriol) can significantly prolong the duration of stay on the rotating stick in mice with models of fibular muscular atrophy;

figure 6 shows that calcipotriol has no effect on mouse body weight;

FIG. 7 is a graph of calcipotriol that can significantly increase the rate of sural innervation in mice model for sural muscular atrophy;

figure 8 is that calcipotriol can significantly increase the rate of large diameter axons around the peripheral nerve of mice model for fibular muscular atrophy.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but it will be understood by those skilled in the art that the following embodiments and examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The specific conditions are not specified, and the process is carried out according to conventional conditions or conditions suggested by manufacturers. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The inventor researches find that Vitamin D Receptor (VDR) is a transcription regulator of VEGF, and the activation of VDR can significantly up-regulate VEGF expression, so that the target VDR is expected to improve the neuropathy of CMT. According to the invention, the VDR agonist provided by the invention can obviously up-regulate the transcription level of VEGF, improve the disease phenotype of CMT, can be used for treating CMT, and provides a reference for developing effective treatment and prevention of CMT neuropathy.

In some preferred embodiments, the motor neuron disease comprises fibular muscular dystrophy.

In some preferred embodiments, the vitamin D receptor agonist includes, but is not limited to, calcipotriol, preferably calcipotriol.

The mechanism by which vitamin D receptor agonists (calcipotriol) effect treatment of fibular muscular dystrophy is shown in figure 1. According to the research of the invention, calcipotriol can enhance VEGF/Nrp1 signals in motor nerves by regulating the expression of VEGF in surrounding tissues, thereby resisting GlyRS ^CMT2D Is effective in improving motor function of CMT mice.

In some preferred embodiments, the product comprises a medicament.

Because the medicine provided by the invention contains vitamin D receptor agonists, the medicine has therapeutic effect on motor neuron diseases.

In some preferred embodiments, the vitamin D receptor agonist is calcipotriol.

In some preferred embodiments, the motor neuron disease is fibula atrophy.

In some preferred embodiments, pharmaceutically acceptable excipients are also included in the raw materials of the medicament.

In some preferred embodiments, the adjuvant includes, but is not limited to, diluents, fillers, excipients, binders, humectants, disintegrating agents, absorption enhancers, surfactants, adsorption carriers, lubricants, or flavoring agents.

In some preferred embodiments, the pharmaceutical dosage form is an oral or injectable formulation.

Preferably, the oral agent comprises: at least one of tablets, granules, dripping pills, soft capsules, suspending agents, solutions and syrups;

preferably, the injection comprises: at least one of dry powder, solution type injection, suspension type injection, and emulsion type injection.

The invention is further illustrated by the following specific examples, however, it should be understood that these examples are for the purpose of illustration only in greater detail and are not to be construed as limiting the invention in any way.

In the following examples, all statistical analyses were performed using GraphPad Prism software. The comparison of the two independent experimental groups adopts t-test, the comparison of the single independent variable group adopts single-factor analysis of variance, and the comparison of the two independent variable groups (mouse model and treatment method) adopts double-factor analysis of variance. All statistical tests were performed on at least 3 biological replicates. P <0.05 was considered significant. The graphs are expressed as mean ± standard deviation unless otherwise indicated.

Example 1 vitamin D receptor agonists can increase the transcript levels of VEGF in C2C12 cells

The method comprises the following steps: C2C12 mouse myoblasts were cultured in DMEM (containing 10% heat-inactivated fetal bovine serum and penicillin-streptomycin) cell culture medium. On the day of the experiment, cells were treated with 10nM calcipotriol or 0.1% dmso (control) for 1 hour, collected for RNA extraction, and then the expression level of VEGF was detected using real-time fluorescent quantitative PCR (RT-QPCR), as shown in fig. 2 (independent replicates, p <0.001 compared to control).

Results: the rapid increase in VEGF expression within 1 hour of calcipotriol treatment of C2C12 cells suggests that VEGF is a direct downstream target gene for VDR.

Example 2 vitamin D receptor agonists can increase VEGF transcription levels in the fibula muscle tissue of CMT model mice

The method comprises the following steps: method for establishing model mice, for post-natal day 6 CMT model mice (carrying GlyRS ^P234KY Mutant genes) were randomly grouped and intraperitoneally injected with physiological saline (0.1% DMSO) and calcipotriol (60. Mu.g/kg), respectively. Mice were sacrificed 24h later and fibular muscle tissue was removed, mRNA from the tissue was extracted using Trizol method, and the transcript levels of VEGF in the tissues of mice from different treatment groups were then detected and counted using RT-QPCR as shown in fig. 3 (number of mice = 4 for each treatment group compared to control group, p<0.05 Shown in the drawings).

Results: calcipotriol can significantly up-regulate VEGF transcript levels in fibular muscle tissue in CMT model mice.

Example 3 behavioral effects of calcipotriol on CMT model mice

The method comprises the following steps: method for establishing model mice, CMT model mice (carrying GlyRS) ^P234KY Mutant gene) and wild type mice were individually grouped, and a control group of calcipotriol (60 μg/kg) was intraperitoneally injected with physiological saline (0.1% DMSO) from day 6 of birth of the animals, administered for five days a week and at rest for two days, and administered to the end of 7 weeks of age. Behavioural tests were performed weekly during dosing to assess the effect of the drug on motor function, including hindlimb extension scoring, stick rotation experiments, and the like. And the influence of the drug on the weight of the mice is detected, so that the toxicity of the drug is examined, and the specific implementation method is as follows:

hind limb skeletal muscle disorders were assessed using hind limb stretch scores: the tail of the mice was lifted and the hind limb extension was observed, and the hind limb extension was rated as 2 points, the hind limb extension was rated as 1.5 points, the hind limb extension was rated as 1 point, the hind limb extension was rated as 0.5 points, the hind limb extension was rated as 0 point, and 3 effective data were obtained each time. The mice in the different treatment groups were examined one hour after drug injection and evaluated for skeletal muscle dysfunction in hind limbs as shown in fig. 4 (showing the results of the evaluation of mice at 7 weeks of age, mice in each treatment group were.

The rotating rod experiment is used for evaluating coordination, balance force and movement function conditions: the mice are placed on a stick rotating instrument, the rotating speed and the acceleration are set to be 0.2 r/s, the falling time of the mice is observed and recorded after the instrument is operated, and each time is repeated three times, and each time interval is more than 30min. The mice in the different treatment groups were tested one hour after drug injection and were evaluated for differences in motor function, as shown in fig. 5 (showing the results of the evaluation of mice at 7 weeks of age, with more than or equal to 10 mice in each treatment group, p <0.05 compared to the control group).

And the final period of the experiment is used for carrying out statistical analysis on the weight of the mice, and the result shows that the carbotriol has no significant effect on the weight of the mice, as shown in fig. 6 (showing the evaluation result of the mice at 7 weeks of age, the number of mice in each treatment group is more than or equal to 10, and the p is less than 0.05 compared with the control group).

Results: calcipotriol can significantly improve peripheral nerve function defects of fibular muscular atrophy, and has no side effect on wild mice.

Example 4 Effect of calcipotriol on peripheral neuropathological morphology of CMT model mice

The method comprises the following steps: animal-related grouping and administration treatments were performed as in example 3, and tissue of 7-week-old CMT mice and littermates WT mice were harvested after the end of the behavioral assessment for histopathological examination, including neuromuscular junction innervation, neurite cross-section size and number, etc., as follows:

neuromuscular junction innervation analysis: neuromuscular junctions are points of contact of motor neuron axon terminals on skeletal muscle fibers. Signals transmitted from nerve fibers are transmitted to muscle fibers through joints to regulate contraction and tension of muscles. Muscle atrophy occurs due to nerve fiber degeneration, and is one of the clinical pathological characteristics of fibular muscle atrophy. In the experiment, cervical dislocation of mice in different treatment groups is killed, fibula muscles of the mice are dissected and fixed, and then the mice are sectioned for tissue immunofluorescence staining. The post-synaptic acetylcholine receptor clusters (red) were labeled with R-BTX, NF145 and synaptophysin antibodies to label the nerve fibers. After staining, morphology and co-localization of the nerve fibers and the acetylcholine receptor clusters were observed and counted using a confocal microscope, and at least 5 non-overlapping fields of view were selected for each mouse, containing more than 100 neuromuscular junctions, and the innervation of the muscles was counted, as shown in fig. 7 (a is a neuromuscular junction morphology map; b is a statistic of neuromuscular junction innervation of different morphologies; the number of mice in the group=3, p <0.01 compared to the control group).

Statistics of nerve axon diameter: peripheral nerve synaptosis atrophy is one of the clinical pathological phenomena of fibular atrophy, and thus the improvement of fibular atrophy by drugs is evaluated by diameter of peripheral nerve synapses: the sciatic nerve of the mice was harvested, fixed with 1% osmium acid for 1 hour, and then fixed/stained with 2% uranium acetate aqueous solution for 30 minutes. After dehydration of ethanol at different gradients, the embedding medium+acetone (3:1) was used overnight. Embedding is then carried out at 30-37℃using an embedding agent, and slicing is carried out. Dyeing was performed using dye A (methyl blue 0.52g, blue 0.08g, glycerol 40ml, methanol 40ml,1 Xphosphate buffer 120ml (pH 6.9), purified water 200 ml) at 66℃for 30 minutes, followed by dyeing B (accessory red base 0.2g and 50% ethanol 20ml,380ml purified water) at room temperature for 10-15 minutes. After cleaning and airing, using an electron microscope (790 times mirror) to photograph the cross section of the nerve (more than 5 non-overlapping fields of view are selected); the neurite diameters in each field were counted using ImageJ software and the proportion of neurites with a diameter of the entire cross-section greater than 2 μm was calculated as shown in fig. 8 (a is neurite cross-section; b is neurite proportion count (diameter > 2 μm); number of mice per group = 3, p <0.05 compared to control).

Results: calcipotriol can significantly improve peripheral neuropathological changes in mice with fibular muscle atrophy, and has no side effects on wild mice.

In conclusion, the invention discovers that the vitamin D receptor agonist calcipotriol can obviously up-regulate VEGF transcription level in a fibular muscular atrophy animal model, and improve fibular muscular atrophy peripheral nerve and muscle lesions and motor nerve dysfunction. Moreover, the medicine has no obvious toxicity to normal mice. Thus, calcipotriol has a significant effect in treating muscular dystrophy by up-regulating VEGF transcript levels.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. Use of a vitamin D receptor agonist in the manufacture of a product for the treatment of motor neuron diseases.

2. The use according to claim 1, wherein the vitamin D receptor agonist comprises calcipotriol.

3. The use according to claim 1, wherein the motor neuron disease comprises fibular muscular atrophy.

4. The use according to claim 1, wherein the product comprises a medicament.

5. A medicament for treating motor neuron disease, comprising a vitamin D receptor agonist.

6. The medicament of claim 5, wherein the vitamin D receptor agonist comprises calcipotriol.

7. The medicament of claim 5, wherein the motor neuron disease comprises fibular muscular dystrophy.

8. The medicine according to claim 5, wherein the raw materials of the medicine further comprise pharmaceutically acceptable auxiliary materials.

9. The medicament of claim 8, wherein the adjuvant comprises at least one of a diluent, a filler, an excipient, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, and a flavoring agent.

10. The medicament according to claim 5, wherein the medicament is an oral or injectable preparation.