Application of fenoterol and pharmaceutically acceptable salt thereof in preparation of medicine for treating diabetic peripheral neuropathy

Abstract

The invention discloses an application of fenoterol hydrobromide and pharmaceutically acceptable salt thereof in preparation of a medicine for treating diabetic peripheral neuropathy. The fenoterol hydrobromide and the pharmaceutically acceptable salt thereof have obvious effects of promoting growth of peripheral sensory neurite and protecting myelin sheath injury, and can improve symptoms of slow nerve conduction speed and sensory deficiency induced by diabetes mellitus.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical technology, in particular to the application of fenoterol and pharmaceutically acceptable salts thereof in the preparation of a drug for treating diabetic peripheral neuropathy. BACKGROUND

[0002] Diabetic peripheral neuropathy (DPN) is a kind of neurodegenerative disease caused by diabetes, which refers to the symptoms related to peripheral nerve dysfunction in diabetic patients under the exclusion of other causes. The main pathological features are different degrees of axonal degeneration, demyelination and neuronal apoptosis in peripheral nerves, among which distal symmetrical polyneuropathy is the most common, mainly manifested as symmetrical numbness, abnormal pain sensation and hyperalgesia from distal to proximal limbs, including burning sensation, needle prick sensation, electric shock sensation and stepping cotton sensation, etc., showing a glove and sock-like distribution, which is more obvious at night, with slow and concealed onset. It develops into stubborn and irreversible nerve damage, leading to the insensitivity or even disappearance of patients' sensation to pain, temperature, etc., thus causing secondary foot damage, ulceration, further aggravation of infection, osteomyelitis, gangrene, etc., and eventually leading to amputation.

[0003] Although many studies have been carried out on the treatment of diabetic peripheral neuropathy, there is still no effective treatment method in clinic. Therefore, it is of great practical value and practical significance to find new strategies and drugs for treating diabetic peripheral neuropathy.

[0004] At present, the drugs for treating DPN in clinic can only alleviate the symptoms of patients, and cannot block the progress of diabetic peripheral neuropathy. Therefore, there is an urgent need to find new anti-diabetic peripheral neuropathy drugs that can block the pathological process of diabetic peripheral neuropathy. The pathogenesis of diabetic peripheral neuropathy is complex, and is the result of multiple factors, mainly including mitochondrial dysfunction, neural inflammation, vascular injury, lack of neurotrophic factors, abnormal cytokines, oxidative stress, etc.

[0005] Diabetic peripheral neuropathy (DPN) affects sensory neurons, causing hyperesthesia or hypoesthesia, primarily manifested as a significant decrease in thermal and mechanical pain sensation, as well as a decline in motor and sensory nerve conduction velocities. Pathological sections show that sensory nerve endings in the skin of diabetic patients are curled, twisted, locally swollen, and vacuolated, eventually disappearing. DPN pathology also involves axonal atrophy and degeneration, even disappearance; segmental or diffuse myelin sheath shrinkage or demyelination; and changes in the internode length of Ranvier's nodes caused by myelin regeneration. Current research has found segmental demyelination of major myelinated nerves such as the sciatic, sural, and tibial nerves in DPN patients. This pathological change can cause shortening of the internode length of Ranvier's nodes, slowing nerve conduction velocity, and resulting in symptoms such as hypoesthesia and weakness. Therefore, it is believed that protecting and promoting the growth of sensory neuron processes and reducing peripheral nerve myelin sheath damage can achieve a therapeutic effect against diabetic peripheral neuropathy.

[0006] Fennoteukol hydrobromide is mainly used clinically to treat asthma and bronchitis. However, there are no reports on its use in diabetic peripheral neuropathy. Summary of the Invention

[0007] This invention provides the use of fenoterol or a pharmaceutically acceptable salt thereof in the preparation of medicaments for treating peripheral neuropathy.

[0008] In some implementations, fenoterol or a pharmaceutically acceptable salt thereof is used as the sole active ingredient in the preparation of a medicament for treating peripheral neuropathy.

[0009] In some embodiments, the pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt.

[0010] In some embodiments, the inorganic acid salt is a hydrochloride, hydrobromide, or sulfate, preferably a hydrobromide.

[0011] In some embodiments, the organic acid salt is fumarate, maleate, citrate, or malate.

[0012] In some embodiments, the peripheral neuropathy is diabetic peripheral neuropathy, chemotherapy-induced peripheral neuropathy, or peripheral neuropathy related to peripheral nerve injury. Preferably, the peripheral neuropathy is diabetic peripheral neuropathy.

[0013] In some embodiments, the drug has the effect of protecting peripheral sensory neurons, improving peripheral sensory loss and / or myelin damage.

[0014] In some implementations, the drug also includes pharmaceutically acceptable excipients.

[0015] In some embodiments, the dosage form of the drug is an inhaled formulation, an oral formulation, or an injection.

[0016] Beneficial effects

[0017] This invention reveals a novel mechanism of action and pharmacological effects of fennotefuranohydrobromide, which protects peripheral sensory neurons and improves peripheral sensory loss and / or myelin sheath damage. This compound can be used to treat diabetic peripheral neuropathy and neuropathy associated with peripheral sensory neuron damage, peripheral sensory loss, and / or myelin sheath damage. Attached Figure Description

[0018] Figure 1 Changes in mechanical pain threshold among different groups.

[0019] Figure 2 Changes in thermal pain threshold among different groups.

[0020] Figure 3 Changes in motor nerve transmission speed in different groups.

[0021] Figure 4 Changes in the speed of sensory nerve transmission in different groups.

[0022] Figure 5 Fluorescence images showing the growth promotion of peripheral sensory neuron processes in different groups.

[0023] Figure 6 Quantitative graph showing the relative growth of protrusions in different groups.

[0024] Figure 7 Electron micrographs of the myelin sheath of the sciatic nerve in different groups.

[0025] about Figures 1-4 and Figure 6 Explanation of statistical difference markers: Model group vs. control group: *, **, *** represent P<0.05, P<0.01, P<0.001 respectively; Fenoterol hydrobromide (0.5 mg / kg) treatment group vs. model group: #, ##, ### represent P<0.05, P<0.01, P<0.001 respectively; Fenoterol hydrobromide (1 mg / kg) treatment group vs. model group: &, &&, &&& represent P<0.05, P<0.01, P<0.001 respectively. One-way ANOVA. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments, but these embodiments should not be construed as limiting the present invention.

[0027] Example 1: Fenoterol hydrobromide improves sensory loss and nerve conduction velocity in diabetic peripheral neuropathy.

[0028] The effects of fenoterol hydrobromide on pain response and nerve conduction velocity in diabetic peripheral neuropathy were investigated in STZ-induced type 1 diabetic mice. The results showed that fenoterol hydrobromide significantly improved sensory loss and nerve conduction velocity in diabetic peripheral neuropathy.

[0029] 1. Experimental Principle

[0030] This experiment is based on the withdrawal reflex in rodents when their claws are subjected to mechanical stimulation. Vonfrey filaments can provide a stimulating force ranging from 0.008g to 300g, with the thickness and extension length of the nylon filament determining the magnitude of the stimulating force. The experiment involved selecting an appropriate nylon filament thickness and adjusting the extension length to vertically stimulate the skin. The stimulating force could be adjusted by changing the extension length and replacing the nylon filament until it bent, thus assessing the mice's response to pain.

[0031] 2. Experimental Materials and Methods

[0032] DPN animal model: C57BL / 6J mice were purchased from Vital River Pharmaceuticals in Beijing. After one week of acclimatization, they were intraperitoneally injected with STZ (150 mg / kg). Blood glucose was measured 4-7 days later, and mice with random blood glucose levels greater than 16 mmol / L were selected. After successful model establishment, body weight, blood glucose, mechanical pain, and thermal pain were measured weekly. Six weeks after STZ injection, mice were grouped according to body weight and blood glucose: control group (non-diabetic mice, n=12), model group (DPN mice, n=12), fenoterol hydrobromide (0.5 mg / kg, n=12), and fenoterol hydrobromide (1 mg / kg, n=12). The control and model groups were intraperitoneally injected with saline, while the fenoterol hydrobromide group was intraperitoneally injected with the corresponding dose of fenoterol hydrobromide. The administration lasted for 4 weeks, and body weight, blood glucose, mechanical pain, and thermal pain were measured weekly. Motor and sensory nerve conduction velocities were measured at weeks 0, 4, and 8 after STZ injection.

[0033] The Von Frey tactile measurement kit was purchased from the company, and the plantar thermal pain device was purchased from UGO.

[0034] Paw retraction test: Select a nylon filament of appropriate thickness, adjust the extension length to a suitable level, and stimulate the skin vertically. The stimulation force can be adjusted by changing the extension length or replacing the nylon filament until the nylon filament bends. Test six times on one mouse to determine the threshold. Based on 50% threshold = (10... ^ (xf+k δ The mechanical pain threshold of mice is calculated as 50% by )) / 10000.

[0035] Thermal pain detection experiment: An infrared instrument is placed on the sole of a mouse's paw and kept still. The time it takes for the mouse's paw to spring back is recorded, and this time is the mouse's thermal pain response time.

[0036] Nerve conduction velocity testing experiment: ① Motor nerve conduction velocity: Stimulating electrodes were placed on the mouse's ankle and sciatic notch, respectively; receiving electrodes were placed on the mouse's foot; and the ground wire was placed on the mouse's tail. Sensory nerve conduction velocity: Stimulating electrodes were placed on the mouse's foot; receiving electrodes were placed on the mouse's sciatic notch; and the ground wire was placed on the mouse's tail. ② Calculation of nerve conduction velocity: Strong stimulation of the distal and proximal ends of the nerve trunk resulted in two compound muscle action potentials being recorded on the muscles innervated by that nerve. The different latencies were measured, and the nerve conduction velocity was calculated by dividing the distance between the distal and proximal ends by the difference in latency between the two points. The calculation formula is: Nerve conduction velocity (m / s) = Distance between two points (cm) × 10 / Latency difference between two points.

[0037] 3. Experimental Results

[0038] The results are as follows Figures 1-4 As shown, fenoterol hydrobromide can increase pain response and improve nerve conduction velocity in mice, indicating that fenoterol hydrobromide has the effect of improving sensory loss and nerve conduction velocity in diabetic peripheral neuropathy.

[0039] Example 2: Fenoterol hydrobromide promotes the growth of peripheral sensory neuron neurites.

[0040] The effect of fennotefuran hydrobromide on promoting the growth of peripheral sensory neurons in peripheral sensory neurons (DRG) was investigated. The experiment showed that fennotefuran hydrobromide has a significant effect on promoting the growth of peripheral sensory neurons.

[0041] 1. Experimental Principle

[0042] This experiment is based on the fact that DPN primarily affects sensory neurons, and the main pathological changes are axonal atrophy, degeneration, and even disappearance. As peripheral sensory neurons, DRGs have a neurite growth state that is closely related to the development process of DPN. The inventors of this invention extracted primary DRG neurons from DPN mice that were given fennotefurantoin hydrobromide (0.5 mg / kg, 1 mg / kg) in Example 1 and incubated them for 24 h. Then, the growth of cell neurites was measured by β-tubulin III fluorescence staining and the length was quantified by ImageJ to evaluate the promoting effect of fennotefurantoin hydrobromide on the neurite growth of peripheral sensory neurons.

[0043] 2. Experimental Materials and Methods

[0044] β-tubulin III used for fluorescent staining was purchased from Sigma, and all cell culture reagents were purchased from Gibco. DRG cells were obtained from the spine of C57BL6 mice.

[0045] β-tubulin III staining assay: DRG cells were seeded at 100 cells / well in 12-well plates. After overnight adhesion, the original culture medium was removed, and each well was fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton for 5 min, washed with PBS, blocked at room temperature for 1 h, and incubated overnight at 4°C with primary antibody (1:1000). Goat anti-mouse antibody (1:200) was incubated at room temperature in the dark for 1 h. Finally, the images were observed under a fluorescence microscope.

[0046] 3. Experimental Results

[0047] The results are as follows Figure 5 As shown, fennotefuranohydrobromide can promote the growth of peripheral sensory neuron neurites in a concentration-dependent manner, such as... Figure 6 As shown, fenoterol hydrobromide can promote the growth of peripheral sensory neuron processes.

[0048] Example 3: Fenoterol hydrobromide protects against sciatic nerve myelin sheath injury.

[0049] This invention investigates the effect of fenoterol hydrobromide on improving peripheral sciatic nerve demyelination in the sciatic nerve. Experiments show that fenoterol hydrobromide has a significant effect on improving peripheral sciatic nerve myelin damage.

[0050] 1. Experimental Principle

[0051] This experiment is based on DPN-induced sciatic nerve demyelination. The sciatic nerve is a peripheral motor nerve, and its morphology and structure are closely related to the development of DPN. The inventors of this invention extracted the sciatic nerve of DPN mice treated with fenoterol hydrobromide (0.5 mg / kg, 1 mg / kg) in Example 1, and then rapidly fixed it and observed it under an electron microscope to evaluate the effect of fenoterol hydrobromide on peripheral sciatic nerve myelin damage.

[0052] 2. Experimental Materials and Methods

[0053] Fresh tissue was fixed with fixative and then fixed with 1% osmium tetroxide at room temperature in the dark for 2 h. After dehydration and embedding in resin, it was polymerized in a 60°C oven for 48 h. After sectioning, it was stained with 2% uranium acetate saturated ethanol solution in the dark for 8 min, washed three times with 70% ethanol and ultrapure water respectively, and then stained with 2.6% lead citrate solution in the dark for 8 min. After that, it was placed in a copper mesh box to dry overnight at room temperature. Finally, it was observed and imaged under a transmission electron microscope.

[0054] 3. Experimental Results

[0055] The results are as follows Figure 7 As shown, fennotefuranohydrobromide can improve sciatic nerve myelin sheath injury in DPN mice.

Claims

1. Use of fenoterol or a pharmaceutically acceptable salt thereof in the preparation of medicaments for treating peripheral neuropathy.

2. The use of fenoterol or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of medicaments for the treatment of peripheral neuropathy.

3. The use according to claim 1 or 2, characterized in that, The pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt.

4. The use according to claim 3, characterized in that, The inorganic acid salt is a hydrochloride, hydrobromide, or sulfate.

5. The use according to claim 3, characterized in that, The inorganic acid salt is hydrobromide.

6. The use according to any one of claims 1-5, characterized in that, The peripheral neuropathy refers to diabetic peripheral neuropathy, chemotherapy-induced peripheral neuropathy, or peripheral neuropathy related to peripheral nerve damage.

7. The use according to any one of claims 1-6, characterized in that, The peripheral neuropathy mentioned is diabetic peripheral neuropathy.

8. The use according to any one of claims 1-7, characterized in that, The drug has the effects of protecting peripheral sensory neurons, improving peripheral sensory loss and / or myelin damage.

9. The use according to any one of claims 1-8, characterized in that, The drug also contains pharmaceutically acceptable excipients.

10. The use according to any one of claims 1-8, characterized in that, The drug is available in the form of an inhaled formulation, an oral formulation, or an injection.

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