A pharmaceutical composition for treating diabetic peripheral neuropathy and a preparation method thereof

Abstract

The application provides a pharmaceutical composition for treating diabetic peripheral neuropathy and a preparation method thereof, and application of the pharmaceutical composition in treating diabetic peripheral neuropathy, and belongs to the field of biological medicine. The core compound and the peripheral compound are prepared into the composition in a specific proportion and by a specific method, so that the diabetic peripheral neuropathy can be effectively relieved, and the composition has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to a pharmaceutical composition for treating diabetic peripheral neuropathy and its preparation method. Background Technology

[0002] Diabetic peripheral neuropathy (DPN) is a common complication of diabetes. The metabolic disease caused by hyperglycemia in diabetes can, due to long-term metabolic imbalances, affect and alter the structure and function of many organs, primarily the vascular system, further leading to complications such as those affecting the nervous system. Because of the diverse symptoms, the classification and diagnosis of diabetic peripheral neuropathy are not entirely standardized. Based on the speed of onset, diabetic neuropathy can be divided into acute and chronic categories: acute neuropathy and chronic sensorimotor neuropathy. Based on the affected location, it can be classified as symmetrical or asymmetrical. Chronic distal symmetric polyneuropathy (DSPN) accounts for approximately 75% of cases and is the most common type of diabetic neuropathy.

[0003] Cucurbit[n]uril (abbreviated CB[n], also known as cucurbit ring) is an extremely important macrocyclic host molecule in supramolecular chemistry. It is named for its resemblance to a gourd (Cucurbitaceae family). In the chemical community, it is considered one of the four major host molecules, along with crown ethers, cyclodextrins, and calixarnes. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a pharmaceutical composition and its preparation method, as well as their application in the treatment of diabetic peripheral neuropathy. By preparing a pharmaceutical composition by combining a core compound and peripheral compounds in a specific ratio using a specific method, it can effectively treat diabetic peripheral neuropathy and has promising clinical application prospects.

[0005] To address this, the present invention adopts the following technical solution: On one hand, the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises a core compound and peripheral compounds; Furthermore, the core compound is a tyrosine kinase inhibitor (TKI), and the peripheral compound is a molecule with a macrocyclic structure. Furthermore, the molar concentration ratio of the core compound to the peripheral compound is 2:1 to 1:2; Furthermore, the molar concentration ratio is 1:1.

[0006] In some embodiments, the tyrosine kinase inhibitor (TKI) is selected from EGFR inhibitors, BCR-ABL inhibitors, ALK inhibitors, BTK inhibitors, or KIT inhibitors.

[0007] In some specific embodiments, the EGFR inhibitor is selected from Gefitinib, Erlotinib, or Osimertinib; the BCR-ABL inhibitor is selected from Nilotinib, Ponatinib, or Dasatinib; the ALK inhibitor is selected from Alectinib, Crizotinib, Lorlatinib, or Brigatinib; the BTK inhibitor is selected from Ibrutinib or Zanubrutinib; and the KIT inhibitor is selected from Bezuclastinib, Sunitinib, Avapritinib, or Ripretinib.

[0008] In some specific embodiments, the tyrosine kinase inhibitor TKI is selected from the KIT inhibitor, and the KIT inhibitor is selected from Bezuclastinib.

[0009] In some embodiments, the peripheral compound is selected from crown ethers, cyclodextrins, cyclosporines, calixarenes, or cucurbituril.

[0010] In some specific embodiments, the crown ether is selected from 12-crown-4, 15-crown-5, 18-crown-6, 21-crown-7 or 24-crown-8; the cyclopaste is selected from α-CD, β-CD or γ-CD; the calixarene is selected from calix[4]arene, calix[6]arene, calix[8]arene, 4-sulfonylcalix[4]arene, 4-sulfonylcalix[6]arene or 4-sulfonylcalix[8]arene; and the cucurbita is selected from cucurbita[5]urene, cucurbita[6]urene, cucurbita[7]urene, cucurbita[8]urene or cucurbita

[10] urene.

[0011] In some specific embodiments, the peripheral compound is selected from cucurbituril, which is selected from cucurbituril[7].

[0012] In some particularly preferred embodiments, the pharmaceutical composition is composed of a core compound selected from Bezuclastinib and a peripheral compound selected from cucurbita[7]urea in a molar ratio of 1:1.

[0013] In some embodiments, the pharmaceutical composition further includes pharmaceutically acceptable excipients; In some specific implementations, the pharmaceutically acceptable excipient may be selected from a solvent; In some specific embodiments, the solvent is selected from one or more of PBS, DMSO, or DMF.

[0014] On the other hand, the present invention provides a method for preparing a pharmaceutical composition, characterized by comprising the following steps: ① The core compound and peripheral compound are dissolved in solvents respectively to prepare the core compound mother liquor and peripheral compound mother liquor; ② Dilute the mother liquor of the core compound and the mother liquor of the peripheral compound with solvents to prepare the working solution of the core compound and the working solution of the peripheral compound, respectively; ③ Weigh the core compound working solution and the peripheral compound working solution according to the molar concentration ratio of the drug composition formulation, place them in a reaction vessel, mix them evenly, heat and sonicate; ④ After ultrasound is completed, store and keep for future use.

[0015] In some embodiments, the solution is selected from one or more of PBS, DMSO, or DMF.

[0016] In some implementations, the heating conditions in step ③ are 30°C to 60°C, and the ultrasonic time is 15 to 45 minutes; In some specific embodiments, the heating condition is 40°C and the ultrasonic time is 30 minutes.

[0017] In some preferred embodiments, the method for preparing the pharmaceutical composition includes the following steps: ① Dissolve TKI in DMSO and cucurbituril in PBS to prepare the core compound mother liquor and the peripheral compound mother liquor; ② Dilute the mother liquor of the core compound and the mother liquor of the peripheral compound with PBS to prepare the working solution of the core compound and the working solution of the peripheral compound, respectively; ③ Weigh the core compound working solution and the peripheral compound working solution according to the molar concentration ratio of 2:1 to 1:2, place them in the reaction vessel, mix them evenly, heat at 30℃ to 60℃, and sonicate for 15 to 45 minutes. ④ After ultrasound is completed, store and keep for future use.

[0018] In some particularly preferred embodiments, the method for preparing the pharmaceutical composition includes the following steps: ① Dissolve Bezuclastinib in DMSO and cucurbituril[7] in PBS to prepare the core compound mother liquor and the peripheral compound mother liquor; ② Dilute the mother liquor of the core compound and the mother liquor of the peripheral compound with PBS to prepare the working solution of the core compound and the working solution of the peripheral compound, respectively; ③ Weigh the core compound working solution and the peripheral compound working solution according to a molar concentration ratio of 1:1, place them in a reaction vessel, mix them evenly, heat at 40°C, and sonicate for 30 minutes; ④ After ultrasound is completed, store and keep for future use.

[0019] In other respects, the present invention relates to the use of any of the pharmaceutical compositions described in this invention, or pharmaceutical compositions prepared by any of the preparation methods described in this invention, in the preparation of a medicament for treating diabetic peripheral neuropathy.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects: This invention prepares a pharmaceutical composition by combining a core compound and peripheral compounds in a specific ratio using a specific method. This composition can effectively alleviate diabetic peripheral neuropathy and has good prospects for clinical application. Attached Figure Description

[0021] Figure 1 Bezuclastinib (BZC) structure Figure 2 . Cucurbita[7]urea (CB[7]) structural formula Figure 3 Relative mRNA expression levels of MBP, MPZ, and PMP22 in different cell models Figure 4 Changes in mechanical pain threshold among different groups Figure 5 Changes in thermal pain threshold among different groups Figure 6 Changes in motor nerve conduction velocity in different groups Figure 7 Changes in sensory nerve conduction velocity in different groups Detailed Implementation

[0022] The present invention will be further illustrated below with reference to specific embodiments. Unless otherwise specified, the raw materials, reagents or devices used in the following embodiments can be obtained from conventional commercial sources or by existing known methods.

[0023] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0024] Example 1: Preparation of the Pharmaceutical Composition Bezuclastinib (BZC, CAS: 1616385-51-3) was chosen as the core compound, with the following structural formula: Figure 1 As shown.

[0025] Cucurbita[7]urea (CB[7], CAS: 259886-50-5), which has good water solubility and low cytotoxicity, was selected as a peripheral compound for drug composition optimization. The structural formula of CB[7] is as follows: Figure 2 As shown.

[0026] The BZC@CB[7] pharmaceutical composition was prepared according to the following method: ① Dissolve BZC in DMSO and CB[7] in PBS to prepare the core compound mother liquor and the peripheral compound mother liquor; ② The mother liquor of the core compound and the mother liquor of the peripheral compound were diluted with PBS to prepare a 10 nM BZC core compound working solution and a 10 nM CB[7] peripheral compound working solution; ③ Weigh 1 mL of core compound working solution and 1 mL of peripheral compound working solution according to a molar concentration ratio of 1:1, place them in a 5 mL centrifuge tube in a reaction vessel, mix them evenly, place them in an ultrasonic instrument, and heat and sonicate at 40°C for 30 minutes. ④ After ultrasound is completed, store and keep for future use.

[0027] In the BZC@CB[7] composition prepared above, the BZC equivalent is 5 nM.

[0028] Example 2: Protection of DPN in vitro model by the drug composition 1. Experimental Samples BZC@CB was prepared according to the method in Example 1[7].

[0029] BZC@CB[7] control 1 (BZC@CB[7]-1) was prepared as follows: ① Dissolve BZC in DMSO and CB[7] in PBS to prepare the core compound mother liquor and the peripheral compound mother liquor; ② The peripheral compound mother liquor and the core compound mother liquor were diluted with PBS to prepare 10 nM CB[7] peripheral compound working solution and 10 nM BZC core compound working solution; ③ Weigh 1 mL of peripheral compound working solution and 1 mL of core compound working solution according to a molar concentration ratio of 1:1, place them in a 5 mL centrifuge tube of the reaction vessel, mix them evenly, place them in an ultrasonic instrument, and sonicate at room temperature for 30 minutes. ④ After ultrasound is completed, store and keep for future use.

[0030] BZC@CB[7] control 2 (BZC@CB[7]-2) was prepared as follows: ① Dissolve CB[7] in PBS and BZC in DMSO to prepare the peripheral compound mother liquor and the core compound mother liquor; ② The peripheral compound mother liquor and the core compound mother liquor were diluted with PBS to prepare 10 nM CB[7] peripheral compound working solution and 10 nM BZC core compound working solution; ③ Weigh 1 mL of peripheral compound working solution and 1 mL of core compound working solution according to a molar concentration ratio of 1:1, place them in a 5 mL centrifuge tube in the reaction vessel, mix well, and heat at 40°C for 30 minutes. ④ After heating, store and keep for later use.

[0031] BZC@CB[7] control 3 (BZC@CB[7]-3) was prepared as follows: ① Dissolve CB[7] in PBS and BZC in DMSO to prepare the peripheral compound mother liquor and the core compound mother liquor; ② The peripheral compound mother liquor and the core compound mother liquor were diluted with PBS to prepare 10 nM CB[7] peripheral compound working solution and 10 nM BZC core compound working solution; ③ Weigh 1 mL of peripheral compound working solution and 1 mL of core compound working solution according to a molar concentration ratio of 1:1, place them in a 5 mL centrifuge tube in the reaction vessel, mix well, and let stand at room temperature for 30 minutes. ④ Store and use after 30 minutes.

[0032] In the BZC@CB[7], BZC@CB[7]-1, BZC@CB[7]-2 and BZC@CB[7]-3 prepared above, the BZC equivalent is 5 nM.

[0033] BZC control was prepared as follows: ① Dissolve BZC in DMSO to prepare the mother liquor of the core compound; ② Dilute the mother liquor of the core compound with PBS to prepare a 10 nM working solution of the BZC core compound; ③ Weigh 1 mL of the core compound working solution and 1 mL of PBS respectively, place them in a 5 mL centrifuge tube of the reaction vessel, mix them evenly, place them in an ultrasonic instrument, and sonicate at 40°C for 30 minutes. ④ After ultrasound is completed, store and keep for future use.

[0034] CB[7] control was prepared as follows: ① Dissolve CB[7] in PBS to prepare a mother liquor of peripheral compounds; ② Dilute the mother liquor of the peripheral compound with PBS to prepare a 10 nM working solution of the CB[7] peripheral compound; ③ Weigh 1 mL of peripheral compound working solution and 1 mL of PBS respectively, place them in a 5 mL centrifuge tube of the reaction vessel, mix them evenly, place them in an ultrasonic instrument, and sonicate at 40°C for 30 minutes; ④ After ultrasound is completed, store and keep for future use.

[0035] 2. Experimental Methods 1) Construction of SC / DRG cell model Dorsal root neurons (DRG) were divided into groups of 5 × 10 3 / wells were seeded into 24-well plates and cultured in serum-free medium for 7 days; DRG cells were washed 3 times with PBS, and 100 μL of SC cell suspension (about 50,000 cells) was added to the center of the DRG cells. After co-culturing for 24 hours, the medium was replaced with serum-free medium, and the medium was changed every other day for 6 days. Then the medium was replaced with myelinating medium (Neurobasal medium + 20 mL / L B27 + 50 ng / mL NGF + 50 μg / mL vitamin C), and half of the medium was changed every other day until obvious myelin segments were formed, thus obtaining the SC / DRG co-culture model.

[0036] 2) Construction and processing of DPN in vitro model A high-glucose-induced Schwann cell-dorsal root neuron (SCs / DRG) in vitro model of diabetic peripheral neuropathy (DPN) was constructed. The specific steps are as follows: The SC / DRG co-culture model was further cultured in a medium with a glucose content of 5.5 mM as a blank control group. The SC / DRG co-culture model was further cultured in a medium with a glucose content of 50 mM, serving as the model group. The SC / DRG co-culture model was further cultured in a medium containing 50 mM glucose and 5 mg / mL methylcobalamin as a positive control group (MeCbl). The SC / DRG co-culture model was further cultured in a medium containing 50 mM glucose and 0.1 mL BZC@CB[7], which served as the experimental group (BZC@CB[7]). The SC / DRG co-culture model was further cultured in a medium containing 50 mM glucose and 0.1 mL BZC@CB[7]-1 as the control group-1 (T-1); The SC / DRG co-culture model was further cultured in a medium containing 50 mM glucose and 0.1 mL BZC@CB[7]-2 as the control group-2 (T-2); The SC / DRG co-culture model was further cultured in a medium containing 50 mM glucose and 0.1 mL BZC@CB[7]-3, and used as the control group-3 (T-3); The SC / DRG co-culture model was further cultured in a medium containing 50 mM glucose and 0.1 mL BZC, serving as the control group-4 (BZC). The SC / DRG co-culture model was further cultured in a medium containing 50 mM glucose and 0.1 mL CB[7], and used as the control group -5 (CB[7]); 3. Determination of expression levels of related genes and proteins Cells were collected after culturing for 72 hours in each treatment group. Total RNA was extracted using the TRIzol method and subjected to RT-PCR. The relative expression levels of myelin basic protein (MBP), myelin 0 (MPZ), and peripheral myelin 22 (PMP22) were detected using β-actin as an internal reference gene.

[0037] 3. Experimental Results Experimental results are as follows Figure 3 As shown, compared with the model group, each experimental group was able to upregulate the expression levels of MBP, MPZ, and PMP22, promoting myelin repair. However, the control groups obtained by heating alone, ultrasound alone, or simple mixing alone were unable to upregulate the expression levels of MBP, MPZ, and PMP22, or promote myelin repair.

[0038] Example 3: The pharmaceutical composition improves sensory loss and nerve conduction velocity in diabetic peripheral neuropathy. The effects of BZC@CB[7] on pain response and nerve conduction velocity in diabetic peripheral neuropathy were investigated in STZ-induced type 1 diabetic mice.

[0039] 1. Experimental Materials and Methods DPN animal model: C57BL / 6J mice were purchased from Beijing Vital River. After one week of acclimatization, they were injected intraperitoneally with STZ (150 mg / kg). Blood glucose was measured 4-7 days later, and mice with random blood glucose greater than 16 mmol / L were selected. After successful modeling, body weight, blood glucose, mechanical pain and thermal pain were measured weekly. After 6 weeks of STZ injection, the mice were divided into control group (non-diabetic mice, 6 mice), model group (DPN mice, 6 mice), and BZC@CB[7] group (0.5 mL / kg, 6 mice). The control group and model group were injected intraperitoneally with physiological saline, and the BZC@CB[7] group was injected intraperitoneally with the corresponding dose of BZC@CB[7] solution. The administration was carried out 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 0, 4 and 8 weeks after STZ injection.

[0040] The Von Frey tactile measurement kit was purchased from the company, and the plantar thermal pain device was purchased from UGO.

[0041] Claw retraction test: Select a nylon filament of appropriate thickness, adjust the appropriate extension length, and stimulate the skin vertically. The stimulation force can be adjusted by adjusting the extension length and changing the nylon filament until the nylon filament bends. Test six times on one mouse to determine the threshold. The 50% mechanical pain threshold of the mouse is calculated according to 50% threshold=(10^(xf+kδ)) / 10000.

[0042] 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.

[0043] 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.

[0044] 3. Experimental Results Experimental results are as follows Figures 4 to 7 As shown, BZC@CB[7] can increase the pain response and improve the nerve conduction velocity in mice, indicating that BZC@CB[7] has the effect of improving sensory loss and nerve conduction velocity in diabetic peripheral neuropathy.

[0045] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, The pharmaceutical composition consists of a core compound and peripheral compounds; The core compound is a tyrosine kinase inhibitor (TKI), and the peripheral compounds are molecules with macrocyclic structures.

2. The pharmaceutical composition according to claim 1, wherein, The tyrosine kinase inhibitor TKI is selected from the KIT inhibitor, and the KIT inhibitor is selected from Bezuclastinib.

3. The pharmaceutical composition according to claim 2, wherein, The peripheral compound is selected from cucurbituril, which is selected from cucurbituril[7].

4. The pharmaceutical composition according to claim 3, wherein, The pharmaceutical composition further includes a pharmaceutically acceptable excipient; the pharmaceutically acceptable excipient is selected from a solvent; the solvent is selected from one or more of PBS, DMSO or DMF.

5. A method for preparing the pharmaceutical composition according to any one of claims 1-4, characterized in that, Includes the following steps: ① The core compound and peripheral compound are dissolved in solvents respectively to prepare the core compound mother liquor and peripheral compound mother liquor; ② Dilute the mother liquor of the core compound and the mother liquor of the peripheral compound with solvents to prepare the working solution of the core compound and the working solution of the peripheral compound, respectively; ③ Weigh the core compound working solution and the peripheral compound working solution according to the molar concentration ratio of 2:1 to 1:2, place them in the reaction vessel, mix them evenly, heat at 30℃ to 60℃, and sonicate for 15 to 45 minutes. ④ After ultrasound is completed, store and keep for future use.

6. Use of the pharmaceutical composition of any one of claims 1-4, or the pharmaceutical composition prepared by the method of claim 5, in the preparation of a medicament for treating diabetic peripheral neuropathy.

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