Use of isoleufolin and its derivatives to promote nerve repair

Abstract

A new use of Isorhoifolin and its derivatives. Isorhoifolin and its derivatives can promote the regeneration of damaged brain neurons and retinal nerves.

Description

[Technical Field]

[0001] The present invention provides the use of isorhoifolin and its derivatives to promote nerve repair. [Background technology]

[0002] Currently, approximately 70 million people worldwide suffer from neurological injuries such as traumatic brain injury (TBI) each year. Common treatments include physical therapy, hyperbaric oxygen therapy, transcranial magnetic stimulation, and transcranial direct current stimulation (TCS), which are non-invasive treatments that can improve depression and cognitive function after TBI. However, there are currently no effective drugs that can promote neuronal regeneration after brain injury.

[0003] Traumatic brain injury (TBI) is an injury caused by an external force striking the brain, with approximately 70 million diagnosed cases worldwide each year. Traumatic brain injury damages cranial nerves, resulting in deficits in the patient's behavioral or cognitive function. Because the central nervous system is difficult to regenerate or recover from once damaged, current medical science does not offer a treatment that effectively promotes neuronal regeneration after TBI. Furthermore, many patients with brain injury develop brain lesions after a certain period of time, or develop neurodegenerative diseases in the future. Therefore, the use of drugs that promote regeneration of cranial nerves as quickly as possible after brain injury is a therapeutic solution.

[0004] In addition, when selecting therapeutic drugs, because the cerebrum has a blood-brain barrier, common drugs cannot easily pass through the blood-brain barrier to reach an effective dose, so administering effective drugs is an important issue when treating brain diseases. Summary of the Invention [Problem to be solved by the invention]

[0005] In view of the above, there is currently a great need for the development of drugs that can treat neuronal damage, as well as for administration methods that can cross the blood-brain barrier. [Means for solving the problem]

[0006] Although isorhoifolin is a known compound, there have been few studies on its use as a drug for treatment. Furthermore, compositions containing this compound have been used to treat venous problems and hemorrhoids, but there has been no application for treating nerve-related diseases. Therefore, as a result of further research, the present inventors have discovered that isorhoifolin and its derivatives can effectively promote nerve repair and regeneration, can cross the blood-brain barrier, and can be used to develop potential drugs for treating traumatic brain injury.

[0007] The purpose of this Summary is to provide an overview of the contents of the present disclosure so that the reader can have a basic understanding of the contents of the present disclosure. This Summary is not a complete summary of the contents of the present disclosure, and is not intended to point out the key / major assemblies of the embodiments of the present invention, nor to define the scope of the present invention.

[0008] The nervous system of the human body is divided into the central nervous system and the peripheral nervous system, both of which are composed of neurons. The trigeminal nerve is a cranial nerve among the peripheral nerves and is connected to the pons (central nervous system). Therefore, isorhoifolin and its derivatives of the present invention can pass through the olfactory epithelial cells through the nasal mucosa, enter the olfactory and trigeminal nerve pathways, and enter the cerebrum, while simultaneously achieving the effect of repairing nerves throughout the body through the peripheral nerves.

[0009] In view of the above, the present invention focuses on cranial nerves, which are difficult to repair, and thereby achieves the effect of making it possible to repair both central and peripheral nerves within the nervous system.

[0010] The "central nervous system" comprises the brain and spinal cord. The "central nervous system" as used herein includes, but is not limited to, the rhinencephalon, amygdala, hippocampus, neocortex, lateral ventricles, epithalamus, thalamus, hypothalamus, ventral thalamus, pituitary gland, pineal gland, third ventricle, tectum, cerebral peduncle, pretectal area, aqueduct, pons, cerebellum, medulla oblongata, and spinal cord.

[0011] The "peripheral nerves" consist of the somatic nervous system and the autonomic nervous system. As used herein, the "peripheral nerves" include, but are not limited to, sensory nerves, motor nerves, cranial nerves, spinal nerves, sympathetic nerves, parasympathetic nerves, and the enteric nervous system.

[0012] In view of the above issues, The present invention provides A pharmaceutical composition for use in the treatment of nerve damage, comprising isorhoifolin and its derivatives. to provide.

[0013] In some embodiments, the derivative of Isorhoifolin is Narirutin.

[0014] In some embodiments, the chemical structure of isorhoifolin is represented by formula (1):

[0015] [ka]

[0016] In some embodiments, the chemical structure of Narirutin, a derivative of Isorhoifolin, is represented by formula (2):

[0017] [ka]

[0018] In some examples, isorhoifolin and its derivatives, when used alone, can promote the regeneration of 10-30% of injured hippocampal neurons and promote neuronal regeneration in three-dimensional brain tissue slices, demonstrating that the compounds can effectively promote neuronal regeneration and can be used to repair neuronal damage.

[0019] In some embodiments, isorhoifolin and its derivatives can cross the blood-brain barrier of the user and enter the cerebrum, thereby promoting the repair of damaged neurons.

[0020] In some embodiments, isorhoifolin and its derivatives can cross the blood-brain barrier and enter the cerebrum, thereby promoting the repair, regeneration, or increase in number of cranial nerves.

[0021] In some embodiments, isorhoifolin and its derivatives can promote the regeneration of damaged cortical neurons, damaged hippocampal neurons, and damaged retinal neurons.

[0022] In some embodiments, isorhoifolin has very low toxicity to Neuro2a cells.

[0023] In some embodiments, the effective dose or concentration of isorhoifolin and its derivatives is 1 nM to 864 μM.

[0024] In some embodiments, isorhoifolin and its derivatives have a significant effect on nerve repair, and when administered intranasally, the isorhoifolin compound and its derivatives can penetrate the blood-brain barrier and enter the cerebrum, thereby promoting the repair of damaged neurons, promoting the repair, regeneration, or increase in the number of cranial nerves, and promoting the regeneration of axons of cortical neurons and hippocampal neurons after injury.

[0025] In some embodiments, the pharmaceutical composition can be administered to a subject.

[0026] In some examples, the pharmaceutical composition can be administered to a subject by injection, infusion, intravenous, nasal, or oral administration.

[0027] By referring to the following embodiments, those skilled in the art can easily understand the basic spirit and other objects of the present invention, as well as the technical means and embodiments used in the present invention. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 is a flow chart of an in vitro nerve repair test according to the present invention. [Figure 2] FIG. 2 is a schematic reference diagram for calculating the gap closure rate in the present invention. [Figure 3A] FIG. 3A shows the results of an in vitro hippocampal neuronal repair experiment using isorhoifolin of the present invention. [Figure 3B] FIG. 3B shows the results of an in vitro hippocampal neuronal repair experiment using isorhoifolin of the present invention. [Figure 3C] FIG. 3C shows the results of an in vitro hippocampal neuronal repair experiment using isorhoifolin of the present invention. [Figure 4]FIG. 4 shows the results of an in vitro hippocampal nerve regeneration experiment using narirutin, an isoleufolin derivative of the present invention. [Figure 5] FIG. 5 shows the results of an in vitro cortical nerve regeneration experiment using narirutin, an isoleufolin derivative of the present invention. [Figure 6] FIG. 6 is a flow chart of the ex vivo brain tissue slice experiment of the present invention. [Figure 7] FIG. 7 shows the results of an ex vivo brain tissue slice experiment using isorhoifolin of the present invention. [Figure 8] FIG. 8 shows the results of an ex vivo brain tissue slice experiment using narirutin, an isoleufolin derivative of the present invention. [Figure 9] FIG. 9 shows the results of a cytotoxicity experiment using the isoleufolin compound of the present invention. [Figure 10] FIG. 10 shows a flow chart of the controlled cortical impact brain injury model experiment of the present invention and the results of an experiment on the promotion of recovery of motor coordination after brain injury in mice by the isoleufolin compound. [Figure 11] FIG. 11 is a flow chart showing the promotion of ex vivo retinal neural tissue repair in the present invention. [Figure 12] FIG. 12 shows the results of an ex vivo experiment on the promotion of retinal neural tissue repair by isoleufolin in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0029] In order to provide a more detailed and complete description of the contents of the present disclosure, the following describes illustrative embodiments and specific examples of the present invention. However, these are not the only ways to implement or operate the specific examples of the present invention. Although the embodiments cover the features of multiple specific examples and the methods and applications for operating these specific examples, other specific examples may achieve the same or equivalent effects. It should be understood that these examples are intended to illustrate the present invention and do not limit the scope of the present invention. [Example]

[0030] In vitro nerve repair studies with isorhoifolin and its derivatives

[0031] As shown in Figure 1, in this experimental procedure, rat fetuses were removed on day 18 of pregnancy and the fetal cerebrum was divided into the cerebral cortex and hippocampus. The cerebral cortex and hippocampus were then dissociated into cortical neurons and hippocampal neurons, and the hippocampal neurons were transferred to a 48-well plate. This day was designated Day In Vitro 0 (DIV0). At DIV2, cytosine beta-D-arabinoside (AraC) was added to inhibit glial cell proliferation. At DIV8, neurons were injured using a micropipette tip and various concentrations of isorhoifolin were added. Seventy-two hours after isorhoifolin addition, neurons were labeled with the TUJ1 antibody using immunofluorescence staining to observe the status of neuronal regeneration. Images were taken with a Zeiss Observer Z1 microscope.

[0032] As shown in Figure 2, the degree of axon regeneration was quantified from the gap closure rate. The white dotted line represents the boundary created when neurons were injured by rubbing with a micropipette tip, and the black area in the center represents the area scraped by the micropipette tip. Regenerated neurites grew from both sides of the dotted line toward the center. A line was drawn every 50 μm in the injury area to calculate the gap width. After drawing a total of 10 lines, the average gap length (Lg) between injured borders was calculated. After a certain period of time, a line was drawn every 50 μm in the same injury area to connect the regenerated axons. The average gap length (Ln) between regenerated neurons was calculated, and the gap closure rate was calculated as (Lg - Ln) / Lg. The scale is 100 μm. This experiment was photographed with a Zeiss Observer Z1 microscope.

[0033] As shown in Figures 3A-3C, the white dotted lines indicate the boundaries created by damaging neurons with a micropipette tip. The scale is 100 μm. This experiment was photographed using a Zeiss Observer Z1 microscope. This experiment demonstrated that isoleufolin can promote the growth of hippocampal neurites. Figure 3A shows the experimental results for isoleufolin at 3.5 nM to 34.6 nM, Figure 3B shows the experimental results for isoleufolin at 0.009 μM to 864 μM, and Figure 3C shows the calculated gap closure rates for different concentrations of isoleufolin.

[0034] Narirutin, a derivative of isorhoifolin, promotes hippocampal neuronal regeneration after injury

[0035] As shown in Figure 1, in this experiment, fetal rats were removed on day 18 of pregnancy. The fetal cerebrum was divided into the cerebral cortex and hippocampus, and then the cortical and hippocampal neurons were separated. The hippocampal neurons were then transferred to a 48-well plate. This day was designated Day In Vitro 0 (DIV 0). At DIV 2, cytosine arabinoside (AraC) was added to inhibit glial cell proliferation. At DIV 8, neurons were injured using a micropipette tip, and 1 nM and 10 nM narirutin were added. 0.1% DMSO served as the solvent control. Results are normalized to the 0.1% DMSO group. Seventy-two hours after narirutin addition, neurons were labeled with TUJ1 antibody using immunofluorescence staining to observe the status of neuronal regeneration. Images were taken with a Zeiss Observer Z1 microscope. The scale is 100 μm.

[0036] The degree of axon regeneration was quantified from the gap closure rate, as shown in Figure 4. The experimental results indicated that narirutin can effectively promote the growth of hippocampal neurites.

[0037] Narirutin, a derivative of isorhoifolin, promotes cortical nerve regeneration after injury

[0038] As shown in Figure 1, in this experiment, fetal rats were removed on day 18 of pregnancy. The fetal cerebrum was divided into the cerebral cortex and hippocampus, and then cortical and hippocampal neurons were isolated. The cortical neurons were then transferred to a 48-well plate. This day was designated Day In Vitro 0 (DIV 0). At DIV 2, cytosine arabinoside (AraC) was added to inhibit glial cell proliferation. At DIV 8, neurons were injured using a micropipette tip, and 10 nM and 100 nM narirutin were added. 0.1% DMSO served as the solvent control. Results are normalized to the 0.1% DMSO group. Seventy-two hours after narirutin addition, neurons were labeled with TUJ1 antibody using immunofluorescence staining to observe the status of neuronal regeneration. Images were taken with a Zeiss Observer Z1 microscope. The scale is 100 μm.

[0039] The degree of axon regeneration was quantified from the gap closure rate, as shown in Figure 5. The experimental results indicated that narirutin can effectively promote the growth of cortical neurites. [Example]

[0040] Ex vivo brain tissue slice experiments with isoleufolin and its derivatives

[0041] After testing the effects of compounds on neural regeneration in vitro, we attempted to observe their effects ex vivo (three-dimensional). As shown in Figure 6, we observed the effects of compounds on neural regeneration using a three-dimensional brain tissue slice culture method. For the experiment, we harvested a female rat fetus on day 18 of pregnancy, removed the fetal cerebrum, and embedded it in low-melting agarose gel. Then, we sliced ​​the brain slices using a Leica microtome VT100 to obtain 350 μm-thick brain slices. After injury with a scalpel, the slices were transferred to inserts in a 6-well plate. This culture method allowed the brain slices to be simultaneously exposed to both medium and air. Furthermore, culturing the brain slices allowed us to more realistically reflect the interactions between neurons and other cells in the cerebrum and their three-dimensional structure than in vitro culture. In addition, ddH2O or isorhoifolin was added daily, and after 96 hours of culture, fluorescent immunostaining was performed to label neurons with TUJ1 antibody, glial cells with GFAP antibody, and cell nuclei with DAPI reagent.

[0042] As shown in Figure 7, the TUJ1 antibody labeled neurons, the GFAP antibody labeled glial cells, and the DAPI reagent labeled cell nuclei. The white dotted line indicates the site of injury with a scalpel. The brain section tissue is to the left of the dotted line, and the newly formed axons are to the right of the dotted line. The effect of isoleufolin on promoting nerve regeneration was quantified by calculating the length of the white dotted line and the area of ​​the newly formed axons to the right of the white line, and then dividing the area of ​​the newly formed axons by the length of the white dotted line to calculate neurite length. The scale was 100 μm. Images were taken using a Zeiss LSM800 confocal microscope. The effect of isoleufolin on nerve regeneration was observed in ex vivo brain sections, and the experimental results demonstrated that isoleufolin promotes nerve regeneration.

[0043] Similarly, in the experimental flow for narirutin, a derivative of isorhoifolin, as shown in Figure 6, fetal rat brains were removed, embedded in low-melting-point agarose gel, and sliced ​​into 350 μm-thick tissue sections using a Leica VT100 microtome. The brain sections were then injured with a scalpel and cultured, with daily addition of ddH2O or narirutin. After 96 hours of culture, neurons were labeled with TUJ1 antibody, glial cells with GFAP antibody, and cell nuclei with DAPI reagent using immunofluorescence staining. The scale for this experiment was 100 μm. Images were taken using a Zeiss LSM800 confocal microscope.

[0044] As shown in FIG. 8, the effect of narirutin on nerve regeneration was observed in ex vivo brain slices, and the experimental results showed that narirutin has the effect of promoting nerve regeneration. [Example]

[0045] Cytotoxicity experiments with isoleufolin

[0046] The toxicity of isorhoiofolin to Neuro2a cells was examined using the CellTiter-Glo cell viability assay, which measured ATP content in the cells and allowed us to monitor cell viability after compound addition.

[0047] As shown in Figure 9, the results showed that the IC50 of isoleufolin on Neuro2a cells was in the range of about 11.616 mM, indicating very low toxicity to the cells. [Example]

[0048] Isoleufolin promotes recovery of motor coordination after brain injury in mice

[0049] In the horizontal bar experiment, mice were placed on a 38 cm long brass bar, 2 mm, 4 mm, or 6 mm in diameter, located 49 cm above the ground, and allowed to grasp the bar with their forepaws. The time the mouse spent on the bar was calculated, and the mouse's motor coordination ability was evaluated based on whether or not it reached the platform at the end of the bar. The evaluation criteria were as follows: 1 point for 1-5 seconds, 2 points for 5-10 seconds, 3 points for 10-20 seconds, 4 points for 20-30 seconds, and 5 points for 30 seconds or reaching the platform.

[0050] As shown in Figure 10, pre-training was performed on mice at -5, -3, and -1 days post-injury (Dpi), 5, 3, and 1 day before brain injury, respectively. At 0 Dpi, mice were brain-injured using a controlled cortical impact model. At 0, 2, 4, 6, 8, 10, and 12 Dpi, mice were intranasally administered 14 μg / kg or 140 μg / kg of isoleufolin. Horizontal bar tests were also performed at 1, 3, 6, 10, and 13 Dpi.

[0051] As is clear from Figure 10, the group that received only water (ddH2O) after brain injury showed a loss of motor coordination, but the group that received isoleufolin showed motor coordination similar to that of the sham group (sham operation group), and the recovery of motor coordination was clearly promoted. [Example]

[0052] Isoleufolin promotes ex vivo repair of retinal neural tissue

[0053] The experimental design and flow are shown in Figure 11. Retinal explants were isolated from 8-day-old C57BL / 6 mice. After sacrifice, the eyes were removed, and the retina was separated from the globe using tweezers and microscissors, and the vitreous was removed. Finally, the isolated retina was divided into four pieces, and the tissue edges were trimmed and damaged using microscissors. Each piece was cultured on an 18-mm circular glass plate and then placed in a 12-well plate. The plate was then placed in an incubator at 5% CO2 and 35°C for 5 days. Fresh culture medium was replaced daily, and 9 μM or 90 μM isoleufolin was added. After 5 days of culture, the retinal tissue was fixed in a mixture of 0.1% glutaraldehyde and 4% paraformaldehyde for 1 hour at room temperature. The sections were then stained with the primary antibody axonal marker beta-III-tubulin (TUJ1) and DAPI to label neurons and cell nuclei, and then photographed using a super-resolution upright confocal microscope (LSM-800, Carl Zeiss). The tissue boundary was selected and the perimeter and extracellular nerve fiber area were calculated using the image analysis program ImageJ. The nerve fiber area was then divided by the perimeter of the tissue boundary to calculate the nerve fiber length per perimeter.

[0054] As shown in Figure 12, the group that received isoleufolin after damaging retinal neurons showed a clear post-injury recovery effect compared to the group that received only water (ddH2O).

[0055] The above experimental data are preliminary experimental results obtained under specific conditions, and are only intended to facilitate understanding or reference of the technical content of the present invention, and further related experiments need to be conducted. The experimental data and results are not intended to limit the scope of the present invention.

[0056] The above preferred embodiments are merely illustrative of the present invention and its technical features, and the techniques of the embodiments may be implemented by appropriately making various substantially equivalent modifications and / or substitutions. Therefore, the scope of the present invention is limited to the scope defined in the claims.

Claims

1. A pharmaceutical composition for use in the treatment of nerve damage, comprising isorhoifolin and its derivatives.

2. 2. The pharmaceutical composition for treating nerve damage according to claim 1, wherein the derivative of isorhoifolin is narirutin.

3. The pharmaceutical composition for use in treating nerve damage according to claim 1, characterized in that the nerve damage is damage to the central nervous system or peripheral nerves.

4. The pharmaceutical composition for treating nerve damage according to claim 1, characterized in that the nerve damage is neuronal damage.

5. The pharmaceutical composition for treating nerve damage according to claim 1, characterized in that the nerve damage includes damage to cortical neurons, hippocampal neurons, or retinal neurons.

6. The pharmaceutical composition for use in treating nerve damage according to claim 1, characterized in that the treatment is nerve regeneration, nerve number increase, or nerve repair.

7. The pharmaceutical composition for treating nerve damage according to claim 1, wherein the isorhoifolin and its derivatives can penetrate the blood-brain barrier and enter the cerebrum when administered via the nasal cavity.

8. The pharmaceutical composition for treating nerve injury according to claim 1, wherein the effective dose of isorhoifolin and its derivatives is 1 nM to 864 μM.

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