Application of tenofovir disoproxil fumarate

Abstract

The invention discloses an application of tenofovir disoproxil fumarate in preparation of a medicine for preventing or / and treating alpha-synucleinopathy, and also discloses a related combined application of the tenofovir disoproxil fumarate. Specifically, the tenofovir disoproxil fumarate protects neurons by reducing the level of alpha-synuclein in nerve cells and relieving behavior defects induced by alpha-synuclein, realizes effective prevention and treatment of alpha-synucleinopathy, and improves the insufficiency of the efficacy of existing drugs.

Description

Technical Field

[0001] The present invention relates to an application of tenofovir disoproxil fumarate, and particularly to a pharmaceutical application of tenofovir disoproxil fumarate in preventing and treating α-synucleinopathy. Background Art

[0002] α-synuclein (α-Syn) diseases are the most common neurodegenerative diseases, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). α-Syn is an intrinsically regulated protein and is highly expressed in neurons, participating in vesicle transport, synaptic vesicle aggregation, maintaining the synaptic vesicle pool, and neuronal transmission. The misfolding and aggregation processes of α-Syn play an important role in the pathogenesis of Parkinson's disease. Oligomeric α-Syn is a neurotoxic effector substance that can induce neuronal degeneration in dopaminergic neurons through mitochondrial dysfunction and oxidative stress.

[0003] Currently, the drugs for treating α-Syn diseases clinically mainly include cholinergic drugs, drugs for dopamine replacement therapy, dopamine releasers, drugs for inhibiting dopamine catabolism, and antipsychotic drugs for symptomatic treatment, etc. There is still no drug that can radically cure the occurrence and progression of α-Syn diseases. Therefore, it is necessary to find drugs with new pharmacological mechanisms in basic research. Summary of the Invention

[0004] Object of the Invention: The present invention aims to provide a new pharmaceutical application of tenofovir disoproxil fumarate.

[0005] Technical Solution: The tenofovir disoproxil fumarate described in the present invention is applied in the preparation of a drug for preventing and / or treating α-synucleinopathy.

[0006] Preferably, the drug is a drug for inhibiting α-synuclein.

[0007] More preferably, the drug is a drug having any one of the following effects:

[0008] (1) reducing the level of α-synuclein in nerve cells;

[0009] (2) relieving the behavioral defects induced by α-synuclein;

[0010] (3) protecting neurons.

[0011] Preferably, tenofovir disoproxil fumarate is used in combination with other drugs having the effect of inhibiting α-synuclein.

[0012] Tenofovir Disoproxil fumarate (TDF) is an oral prodrug of tenofovir, an acyclic nucleotide analogue closely related to adefovir dipivoxil, and has been approved for the treatment of human immunodeficiency virus infection. Tenofovir Disoproxil fumarate also has effective antiviral activity against wild-type hepatitis B virus and lamivudine-resistant hepatitis B virus infection. There has been no report on the association between Tenofovir Disoproxil fumarate and α-Syn disease so far.

[0013] The Tenofovir Disoproxil fumarate described in the present invention is used in the preparation of a drug for inhibiting α-synuclein.

[0014] Preferably, Tenofovir Disoproxil fumarate is used in combination with other drugs for inhibiting α-synuclein.

[0015] The Tenofovir Disoproxil fumarate described in the present invention is used in the preparation of a drug for preventing and / or treating neurodegenerative diseases caused by α-synuclein aggregation.

[0016] Preferably, the drug is a drug having any one of the following effects:

[0017] (1) reducing the content of α-synuclein in nerve cells;

[0018] (2) relieving the behavioral defects induced by α-synuclein;

[0019] (3) protecting neurons.

[0020] Preferably, Tenofovir Disoproxil fumarate is used in combination with other drugs for preventing and / or treating neurodegenerative diseases caused by α-synuclein aggregation.

[0021] The pharmaceutical composition with Tenofovir Disoproxil fumarate as the active ingredient described in the present invention is used in the preparation of a drug for preventing and / or treating α-synucleinopathy, a drug for inhibiting α-synuclein, and a drug for preventing and / or treating neurodegenerative diseases caused by α-synuclein aggregation.

[0022] Preferably, the combination therapy described in the present invention comprises the use of a drug with Tenofovir Disoproxil fumarate as the sole active ingredient in combination with a drug selected from any one of the following:

[0023] a drug for preventing and / or treating α-synucleinopathy, a drug with an active molecule capable of inhibiting α-synuclein as the sole active ingredient, a drug for inhibiting α-synuclein, and a drug for preventing and / or treating neurodegenerative diseases caused by α-synuclein aggregation.

[0024] It also includes a drug combination in the form of a compound drug with tenofovir disoproxil fumarate and an active molecule selected from any of the following as active ingredients:

[0025] An active molecule for preventing or / and treating α-synucleinopathy, an active molecule capable of inhibiting α-synuclein, an active molecule of α-synuclein inhibitor, an active molecule for preventing or / and treating neurodegenerative diseases caused by α-synuclein aggregation.

[0026] Among them, the said active ingredients also include pharmaceutically acceptable salts, deuterated compounds, and various crystal forms of the active ingredients.

[0027] "Pharmaceutically acceptable salt" refers to a salt of a compound, prepared from a compound with specific substituents and a relatively non-toxic acid or base. When a compound contains a relatively acidic functional group, the base addition salt can be obtained by contacting the free form of such a compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts or similar salts. When a compound contains a relatively basic functional group, the acid addition salt can be obtained by contacting the free form of such a compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, and the inorganic acids include, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonate or bicarbonate), phosphoric acid (forming phosphate, monohydrogen phosphate, dihydrogen phosphate), sulfuric acid (forming sulfate or bisulfate), hydroiodic acid, phosphorous acid, etc.; and organic acid salts, and the organic acids include, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid and similar acids; organic acid salts also include salts of amino acids (such as arginine, etc.), glucuronic acid and other organic acids. When certain specific compounds contain both basic and acidic functional groups, they can be converted into either base or acid addition salts. Preferably, the salt is contacted with a base or acid in a conventional manner, and then the parent compound is separated to regenerate the free form of the compound. The free form of the compound is different from its various salt forms in certain physical properties, such as solubility in polar solvents.

[0028] "Pharmaceutically acceptable salt" can be synthesized from a parent compound containing an acid radical or a base group by conventional chemical methods. Generally, the preparation method of such salts is: in water or an organic solvent or a mixture of both, these compounds in the free acid or base form are reacted with a stoichiometric amount of the appropriate base or acid. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

[0029] "Deuterated compound" means that each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom.

[0030] The drug contains tenofovir disoproxil fumarate and a pharmaceutically acceptable carrier. The specific dosage forms are selected from capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories, and patches. The carriers that can be arbitrarily mixed can be changed according to the dosage form, administration form, etc. Among them, pharmaceutically acceptable carriers are, for example, excipients, binders, disintegrants, lubricants, flavoring agents, fragrances, coloring agents, or sweetening agents, etc.

[0031] "Pharmaceutically acceptable carrier" can be an auxiliary material widely used in the field of drug production. The auxiliary materials are mainly used to provide a safe, stable, and functional drug composition, and can also provide methods to enable the active ingredient to dissolve at the desired rate after the subject receives the administration, or to promote the effective absorption of the active ingredient after the subject receives the administration of the composition. The medicinal auxiliary materials can be inert fillers or provide certain functions, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The medicinal auxiliary materials can include one or more of the following auxiliary materials: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption retardants, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, coloring agents, flavoring agents, and sweetening agents.

[0032] The drug can be prepared by any method known to those skilled in the art according to the disclosed content. For example, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding, or freeze-drying processes.

[0033] The drug can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The drug can also be in a controlled-release or sustained-release dosage form (such as liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of preparations for parenteral administration include, but are not limited to, injection solutions, dry powder preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, injection suspensions, and injection emulsions. Examples of other suitable preparations of the drug composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalants; liquid dosage forms suitable for parenteral administration; suppositories, and lozenges.

[0034] Preferably, the dosage is 10-80 mg·kg -1 , more preferably 50-80 mg·kg -1 ; the administration route is oral administration.

[0035] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0036] The present invention discovers a new pharmaceutical application of tenofovir disoproxil fumarate, protects neurons by reducing the level of α-synuclein in nerve cells and alleviating the behavioral defects induced by α-synuclein, realizes the effective prevention and treatment of α-synucleinopathy, and improves the deficiencies of the efficacy of existing drugs. Description of the drawings

[0037] Figure 1 shows the inhibitory effect of tenofovir disoproxil fumarate on α-Syn; A: Immunoblotting detection results of A53T-αSyn level; B: Histogram of α-Syn content statistical results;

[0038] Figure 2 shows the improvement effect of tenofovir disoproxil fumarate on the behavioral defects of α-Syn disease; A: Movement trajectory diagrams of mice in each group, B: Histogram of total travel distance statistics of mice in each group within 6 minutes;

[0039] Figure 3 shows the neuroprotective effect of tenofovir disoproxil fumarate under nerve injury of α-Syn disease; A: Immunohistochemical staining diagrams of Th-positive neurons in the substantia nigra region of mouse brain tissues in each group; B: Histogram of Th-positive neuron number statistics in the substantia nigra region of mouse brain tissues in each group. Detailed implementation manners

[0040] The technical solution of the present invention will be further described below in conjunction with the embodiments.

[0041] Example 1: Inhibitory effect of tenofovir disoproxil fumarate on α-Syn in SH-SY5Y nerve cells overexpressing α-Syn

[0042] 1. Experimental method

[0043] (1) Transfect SH-SY5Y cells with α-Syn overexpression plasmid

[0044] The SH-SY5Y cells were seeded in 6-well plates and cultured in an incubator. When the cell growth density reached about 60%, the transfection experiment could be carried out. Take Opti-MEM medium (125 μl / well) and add it to centrifuge tube A. Add the pCMV3-A53T-αSyn-His plasmid at 1000 ng / well to the tube, mix gently, and incubate at room temperature for 5 minutes. Take another Opti-MEM medium (125 μl / well) and add it to centrifuge tube B. Add the transfection reagent Lipofectamine TM 3000 at 7.5 μl / well, mix gently, and incubate at room temperature for 5 minutes. Mix the solutions in centrifuge tube A and centrifuge tube B together, mix well, and incubate at room temperature for 20 minutes. Replace the medium with DMEM high-glucose complete medium, 2 ml per well, and then add 250 μl of the above transfection mixture to each well. After shaking well, place it in the cell culture incubator and culture for 6 hours.

[0045] (2) Effects of TDF on α-Syn in SH-SY5Y neuronal cells overexpressing α-Syn

[0046] After 6 hours of transfection of SH-SY5Y cells, a solvent control group and a TDF (10 μM) experimental group were set up. After 24 hours of drug treatment, take out the 6-well plates and extract the cell protein samples of each group with SDS lysis buffer. Through the gel electrophoresis device, add 30 μg of total protein amount per well, electrophorese to separate the proteins, and the conditions are 80 V for 30 minutes; 120 V for 60 minutes. After electrophoresis, place it in the order of "filter paper - NC membrane - separation gel - filter paper", insert it into the wet transfer tank after exhausting the air bubbles, add wet transfer solution and ice cubes, and the wet transfer conditions are a constant current of 350 mA for 1.5 hours. After the transfer is completed, take out the NC membrane and put it into a freshly prepared 5% skimmed milk powder solution, shake slowly, and block at room temperature for 1 hour. After blocking, cut the target band. Wash the target band with TBST buffer. Prepare an α-Syn primary antibody incubation solution with antibody diluent, place the cut band in the primary antibody incubation solution, shake slowly on a shaker at 4 °C overnight. The next day, wash the band with TBST buffer 3 times, 10 minutes each time. Prepare a secondary antibody incubation solution with antibody diluent, place the band in the prepared secondary antibody dilution solution, place it on a shaker, shake slowly, and incubate at room temperature for 1 hour. After incubation, wash the band with TBST buffer 3 times, 15 minutes each time. At the same time, prepare the ECL chemiluminescence solution, place the band in the gel imager, expose and image, and perform relative quantitative analysis of the gray value of the developed band with imagej software. To eliminate the differences caused by the quantity and quality of proteins, protein expression was normalized relative to GAPDH.

[0047] 2. Experimental results

[0048] As shown in Table 1 and Figure 1 shown (TDF vs. DMSO, Student's t-test).

[0049] Table 1. Effects of TDF on α-Syn in SH-SY5Y neurons overexpressing α-Syn

[0050]

[0051] As can be seen from Table 1 and Figure 1 it can be seen that after overexpression of α-Syn in SH-SY5Y neurons, continued treatment of SH-SY5Y with TDF can reduce α-Syn expression.

[0052] Example 2: Improvement of behavioral defects and neuroprotection of α-Syn-induced mouse α-Syn disease model by tenofovir disoproxil fumarate

[0053] 1. Experimental method

[0054] (1) Feeding of C57BL / 6J mice and construction of A53T-αSyn-induced mouse α-Syn disease model

[0055] Maintain a 12 / 12 hour light / dark cycle within a temperature range of 22-24 °C, and continuous access to food and water is available. Three-month-old mice were stereotaxically injected with empty AAV5 (empty vector) or human mutant A53T-αSyn expressing AAV5. Using a microsyringe, the virus at a concentration of 10 12 genomic particles / ml was delivered into the right substantia nigra region of the mice at a rate of 0.5 μL / min. The injection coordinates were as follows: AP - 3.1 mm; ML - 1.4 mm; DV - 4.4 mm. For the blank control group and the A53T-αSyn injection group, the mice in the treatment group were gavaged with 80 mg·kg -1 TDF daily starting from one day after A53T-αSyn injection for 27 days.

[0056] (2) Improvement of spontaneous activity of A53T-αSyn-induced mouse α-Syn disease model by TDF

[0057] Spontaneous activity was detected after the administration. An open field area was assembled with plywood (50 cm × 50 cm × 45 cm), and the inner surface was painted white. During the test, only one mouse was placed in the peripheral area of the open field each time. The mice were allowed to explore the open field for 6 minutes, recorded by a camera, and the total distance of the mice's movement was analyzed and calculated using SMART v3.0.04 software. After 6 minutes, the mice were returned to the cage. Then the open field area was wiped and cleaned with 75% ethanol, and after drying, the next mouse was placed.

[0058] (3) Neuroprotective effect of TDF on substantia nigra neurons in α-Syn-induced mouse a-Syn disease model

[0059] After the behavioral test, fresh mouse brain tissues were collected and fixed in fixative for 24 hours. The tissues were taken out from the fixative liquid, and the target tissues were trimmed with a scalpel in a fume hood. Then, the trimmed tissues and labels were put into dehydration cassettes. The dehydration cassettes were placed in a dehydrator to dehydrate with gradient alcohol. 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, absolute ethanol I for 30 minutes, absolute ethanol II for 30 minutes, ethanol-benzene for 5 - 10 minutes, xylene II for 5 - 10 minutes, melted paraffin I at 65°C for 1 hour, melted paraffin II at 65°C for 1 hour, melted paraffin III at 65°C for 1 hour. The wax-impregnated tissues were embedded in an embedding machine. The melted wax was put into an embedding frame. Before the wax solidified, the tissue paper was taken out from the dehydration cassette according to the embedding surface, put into a tissue paper frame and labeled with the corresponding label. Tissue sections measured at 30 μm were incubated with polyclonal anti-tyrosine hydroxylase (Th) at 4°C for 24 hours. HRP-labeled goat anti-rabbit IgG (H+L) was used as the secondary antibody and applied to the sections for incubation at room temperature for 30 minutes. Using Stereo Investigator software, images were captured and the number of positive cells was quantified under a microscope.

[0060] 2. Experimental Results

[0061] As shown in Table 2 and Figure 2 (Vector_cmcNa vs. Vector_TDF, Vector_cmcNa vs. A53T-αSyn_cmcNa, A53T-αSyn_cmcNa vs. A53T-αSyn_TDF, two-way ANOVASidak's multiple comparisons test).

[0062] Table 2. Total distance of movement of mice in each group

[0063]

[0064] As can be seen from Table 2 and Figure 2 it can be seen that compared with the blank control mice, the total travel distance of the mice treated with A53T-αSyn within 6 minutes was significantly reduced, indicating a defect in spontaneous activity. After administration of TDF, the travel distance of the mice increased greatly, indicating that the impairment of spontaneous activity in the mice was alleviated.

[0065] As shown in Table 3 and Figure 3Shown (Vector_cmcNa vs. Vector_TDF, Vector_cmcNa vs. A53T-αSyn_cmcNa, A53T-αSyn_cmcNa vs. A53T-αSyn_TDF, two-way ANOVA Sidak's multiple comparisons test).

[0066] Table 3. Statistics of Th-positive neurons in the substantia nigra of mice in each group

[0067]

[0068] As can be seen from Table 3 and Figure 3 it can be seen that compared with the blank control mice, the number of Th-positive neurons in the substantia nigra of mice treated with A53T-αSyn was significantly reduced, indicating that neurons were damaged. After administration of TDF, the number of Th-positive neurons in mice increased significantly, indicating that TDF has a protective effect on neurons in the substantia nigra of the α-Syn-induced mouse α-Syn disease model.

[0069] In summary, TDF can effectively reduce the content of α-Syn and improve the behavioral defects and neuronal protection in the α-Syn-induced mouse α-Syn disease model, and can be used to prepare drugs for the prevention and treatment of α-Syn disease.

Claims

1. Use of tenofovir disoproxil fumarate in the preparation of a medicament for preventing and / or treating α-synuclein disease.

2. The use according to claim 1, characterized in that The drug is a drug that inhibits α-synuclein.

3. The use according to claim 2, characterized in that The drug is a drug having any of the following effects: (1) Reduce the level of α-synuclein in nerve cells; (2) alleviate α-synuclein-induced behavioral deficits; (3) Protect neurons.

4. The use according to claim 1, characterized in that Tenofovir disoproxil fumarate is used in combination with other drugs that have the effect of inhibiting α-synuclein.

5. Application of tenofovir disoproxil fumarate in the preparation of α-synuclein inhibitor drugs.

6. The use according to claim 5, characterized in that Tenofovir disoproxil fumarate in combination with other α-synuclein inhibitors.

7. Use of tenofovir disoproxil fumarate in the preparation of a medicament for preventing and / or treating neurodegenerative diseases caused by α-synuclein aggregation.

8. The use according to claim 7, characterized in that The drug is a drug having any of the following effects: (1) Reduce the content of α-synuclein in nerve cells; (2) alleviate α-synuclein-induced behavioral deficits; (3) Protect neurons.

9. The use according to claim 7, characterized in that Tenofovir disoproxil fumarate is used in combination with other drugs for preventing and / or treating neurodegenerative diseases caused by α-synuclein aggregation.

10. Use of a pharmaceutical composition comprising tenofovir disoproxil fumarate as an active ingredient in the preparation of a drug for preventing and / or treating α-synucleinopathy, an α-synuclein inhibitor, or a drug for preventing and / or treating neurodegenerative diseases caused by α-synuclein aggregation.

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