Composition, nasal spray for treating Parkinson's disease and preparation method thereof

Abstract

The present invention discloses a composition, a nasal spray for treating Parkinson's disease and a preparation method thereof. The composition is composed of levodopa and epigallocatechin gallate in a mass ratio of 1-3:1. The nasal spray for treating Parkinson's disease is prepared from the following components in weight percentages: 54-72% of glyceryl monooleate, 18-36% of N-methylpyrrolidone, 1.4-2.1% of levodopa, 0.7-1.4% of epigallocatechin gallate 、 Water is added to 100%. The composition can significantly improve the stability of levodopa and synergistically improve its therapeutic effect on Parkinson's disease; the nasal spray can improve the olfactory region deposition rate of the drug, improve its nasal-brain delivery efficiency, and thus improve its therapeutic effect on Parkinson's disease.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to a composition for treating Parkinson's disease, a nasal spray and a preparation method thereof. Background Art

[0002] Parkinson's disease (PD) is a common neurodegenerative disease of the nervous system. There are many causes and mechanisms for the onset of this disease, which is the result of comprehensive pathogenic factors. The main pathological changes are the loss of dopaminergic neurons in the substantia nigra compacta and the formation of Lewy bodies. The main biochemical change is the lack of dopamine in the striatal region, and the imbalance between dopamine and acetylcholine neurotransmitters leads to the relative hyperfunction of acetylcholine. The main symptoms of clinical patients include motor symptoms such as bradykinesia, resting tremor, muscle rigidity and postural balance disorder, and non-motor symptoms such as olfactory disorder, constipation, sleep disorder, etc. Currently, the commonly used treatment methods include drug treatment, surgical treatment, rehabilitation treatment, etc., but it cannot be cured, only the symptoms can be relieved and the quality of life of patients can be improved, and its treatment needs to be emphasized.

[0003] The drugs used for the treatment of PD disease mainly include dopamine drugs (levodopa, dopamine receptor agonists, etc.) and choline drugs (trihexyphenidyl hydrochloride, benzatropine mesylate, procyclidine hydrochloride, etc.). Among them, levodopa (LDA) is the standard therapy for Parkinson's disease and the most effective symptomatic treatment drug in the drug treatment of Parkinson's disease. Levodopa is a prodrug of dopamine, which has no pharmacological activity itself. After entering the central nervous system through the blood-brain barrier, it is converted into dopamine by dopa decarboxylase and exerts its pharmacological effect. However, alone, levodopa can only supplement the dopamine deficiency in the brain and is difficult to reverse or relieve neurodegenerative diseases. Therefore, its therapeutic effect is limited.

[0004] In addition, levodopa is unstable in a humid state and is prone to oxidation. Therefore, the currently marketed preparations are mostly solid preparations such as levodopa tablets, capsules, granules, etc. administered via the oral route. However, after oral administration of levodopa, only 1% can be transported to the striatal tissue in the brain through the neutral amino acid carrier and converted into dopamine to exert its effect. Therefore, the onset is slow, the bioavailability is low, and the dosage is large. More than 95% of levodopa is converted into dopamine by decarboxylase widely distributed in peripheral tissues, resulting in adverse reactions such as cardiovascular diseases and gastrointestinal diseases.

[0005] Nasal administration preparations can directly deliver drugs from the olfactory region to the brain, bypassing the blood-brain barrier, and have significant advantages such as rapid onset, improved brain entry efficiency, reduced dosage, and avoidance of gastrointestinal reactions. Currently, there are already many nasal administration preparations for central nervous system diseases on the market, such as zolmitriptan nasal spray for the treatment of migraine and sumatriptan nasal spray The opioid butorphanol tartrate nasal spray (Butorphanol ), and fentanyl citrate nasal spray Diazepam nasal spray for the treatment of epilepsy Midazolam nasal spray And esketamine nasal spray for the treatment of depression etc. have all been approved by the FDA for marketing.

[0006] For nasal-brain delivery formulations, the absorption amount of the drug in the olfactory region is crucial for its efficacy. The area of the olfactory mucosa is extremely small (1 - 5 cm 2 ), only accounting for 3% - 5% of the total nasal area. How to deposit the drug in the olfactory region and improve the drug delivery efficiency is the key point and difficulty in the research and development of levodopa nasal spray. In addition, since levodopa is extremely easy to oxidize in the air when moist, there is only one enteral suspension that needs to be stored frozen among the currently marketed levodopa liquid preparations One kind. Improving the stability of levodopa is a prerequisite for preparing it into a nasal-brain delivery nasal spray. Summary of the Invention

[0007] Based on this, the present invention provides a composition and its corresponding nasal spray. The composition is composed of epigallocatechin gallate and levodopa, which can significantly improve the stability of levodopa and synergistically improve its therapeutic effect on Parkinson's disease; the nasal spray can increase the deposition rate of the drug in the olfactory region, improve its nasal-brain delivery efficiency, and thus improve its therapeutic effect on Parkinson's disease.

[0008] The present invention includes the following technical solutions.

[0009] On the one hand, the present invention provides a composition for the treatment of Parkinson's disease, which is composed of levodopa and epigallocatechin gallate in a mass ratio of 1 - 3:1.

[0010] In some embodiments, the mass ratio of levodopa to epigallocatechin gallate is 1:1 or 2:1.

[0011] In some embodiments, the mass ratio of levodopa to epigallocatechin gallate is 1.5:1 or 2.5:1.

[0012] On the second hand, the present invention provides the application of the composition in the preparation of drugs for preventing and / or treating Parkinson's disease.

[0013] On the third hand, the present invention provides a drug for the treatment of Parkinson's disease, which is prepared from an active ingredient and a pharmaceutically acceptable excipient, and the active ingredient is the composition of the present invention.

[0014] Fourthly, the present invention provides a nasal spray for treating Parkinson's disease, which is prepared from the following components in percentage by weight:

[0015]

[0016] The mass ratio of levodopa to epigallocatechin gallate is 1-3:1.

[0017] In some embodiments, the nasal spray for treating Parkinson's disease is prepared from the following components in percentage by weight:

[0018]

[0019] In some embodiments, the nasal spray for treating Parkinson's disease is prepared from the following components in percentage by weight:

[0020] Glycerol monooleate 62-64%

[0021] N-methylpyrrolidone 26-28%

[0022] Levodopa 2.0-2.1%

[0023] Epigallocatechin gallate 0.8-1.0%

[0024] Water is added to 100%.

[0025] In some embodiments, the levodopa is micronized levodopa; preferably, the d 90 particle size is less than or equal to 10 μm; preferably less than or equal to 5 μm.

[0026] Fifthly, the present invention provides a preparation method of the nasal spray for treating Parkinson's disease, comprising the following steps:

[0027] 1) Add levodopa to water, emulsify for 5 min-15 min, add epigallocatechin gallate, and stir evenly to obtain liquid A;

[0028] 2) Melt glycerol monooleate and mix it evenly with N-methylpyrrolidone to obtain liquid B;

[0029] 3) Drop liquid A into liquid B and mix evenly to obtain a precursor solution;

[0030] 4) Fill the precursor solution into a nasal spray device to obtain a levodopa nasal spray.

[0031] The present invention has the following beneficial effects:

[0032] During the research on levodopa preparations, the inventors of the present invention unexpectedly discovered that when levodopa and epigallocatechin gallate are used in combination at a specific ratio, not only can the stability of levodopa be improved, but also the neuroprotective effect of epigallocatechin gallate on Parkinson's disease can be enhanced. The synergistic cooperation of the two can effectively supplement the dopamine content in the striatum while delaying the neurodegenerative process, and can synergistically improve the therapeutic effect on Parkinson's disease.

[0033] On this basis, the inventors further found that a nasal spray can be prepared by combining this specific composition with a specific amount of glyceryl monooleate and N-methylpyrrolidone. Before administration, the nasal spray is a flowable, shear-thinning pseudoplastic fluid with good rheological behavior and is easy to spray out from the spray device. After being sprayed out in a mist form through the nasal administration device, it can form a spray with an appropriate spray pattern and spray mode. Its flight stability is good, the spray area and plume angle are small, which can effectively improve the olfactory region deposition rate of levodopa and the nasal-brain delivery efficiency. And after being intercepted in the nasal olfactory region, it can absorb the nasal moisture and quickly phase-transition into a high-viscosity in-situ gel, adhere to the inner wall of the nasal cavity, and release the drug stably, thereby prolonging the residence time of the drug in the nasal cavity, enhancing drug absorption, and further improving the nasal-brain delivery efficiency of levodopa and its therapeutic effect on Parkinson's disease. Brief Description of the Drawings

[0034] Figure 1 It is the qualitative chromatogram of levodopa.

[0035] Figure 2 It is the standard curve of levodopa.

[0036] Figure 3 It is the qualitative determination chromatogram after the combined oxidation and destruction of different antioxidant drugs and levodopa.

[0037] Figure 4 It is the effect of the drug on the activity of SH-SY5Y cells; among them, A is the toxicity of 6-OHDA to SH-SY5Y cells, B is the toxicity of LDA to SH-SY5Y cells, C is the toxicity to SH-SY5Y cells, D is the improvement effect of LDA and / or EGCG on the damage of SH-SY5Y cells induced by 6-OHDA; n = 3, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0038] Figure 5 It is the effect of LDA and / or EGCG on the production of ROS in SH-SY5Y cells induced by 6-OHDA; among them, A is the detection of the DCF content in cells using flow cytometry, B is the data analysis of the DCF positive rate; n = 3, *p < 0.05, **p < 0.01, ***p < 0.001.

[0039] Figure 6 It is the evaluation result of the water absorption and swelling behavior of the levodopa nasal spray.

[0040] Figure 7 It is the shear viscosity of the levodopa nasal spray.

[0041] Figure 8 It is the shear viscosity after the levodopa nasal spray forms a gel.

[0042] Figure 9 It is the spray pattern and spray mode of the levodopa nasal spray.

[0043] Figure 10 It is the droplet size distribution (A) and the proportion of the stable period (B) of the levodopa nasal spray.

[0044] Figure 11 It is a 3D printed nasal cavity model; among them, A is a schematic diagram of the brain structure, and B is a 3D printed segmented nasal cavity model: 1. Nasal vestibule; 2. Inferior nasal meatus; 3. Middle nasal meatus; 4. Superior nasal meatus; 5. Oropharynx; 6. Nasopharynx.

[0045] Figure 12 It is the deposited drug amount (A) of the levodopa nasal sprays of Formulations F6 - F8 in each region of the nasal cavity and the drug deposition rate in the olfactory region of the superior nasal meatus (B); Area1: Nasal vestibule; Area2: Inferior nasal meatus; Area3: Middle nasal meatus; Area4: Superior nasal meatus; Area5: Oropharynx; Area6: Nasopharynx; n = 3, **p < 0.01, ***p < 0.001.

[0046] Figure 13 It is the hemolysis rate of the levodopa nasal spray.

[0047] Figure 14 It is the cytotoxicity of the levodopa nasal spray.

[0048] Figure 15 It is the result of the APO-induced ipsilateral rotation experiment, n = 6, **p < 0.01, ****p < 0.0001. Specific Embodiments

[0049] The technical solutions of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations to the present invention.

[0050] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and do not limit the present invention.

[0051] As used in the present invention, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product or device that includes a series of steps is not limited to the listed steps or modules, but may optionally further include steps not listed, or may optionally further include other steps inherent to these processes, methods, products or devices.

[0052] As used in the present invention, "a plurality of" means two or more. "And / or" describes the relationship between associated objects and indicates that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. The character " / " generally indicates that the associated objects before and after are in an "or" relationship.

[0053] The levodopa used in the following examples is micronized levodopa, and the pulverization method is as follows:

[0054] Use a JET MILL Lab air jet mill, set the inlet pressure and pulverization pressure to 6.5 bar and 7.0 bar respectively, adopt the automatic feeding mode, the feeding speed is 10 rpm, and feed the material for air jet pulverization.

[0055] Measurement of the particle size of micronized levodopa: Use Malvern wet method to measure the particle size of levodopa after micronization, and the test parameters are as follows: background / sample measurement time 10 s, obscuration 8%-20%, stirring speed 2000 rpm, stirring time 5 min.

[0056] The particle size distribution of levodopa before and after micronization is shown in the following table:

[0057]

[0058] The following are specific examples.

[0059] Example 1: Antioxidant protection of levodopa by antioxidant drugs

[0060] 1.1 Establishment of HPLC method

[0061] 1) Chromatographic conditions

[0062] Chromatographic column: TitanK C18 (250 mm × 4.6 mm, 5 μm); mobile phase: water (0.1% TFA): acetonitrile = 90:10; flow rate: 1.0 mL·min -1 ; column temperature: 30 °C; detection wavelength: 280 nm; injection volume: 20 μL.

[0063] 2) Preparation of reference solution: Accurately weigh 20 mg of levodopa reference standard, place it in a 100 mL volumetric flask, add an appropriate amount of mobile phase (0.1% TFA aqueous solution), shake to dissolve, and dilute to the mark with 0.1 mol / L dilute hydrochloric acid solution. Mix well to obtain a reference standard solution of 0.2 mg / mL. -1 Accurately measure 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, and 10 mL of the reference standard solution respectively and place them in 10 mL volumetric flasks. Dilute to the mark with 0.1 mol / L dilute hydrochloric acid solution, mix well. Take 20 μL of each of the above solutions for injection, record the chromatogram, and plot the standard curve.

[0064] 3) Results: The liquid chromatogram is as Figure 1 shown. The retention time of levodopa is 4.5 min; the standard curve is as Figure 2 shown. The concentration has a linear relationship with the peak area (R 2 = 0.9999).

[0065] 1.2 Stability experiment test

[0066] 1) Degradation experiment: Take 8 portions of levodopa with a purity of over 99% in 100 mL volumetric flasks, about 20 mg each. Accurately weigh 20 mg of vitamin A (VA), vitamin C (VC), vitamin E (VE), catechin epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), and epigallocatechin gallate (EGCG) respectively and add them to the above volumetric flasks. Then add 10 mL of 0.1 mol / L dilute hydrochloric acid solution to each volumetric flask, shake to dissolve to obtain test samples A1 - A8 (A1. LDA test sample without antioxidant drug; A2. VA + LDA; A3. VC + LDA; A4. VE + LDA; A5. EC + LDA; A6. ECG + LDA; A7. EGC + LDA; A8. EGCG + LDA). Conduct oxidation destruction tests under the same conditions. Test conditions: Add 5.0 mL of 30% hydrogen peroxide solution, let stand at room temperature for 24 h, then dilute to the mark with 0.1 mol / L dilute hydrochloric acid solution, mix well. Filter the solution through a 0.45 μm microporous membrane, take the subsequent filtrate, and detect the content of levodopa by HPLC.

[0067] 2) Results: The chromatogram is shown in Figure 3 . Levodopa is extremely unstable in an oxidative environment. VA has almost no protective effect on levodopa, while other components show obvious antioxidant effects.

[0068] The stability results are shown in Table 1: The levodopa content in each group is as shown in Table 1. The levodopa content in the standard solution has no obvious change, indicating that the levodopa solution is stable at room temperature for 24 h. Among them, under the oxidative destruction condition, in the levodopa solution group (A0) and the samples added with VA (A1), the levodopa concentration undergoes severe oxidative degradation, and the content drops sharply, being lower than the limit of quantification (LOQ is 5 μg / mL). In the samples of groups A3 - A8 added with antioxidant drugs, the degradation of levodopa is less. Among them, the levodopa content in the catechol drug compound groups (A6 - A8) is significantly higher than that in the vitamin compound groups (A2 - A4). Catechol drugs show a good effect in improving the stability of levodopa, and among them, EGCG has the best effect in improving the stability of levodopa.

[0069] Table 1 Levodopa content in the compound of different drugs and levodopa after oxidative destruction

[0070]

[0071]

[0072] Example 2 Neuroprotective effect of the composition of levodopa and EGCG on the Parkinson's disease model

[0073] SH - SY5Y is a dopaminergic neuronal cell line commonly used in the field of neuroscience. 6 - OHDA can induce a significant increase in the ROS level of cells, leading to the oxidation of lipids in the cell membrane, thereby simulating DA neuron cell damage. The specific steps are as follows:

[0074] Construction of the Parkinson's disease cell model: Take SH - SY5Y cells in the logarithmic growth phase, adjust the cell suspension concentration to 1×10 5 / mL with MEM / F12 medium, and inoculate into a 96 - well plate, 100 μL per well. After inoculation, place the cells in an incubator for culture. Dissolve 10 mg of 6 - OHDA in 10 mL of MEM / F12 medium to prepare a stock solution with a concentration of 4000 μM. After filtering through a 0.22 - μm filter membrane, it is reserved for use. Then, dilute the stock solution with the medium into gradient concentrations of 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, and 800 μM in sequence. Discard the supernatant in the 96 - well plate, add blank medium and the above - mentioned various concentration 6 - OHDA dilution solutions to the wells, and incubate for 24 h. Discard the supernatant, and wash each well 2 times with 100 μL of PBS. Finally, add 10 μL of MTT + 90 μL of MEM / F12 medium mixture to each well, and incubate in an incubator for 4 h. Discard the supernatant again, add 200 μL of dimethyl sulfoxide to each well, shake in the dark on a shaker for 10 min, and after fully shaking, use a multifunctional microplate reader to measure at a wavelength of 490 nm. The cell survival rate is calculated by the following formula:

[0075]

[0076] Effect of LDA and EGCG on cell viability: SH-SY5Y cells in the logarithmic growth phase were taken, and the cell suspension concentration was adjusted to 1×10 5 / mL with MEM / F12 medium and inoculated into 96-well plates at 100 μL per well. After inoculation, the cells were cultured in an incubator. An appropriate amount of LDA or EGCG was dissolved in 10 mL of MEM / F12 medium to prepare a stock solution with a concentration of 80 mg / mL. After filtration through a 0.22-μm filter membrane, it was reserved for use. Then, the stock solution was diluted successively with the medium into gradient concentrations of 0.625, 1.25, 2.5, 5, 10, 20, 40, and 80 mg / mL. The supernatant in the 96-well plates was discarded, and blank medium and the drug dilution solutions at the above various concentrations were added to the wells and incubated for 24 h. The supernatant was discarded, and each well was washed twice with 100 μL of PBS. Finally, a mixture of 10 μL of MTT + 90 μL of MEM / F12 medium was added to each well, and the cells were incubated in an incubator for 4 h. The supernatant was discarded again, and 200 μL of dimethyl sulfoxide was added to each well and dissolved on a shaker in the dark for 10 min. After thorough mixing, a multifunctional microplate reader was used to measure the absorbance at a wavelength of 490 nm. The cell survival rate was calculated using the following formula:

[0077]

[0078] Effect of the combination of LDA and EGCG on the viability of PD cell models: 6-OHDA at the concentration corresponding to a 50% cell survival rate in the "Construction of Parkinson's disease cell models" was selected as the inducer, and a Parkinson's disease cell model was constructed according to the method of this example and reserved for use. Using MEM / F12 medium as the solvent, drugs with corresponding concentrations were prepared according to Table 2, and after filtration through a 0.22-μm filter membrane, they were reserved for use. The supernatant in the 96-well plates was discarded, and the drug solutions shown in Table 2 below were added to the wells and incubated for 24 h. The supernatant was discarded, and each well was washed twice with 100 μL of PBS. Finally, a mixture of 10 μL of MTT + 90 μL of MEM / F12 medium was added to each well, and the cells were incubated in an incubator for 4 h. The supernatant was discarded again, and 200 μL of dimethyl sulfoxide was added to each well and dissolved on a shaker in the dark for 10 min. After thorough mixing, a multifunctional microplate reader was used to measure the absorbance at a wavelength of 490 nm. The cell survival rate was calculated using the following formula:

[0079]

[0080] Table 2 Experimental groups

[0081]

[0082]

[0083] The results are asFigure 4 As shown: The results of the cytotoxicity experiment indicate that the cytotoxic effect of 6-OHDA on SH-SY5Y cells shows concentration dependence. The higher the concentration, the greater its cytotoxicity. When the concentration is 400 μM, the cell survival rate is approximately 50% ( Figure 4 in A of Figure 4 ). Therefore, in this example, 400 μM is selected as the modeling concentration dose to construct a PD cell model. LDA and EGCG also show concentration dependence on the cytotoxic effect. When the concentrations of LDA and EGCG are lower than 20 mg / mL, the cytotoxicity is relatively low and can be used as the safe drug administration concentration range ( Figure 4 in B and C of

[0084] Example 3 Analysis of the ROS Level in SH-SY5Y Cells by Flow Cytometry

[0085] The progression of PD disease is usually related to oxidative stress. Oxidative stress causes a large accumulation of intracellular peroxides, which damage dopaminergic neuron cells and ultimately lead to a decrease in cell numbers. In this experiment, the ROS level in PD model cells was measured by flow cytometry. The principle is that the DCFH-DA probe itself does not have fluorescence. When it enters the cell, it can be hydrolyzed by related esterases into DCFH, which cannot penetrate the cell membrane. The DCFH retained in the cell can be oxidized by intracellular ROS into 2,7-dichlorofluorescein (DCF), and its fluorescence intensity is proportional to the intracellular ROS level.

[0086] The specific steps are as follows:

[0087] (1) Seeding plates: SH-SY5Y cells were seeded in 6-well plates at a density of 8×10 5 cells / well.

[0088] (2) Adding drugs and culturing: After incubating in the 6-well plate incubator for 24 h until the cells adhered, the supernatant was removed, and then grouped and dosed according to Table 3, and cultured for another 24 h.

[0089] (3) Cell collection: The next day, transfer the cell suspension in the 6-well plate to a labeled sterile centrifuge tube. Add 300 μL of phenol red-free trypsin to each well for digestion. After terminating the digestion, continue to collect the cell suspension into the corresponding centrifuge tube. Centrifuge for 3 minutes to collect the cells, and wash the cells with serum-free medium. Repeat the above washing and centrifugation steps 3 times.

[0090] (4) Probe loading: Dilute the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe reagent in the ROS detection kit with serum-free culture medium at a ratio of 1:1000 to make the final concentration 10 μM. Resuspend the cells in each tube with 1 mL of the diluted DCFH-DA solution and incubate in a 37 °C cell culture incubator for 30 minutes. Invert and mix every 3 - 5 minutes to ensure full contact between the probe and the cells. Wash the cells three times with PBS to fully remove the DCFH-DA that has not entered the cells.

[0091] (5) Detection: Resuspend the cell pellet in each tube with 500 μL of PBS solution and place it on ice for later use. Filter the cell suspension through a filter cloth into a flow cytometry tube. Place the blank group flow cytometry tube in an analytical flow cytometer for detection to determine the detection parameters and range, and then detect the fluorescence intensity of other groups of cells (Table 3) respectively.

[0092] Table 3 Experimental groups

[0093]

[0094]

[0095] The results are as Figure 5 shown: Compared with the blank group, the fluorescence intensity of the cells in the PD model constructed by 6-OHDA induction was significantly increased, indicating that 6-OHDA can significantly increase the intracellular ROS level. Compared with the model group, the fluorescence intensity of the LDA group, EGCG group, and LDA-EGCG combination administration group all decreased, indicating that both LDA and EGCG can improve the excessive accumulation of intracellular ROS to a certain extent. The order of ROS scavenging effect is: LDA-EGCG combination administration group > EGCG group > LDA group. Combining the results of the cytotoxicity experiment, it can be seen that EGCG can play a protective role on nerve cells by inhibiting oxidative stress and alleviating nerve cell damage, and the combination of LDA and EGCG can enhance this protective effect.

[0096] Example 4 Preparation of nasal spray

[0097] The dosage of each component of the levodopa nasal spray provided in this example is as follows (by weight percentage):

[0098] Glycerol monooleate (GMO): 63%;

[0099] N-methylpyrrolidone (NMP): 27%;

[0100] Levodopa: 2%;

[0101] EGCG: 1%;

[0102] Water: 7%;

[0103] The preparation method thereof comprises the following steps:

[0104] 1) Add the prescription amount of levodopa fine powder into the prescription amount of water under mechanical stirring. After mixing evenly, disperse it for 10 min at 13000 rpm by using a high-shear emulsifier, add the prescription amount of EGCG, and stir for 10 min to obtain liquid A;

[0105] 2) Melt glyceryl monooleate in a 45°C water bath and mix it evenly with N-methylpyrrolidone to obtain liquid B;

[0106] 3) Under vortexing, slowly drop liquid A into liquid B and mix evenly by vortexing to obtain the precursor solution;

[0107] 4) Fill the precursor solution into a nasal spray device to obtain the levodopa nasal spray.

[0108] Example 5

[0109] The prescription composition of the levodopa nasal spray provided in this example is shown in Table 4, and the specific preparation method is the same as that of Example 4.

[0110] In this example, by adjusting the contents of glyceryl monooleate and N-methylpyrrolidone, a series of levodopa nasal sprays with different gelling behaviors and viscosities are obtained.

[0111] Table 4 Prescription of nasal spray and contents of each component (by weight percentage (%))

[0112]

[0113] Example 6 Investigation on the gelling behavior of nasal spray

[0114] Investigate the gelling behavior of the levodopa nasal spray prepared in Example 5:

[0115] Take 1 mL of levodopa nasal spray and add it to deionized water preheated to 37 ± 0.5°C in a water bath. Observe whether the nasal spray can form a gel when encountering water, and use a stopwatch to record the time required for the nasal spray to be added to the water until it completely forms a gel.

[0116] The states and gelling times of the nasal sprays of each prescription are shown in Table 5.

[0117] Table 5 States and gelling behaviors of levodopa nasal sprays

[0118]

[0119] Note: √ indicates a homogeneous, transparent, and flowable state; × indicates a turbid and non-flowable state.

[0120] The gel strength is divided into four levels. + indicates very weak gel strength, ++ indicates weak gel strength, +++ indicates strong gel strength, and ++++ indicates very strong gel strength. a It indicates that GMO solids precipitate out in the formed gel.

[0121] The results show that with the increase in the content of NMP in the formulation, the gelation time is prolonged. Levodopa nasal sprays with a mass ratio of GMO to NMP higher than 6:4 can form liquid crystal gels with relatively high strength, and the gelation time is less than 5 s. However, in the gel formed by the levodopa nasal spray with a mass ratio of GMO to NMP of 9:1, some GMO precipitates out. This may be because the content of NMP is relatively low and cannot completely dissolve GMO, and it is difficult for semi-solid GMO to completely self-assemble with water to form a uniform liquid crystal gel. Therefore, to ensure the ejection of the levodopa nasal spray from the dosing device and its adhesion in the nasal cavity, it is preferable to use the levodopa nasal sprays prepared from Formulations F6 - F8 with a short gelation time and good precursor fluidity.

[0122] Water absorption and swelling behavior of the levodopa nasal spray in Example 7

[0123] Good water absorption is the basis for the levodopa nasal spray to absorb nasal mucus, form a gel, and adhere to the nasal mucosa surface. Therefore, the water absorption and swelling behavior of the levodopa nasal sprays prepared from Formulations F6 - F8 was investigated by the weighing method. Weigh a 10 mL centrifuge tube and record the weight m0. Add 5 mL of PBS solution to the EP tube, and use a pipette to aspirate 200 μL of the levodopa nasal spray and drop it into the PBS solution. Weigh the EP tube before and after adding the levodopa nasal spray, and subtract to obtain the mass m1 of the dropped levodopa nasal spray. Subsequently, place the EP tube in a shaker, keep it at a constant temperature of 33 ± 0.5 °C, and the shaking frequency is 100 rpm. Take out the EP tube after 0.5, 1, 2, 3, 6, 12, 24, and 48 h respectively, drain the PBS solution, carefully absorb the residual water in the tube with a cotton swab, and weigh the total weight m of the centrifuge tube and the gel t , and measure 3 replicates in parallel. Calculate the water absorption of the gel according to the following formula:

[0124]

[0125] The results are as Figure 6 shown. The levodopa nasal sprays prepared from F6 - F8 can all rapidly absorb water, quickly swell, form a gel, and have good water absorption.

[0126] Example 8 Rheological Behavior of Levodopa Nasal Spray and Its Gel

[0127] The shear viscosity of levodopa nasal spray at different shear rates was measured using a Kinexus Lab+ rotational rheometer. The measuring fixture model was CP1 / 60, the measuring temperature was 25 ± 0.5 °C, and the shear rate was 0.1 s -1 ~100 s -1 .

[0128] The levodopa nasal spray was dropped into excess water to form a gel. After reaching swelling equilibrium in 24 h, the excess water was drained, the gel was taken out, and the shear viscosity of the gel was measured at a temperature of 33 ± 0.5 °C in the same way.

[0129] The results are as Figure 7 and Figure 8 shown. The viscosity of levodopa nasal spray decreases with the increase of shear rate, showing typical shear thinning properties and being a pseudoplastic fluid. During the spraying process of the dosing device, the shear rate increases suddenly, and the viscosity of levodopa nasal spray decreases rapidly, which is beneficial for spraying and administration. The gel formed after the levodopa nasal spray absorbs water shows high viscosity and low fluidity, can adhere smoothly to the surface of the nasal mucosa, prolong the residence time, and enhance drug absorption.

[0130] Example 9 Evaluation of Spray Pattern and Spray Mode of Levodopa Nasal Spray

[0131] The spray pattern and spray morphology of levodopa nasal sprays of Formulations F6 - F8 were evaluated through a Spray droplet online test system. The trigger distance was 6 cm, the actuator speed was 70 mm / s, the actuator acceleration was 5000 mm / s 2 , and the actuator holding time was 250 ms.

[0132] The results are as Figure 9 and Table 6 shown. With the increase of the proportion of GMO, the spray area of levodopa nasal spray decreases, and the plume angle decreases. Comparatively, the plume angles of Formulations F7 and F8 are narrower and the spray areas are smaller, which is beneficial for reducing the ineffective deposition in the nasal vestibule and increasing the drug deposition in the olfactory region.

[0133] Table 6 Spray Morphology and Spray Mode of Levodopa Nasal Spray

[0134]

[0135] Example 10 Droplet Size Distribution of Levodopa Nasal Spray

[0136] The Sympatec laser particle size analyzer (HELOS&SPRAYER TM)Measure the droplet size of levodopa nasal sprays of each formulation. The lens is R4 (0.5 / 1, 8 - 350 μm), the trigger pressure is 60 N, and the measurement time is 300 ms.

[0137] The spraying process of the nasal spray can be divided into three stages: the formation stage, the stable stage, and the dissipation stage. The formation stage is the stage when the spray is formed, the droplet concentration increases rapidly, and the droplet size also increases rapidly. During the stable stage, the droplet size reaches its peak and remains stable. During the dissipation stage, the droplet concentration drops rapidly, and the droplet size fluctuates greatly. The test results are as Figure 10 shown. As the proportion of GMO in the formulation of levodopa nasal spray increases, the proportion of the stable spraying stage increases, and the droplet size increases. However, when the proportion of GMO is too large, the stable stage shortens, the spraying stability decreases, and the fluctuation of the droplet size also increases. Therefore, Formulation F7, which has a longer stable stage, a stable droplet size, and very few droplets below 10 μm, is selected as the most preferred formulation.

[0138] Example 11: Test the nasal deposition rate of levodopa nasal spray

[0139] Although nasal drug delivery has the potential for brain delivery, it is hindered by the nasal physiological barrier. After nasal spraying, the drug is mainly retained in the nasal vestibule and respiratory region, and only a very small amount of the drug can be deposited in the olfactory region of the superior nasal meatus, which seriously affects the nasal-brain delivery efficiency. The 3D-printed nasal model can be used as a tool for in vitro research of nasal spray preparations. Its advantage lies in that it can completely restore the complex internal structure of the human nasal cavity and can separate each region independently, facilitating the quantification of the drug amount in each nasal region, making the spray deposition in each part of the nasal cavity visible, and thus facilitating the optimization of the formulation. In order to quantitatively study the nasal delivery and deposition pattern of levodopa nasal spray in the olfactory region of the superior nasal meatus, based on the magnetic resonance images of adults, the nasal cavity was three-dimensionally reconstructed and a 3D-printed segmented nasal model was made, which was divided into six parts: the nasal vestibule, the superior nasal meatus, the middle nasal meatus, the inferior nasal meatus, the oropharynx, and the nasopharynx according to the anatomical and physiological characteristics

[125] ( Figure 11 )Subsequently, spray administration was carried out at a sagittal angle of 45° at the lower left corner of the nasal vestibule. The levodopa nasal sprays in six parts were collected respectively. After filtering through a 0.22 μm microporous filter membrane into a sample vial, the drug deposition amounts of levodopa nasal sprays of F6 - F8 in different nasal parts were quantitatively detected by HPLC.

[0140] In this example, a 3D-printed nasal model was used to test the drug deposition rate of levodopa nasal spray in different nasal parts. The results are as Figure 12 shown. The drug is mainly distributed in the nasal vestibule, the superior, middle, and inferior nasal meatus and other regions, and the drug distribution in the middle nasal meatus is the most obvious. In addition, as the proportion of GMO increases, the spraying angle becomes smaller, and the deposition proportion of the nasal spray in the olfactory region of the superior nasal meatus first increases and then decreases ( Figure 12B) among them. According to the deposition rate results in the olfactory region of the superior nasal meatus, F7 with the highest deposition proportion in the olfactory region of the superior nasal meatus is the optimal formulation.

[0141] Example 12 Safety Evaluation of Levodopa Nasal Spray

[0142] A series of evaluations on the safety of levodopa nasal spray were carried out through hemolytic toxicity experiments and cytotoxicity experiments on human nasal mucosa epithelial cells.

[0143] Hemolysis experiment: Take fresh rat blood, centrifuge at 3000 rpm for 10 min, discard the supernatant, and collect the blood cells. Dilute it to 5% with physiological saline as the blood cell test solution. Take a 2.5 mL centrifuge tube, add 0.5 mL of the blood cell test solution and 0.5 mL of levodopa nasal spray, use an equal volume of physiological saline as the negative control, and 1% Triton X-100 as the positive control. After mixing evenly, incubate in a constant temperature shaker at 37 °C for 2 h. Subsequently, centrifuge at 5000 rpm for 10 min, take the supernatant to a 96-well plate, measure the absorbance at 540 nm, and calculate the hemolysis rate.

[0144]

[0145] Cytotoxicity: Take cells in the logarithmic growth phase, digest them with trypsin and count the cell density. Adjust the cell density to 5×10 4 cells / mL using the culture medium. Add 100 μL of the cell suspension to each well of a 96-well plate and culture in a cell culture incubator at 37 °C and 5% CO2 for 24 h. Discard the culture medium in the well plate, add 100 μL of the culture medium containing the extraction solution of levodopa nasal spray to each well, and continue to culture for 24 h. Use the culture medium without drugs as the negative control group with 100% cell survival rate. Remove the culture medium in the well plate, add 110 μL of CCK-8 solution (CCK-8: culture medium = 1:10) to each well, culture in the cell culture incubator for about 1 h, and use an enzyme-linked immunosorbent assay reader to read the absorbance at 450 nm. Calculate the cell survival rate according to the following formula:

[0146] Cell survival rate (%) = (OD Samples -OD Zero ) / (OD Blank -OD Zero ) × 100%;

[0147] As Figures 13 to 14 shown, the hemolysis rate of levodopa nasal spray is lower than 5%, and under the action of its extraction solution, the cell survival rate is between 90% and 120%, indicating that the levodopa nasal spray of the present invention has good safety and the excipients used have good safety.

[0148] Example 13 Test the Efficacy of Levodopa Nasal Spray

[0149] Establishment of an animal model of Parkinson's disease: A stereotaxic apparatus was used to localize the right SNpc, and a single-point injection of 6-OHDA was performed to establish a PD rat model. The specific steps are as follows:

[0150] (1) Weighing and anesthesia: Before the experiment, the rats were weighed and their weights were recorded. Anesthesia was induced by intraperitoneal injection of 1% sodium pentobarbital at a dose of 50 mg / kg.

[0151] (2) Skin preparation and fixation: Most of the hair on the rat's head was removed with a razor, and fine hairs were shaved off with a blade. The surgical instruments and the rat's head skin were disinfected with iodophor. The rat's head skin was longitudinally incised with surgical scissors; the rat was fixed on the stage of the stereotaxic apparatus, keeping the rat's skull horizontal and the anterior midline in the middle position.

[0152] (3) Stereotactic localization: According to the "Stereotaxic Atlas of the Rat Brain", the position of the bregma of the rat was determined using a stereotaxic apparatus for rats and recorded as the coordinate origin. Referring to the atlas, the single-point coordinates were set as A / P (posterior to the center of the bregma): -5.2 mm, L / R (right to the center of the bregma): -1.8 mm, O / V (depth from the surface of the meninges): -7.8 mm for the right SNpc.

[0153] (4) Drilling: A micro handheld cranial drill was used to drill a hole above the accurately located right SNpc, keeping the force applied evenly and constantly to avoid damaging the rat's brain tissue due to excessive force.

[0154] (5) Injection: 6-OHDA was injected through a micro syringe. The needle was inserted slowly at a rate of 1 mm / min. 6 μL of 6-OHDA was injected into each hole at a drug administration rate of 1 μL / min. Then the needle was left in place for 5 minutes and withdrawn slowly at a rate of 1 mm / min.

[0155] (6) Suture and anti-infection treatment: The hole was sealed with bone wax, residual blood was removed, the head skin was sutured, and an appropriate amount of penicillin was applied to the surgical wound. After the animal model was established, the rats were placed on an electric blanket to maintain body temperature, and 50,000 units of sodium penicillin were intraperitoneally injected continuously for three days to prevent wound infection.

[0156] (7) Model evaluation: The degree of nerve damage of 6-OHDA to the substantia nigra-striatum of rats is positively correlated with the number of ipsilateral rotations induced by APO, which is one of the indicators for evaluating the efficacy of PD drugs. Seven days after the model was established, the inducer APO at a concentration of 0.2 mg / mL and a dose of 0.5 mg / kg was intraperitoneally injected to induce ipsilateral rotation in rats. Starting from the 10th minute after injection, an electronic counter was used to measure the behavioral changes of the rats within 30 minutes and record the number of rotations within 30 minutes. If the rats rotated constantly to the left healthy side and the number of rotations was ≥7 r / min, the model was considered successful; if the number of rotations of the rats was <7 r / min or there was no rotation within 15 minutes, the model was considered failed.

[0157] Behavioral evaluation - Rotation test experiment: Rats in 6 experimental groups were administered intranasally according to Table 7 for 4 consecutive weeks. After the administration, the rats were intraperitoneally injected with APO at a concentration of 0.2 mg / mL and a dose of 0.5 mg / kg to induce the rats to rotate to the left side of the brain without injecting 6-OHDA. One rotation was defined as 360°. After 10 minutes of injection, each rat was placed individually in a rat cage without restricting its activities, and an electronic counter was used to count the rotation behavior within 30 minutes. After each test, the rat cage was wiped with 75% ethanol and disposable paper towels to prevent the influence of odor and foreign substances on the results.

[0158] Table 7 Experimental grouping

[0159]

[0160] Among them, the formulation composition and preparation method of the nasal spray solution used in the nasal spray solution group are as follows:

[0161] Levodopa: 2%;

[0162] EGCG: 0.8%;

[0163] Normal saline: Supplemented to 100%;

[0164] Its preparation method includes the following steps:

[0165] 1) Add the prescribed amount of levodopa fine powder to the prescribed amount of normal saline under mechanical stirring. After mixing evenly, disperse it with a high-shear emulsifier at 13,000 rpm for 10 minutes, add the prescribed amount of EGCG, and stir for 10 minutes to obtain;

[0166] 2) Fill the solution into a nasal spray device to obtain a levodopa nasal spray.

[0167] The formulation composition of the levodopa nasal spray used in Nasal Spray Gel Group 1 does not add EGCG compared with F7 in Example 5, and other components, dosages, and preparation methods are the same as F7 in Example 5.

[0168] The levodopa nasal spray used in Nasal Spray Gel Group 2 is the levodopa nasal spray prepared from F7 in Example 5.

[0169] The results are as Figure 15Shown as follows: The rats in the Healthy group did not show obvious circling behavior, while the average number of circles of the rats in the Model group was about 11.4 r / min, indicating severe damage to dopaminergic neurons; after treatment with the LDAPO group, the number of rotation circles was about 9.2 r / min, still higher than the confirmed circle number of the model, 7 r / min, indicating that oral administration of LDA for the treatment of dopaminergic neuron damage had no obvious effect. Compared with the LDAPO group, at 1 / 5 of the dosing dose, the number of healthy-direction rotations of the rats in the LDA-SO IN group was only 55% of that of oral administration, indicating that nasal spray administration improved the nasal-brain delivery efficiency of LDA to a certain extent and the function of dopaminergic neurons was somewhat restored; at the same dose, the curative effect of the LDA IN group was better than that of the LDA-SO IN group, indicating that the nasal spray gel administration method of the present invention further improved the nasal-brain delivery efficiency of LDA compared with conventional nasal spray administration and improved its therapeutic effect; while the number of rotation circles in the LDA-EGCG IN group after adding EGCG for synergistic treatment was further reduced to 2.8 r / min, with the best curative effect, indicating that the synergistic administration of LDA and EGCG could delay disease progression by protecting dopaminergic neurons, thereby further significantly improving the healthy-direction rotation behavior of the PD model rats induced by 6-OHDA and improving the therapeutic effect of the drug.

[0170] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0171] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A nasal spray for treating Parkinson's disease, characterized in that, It is prepared from the following components by weight percentage: The mass ratio of levodopa to epigallocatechin gallate is 1-3:

1.

2. The nasal spray for treating Parkinson's disease according to claim 1, wherein, It is prepared from the following components by weight percentage:

3. The nasal spray for treating Parkinson's disease according to claim 2, wherein It is prepared from the following components by weight percentage:

4. The nasal spray for treating Parkinson's disease according to claim 3, wherein It is prepared from the following components by weight percentage:

5. The nasal spray for treating Parkinson's disease according to any one of claims 1-4, characterized in that, The levodopa is micronized levodopa.

6. The nasal spray for treating Parkinson's disease according to claim 5, characterized in that, The d 90 particle size of the micronized levodopa is less than or equal to 10 μm.

7. A method for preparing a nasal spray for treating Parkinson's disease according to any one of claims 1-6, characterized in that, It includes the following steps: 1) Add levodopa to water, emulsify for 5 min - 15 min, add epigallocatechin gallate, and stir evenly to obtain liquid A; 2) Melt glyceryl monooleate and mix it evenly with N-methylpyrrolidone to obtain liquid B; 3) Drop liquid A into liquid B and mix evenly to obtain the precursor solution; 4) Fill the precursor solution into a nasal spray device to obtain the levodopa nasal spray.

Images