An antidepressant preparation for nasal mucosa administration, its preparation method and application

Abstract

The present invention provides an antidepressant preparation for nasal mucosa administration, its preparation method and application, which relates to the field of medicine. The antidepressant preparation is prepared from the following raw materials: hydroxytyrosol-Angiopep-2 complex, poloxamer, sodium hyaluronate and water; the hydroxytyrosol-Angiopep-2 complex is prepared from hydroxytyrosol and Angiopep-2 with a molar ratio of 1:0.05 to 0.2; the weight-average molecular weight of sodium hyaluronate is 60 to 120 kDa. The antidepressant preparation of the present invention can not only increase the stability of hydroxytyrosol, but also facilitate the delivery of hydroxytyrosol to the brain tissue at a steady rate, directly enter the brain to exert antidepressant efficacy, reduce the number of drug administrations, and better exert the antidepressant activity of hydroxytyrosol. The antidepressant preparation of the present invention has the advantages of long-lasting efficacy and convenient use, and also has the characteristics of good dispersibility, strong adhesion, good stability, and the ability to maintain a local high concentration at the administration site.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to an antidepressant preparation for nasal mucosa administration, a preparation method thereof, and an application thereof. Background Art

[0002] Depression is a common mental illness, and its incidence has been continuously rising in recent years. The clinical manifestations are low mood and pessimism about the world, and severe patients even have suicidal tendencies. At present, oral medications are mainly used in the clinical treatment of depression. However, the mainstream antidepressant drugs have relatively high side effects and a relatively long onset time. It is necessary to develop antidepressant drug preparations with smaller side effects and better dosage forms.

[0003] Hydroxytyrosol (HT) is a polyphenol contained in olives, and its excellent anti-inflammatory and antioxidant effects have attracted much attention. The existing document ZL201811207103.X discloses that hydroxytyrosol and its derivatives have the effects of preventing and treating depression and preventing the recurrence of depression when taken orally and sublingually, and can be used to reduce the incidence of depression and improve the quality of life of depression patients. However, the use of hydroxytyrosol alone for nasal administration in the prevention and treatment of depression has the disadvantages of low bioavailability and poor stability. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides an antidepressant preparation for nasal mucosa administration, a preparation method thereof, and an application thereof, which improve the bioavailability and stability of hydroxytyrosol.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] An antidepressant preparation for nasal mucosa administration, which is prepared from the following raw materials: hydroxytyrosol-Angiopep-2 complex, poloxamer, sodium hyaluronate, and water;

[0007] The hydroxytyrosol-Angiopep-2 complex is prepared from hydroxytyrosol and Angiopep-2 in a molar ratio of 1:0.05 to 0.2;

[0008] The weight-average molecular weight of the sodium hyaluronate is 60 to 120 kDa.

[0009] Preferably, the hydroxytyrosol-Angiopep-2 complex is prepared from hydroxytyrosol and Angiopep-2 in a molar ratio of 1:0.1.

[0010] Preferably, the preparation method of the hydroxytyrosol-Angiopep-2 complex includes:

[0011] Hydroxytyrosol and Angiopep-2 polypeptide were separately dissolved in phosphate buffer solution, and then mixed to obtain a reaction solution to be reacted;

[0012] The reaction solution to be reacted was stirred and reacted at 20-30 °C under a nitrogen atmosphere for 12 h, and then filtered and dialyzed in sequence to obtain a hydroxytyrosol-Angiopep-2 complex.

[0013] Preferably, the pH value of the phosphate buffer solution is 7.4;

[0014] The pore size of the filter membrane used for the filtration is 0.45 μm;

[0015] The dialysis was carried out using a 1 kD dialysis bag, and the dialysis time was 12 h.

[0016] Preferably, the weight-average molecular weight of the sodium hyaluronate is 90 kDa.

[0017] Preferably, the poloxamer includes poloxamer 188 and poloxamer 407, and the mass ratio of poloxamer 188 to poloxamer 407 is 25:5.

[0018] Preferably, the water is deionized water.

[0019] The present invention also provides a preparation method of the antidepressant preparation described in the above technical solution, including the following steps:

[0020] 1) Mix the hydroxytyrosol-Angiopep-2 complex with water to obtain a complex solution, and the mass concentration of hydroxytyrosol in the complex solution is 5%;

[0021] 2) Mix the poloxamer, sodium hyaluronate and water to obtain a thermosensitive gel raw material;

[0022] The mass concentration of poloxamer in the thermosensitive gel raw material is 30%, and the mass concentration of sodium hyaluronate is 0.1%;

[0023] 3) Mix the complex solution obtained in step 1) and the thermosensitive gel raw material obtained in step 2) in equal volume to obtain an antidepressant preparation.

[0024] The present invention also provides the application of the antidepressant preparation described in the above technical solution in the preparation of a drug for treating chronic restraint stress depression.

[0025] The present invention also provides the application of the antidepressant preparation described in the above technical solution in the preparation of a drug for treating chronic unpredictable mild stress depression.

[0026] The beneficial effects of the present invention are:

[0027] (1) After forming a complex of hydroxytyrosol and the brain-targeting Angiopep-2 polypeptide and loading it into a hyaluronic acid and poloxamer thermosensitive gel, not only can the stability of hydroxytyrosol be increased, but hydroxytyrosol is released in an approximate zero-order manner in the nasal cavity, which is beneficial for hydroxytyrosol to be delivered to the brain tissue at a steady rate.

[0028] (2) Through nasal administration, hydroxytyrosol can directly enter the brain to exert antidepressant efficacy.

[0029] (3) The thermosensitive hydrogel of hydroxytyrosol for nasal administration can adhere to the nasal mucosa at body temperature to continuously release the drug, which can reduce the frequency of drug administration, improve the compliance of patients, and better exert the antidepressant activity of hydroxytyrosol.

[0030] (4) The antidepressant preparation of the present invention has the advantages of long-lasting efficacy, convenient use, and increased drug concentration in the brain tissue. It also has the characteristics of good dispersibility, strong adhesion, good stability, and the ability to maintain a local high concentration at the administration site. Description of the Drawings

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0032] Figure 1 is the in vitro release curve of hydroxytyrosol-Angiopep-2 thermosensitive gel;

[0033] Figure 2 is the improvement of depressive symptoms in the chronic restraint stress model by hydroxytyrosol-Angiopep-2 thermosensitive gel;

[0034] Figure 3 is the improvement of behavioral indicators in CUMS rats by hydroxytyrosol-Angiopep-2 thermosensitive gel;

[0035] Figure 4 is the promotion of neurotransmitter levels in serum and brain tissue of CUMS rats by hydroxytyrosol-Angiopep-2 thermosensitive gel. Detailed Embodiments

[0036] The present invention provides an antidepressant preparation for nasal mucosa administration, which is prepared from the following raw materials: hydroxytyrosol-Angiopep-2 complex, poloxamer, sodium hyaluronate, and water; the hydroxytyrosol-Angiopep-2 complex is prepared from hydroxytyrosol and Angiopep-2 at a molar ratio of 1:0.05 to 0.2; the weight-average molecular weight of the sodium hyaluronate is 60 to 120 kDa.

[0037] In the present invention, the hydroxytyrosol-Angiopep-2 complex is preferably prepared from hydroxytyrosol and Angiopep-2 at a molar ratio of 1:0.1. In the present invention, the preparation method of the hydroxytyrosol-Angiopep-2 complex preferably includes: dissolving hydroxytyrosol and Angiopep-2 polypeptide in phosphate buffer respectively, and then mixing to obtain a reaction solution to be reacted; stirring and reacting the reaction solution to be reacted for 12 h under a nitrogen atmosphere at 20-30 °C, and then filtering and dialyzing in sequence to obtain the hydroxytyrosol-Angiopep-2 complex. In the present invention, the Angiopep-2 polypeptide increases the brain targeting of hydroxytyrosol, improves the stability of hydroxytyrosol, is beneficial to the stable delivery of hydroxytyrosol, and increases the drug concentration of hydroxytyrosol in the brain tissue. The present invention has no special limitation on the source of the Angiopep-2 polypeptide, and conventional commercially available products can be used. In the present invention, the pH value of the phosphate buffer is preferably 7.4. In the present invention, the pore size of the filter membrane used for filtration is preferably 0.45 μm. In the present invention, the dialysis is preferably carried out using a 1 kD dialysis bag, and the dialysis time is preferably 12 h. The present invention preferably performs lyophilization after dialysis to obtain the hydroxytyrosol-Angiopep-2 complex. The present invention has no special limitation on the conditions of the lyophilization, and those skilled in the art can operate according to the conventional operation.

[0038] In the present invention, the weight-average molecular weight of the sodium hyaluronate is 60-120 kDa, preferably 90 kDa. In the present invention, the sodium hyaluronate improves the properties of the thermosensitive gel, increases the stability of hydroxytyrosol, improves the drug release characteristics of the hydroxytyrosol gel, increases the nasal mucosa adhesiveness of the thermosensitive gel, and improves the brain targeting.

[0039] In the present invention, the poloxamer preferably includes poloxamer 188 and poloxamer 407, and the mass ratio of poloxamer 188 to poloxamer 407 is preferably 25:5. In the present invention, the poloxamer is used as a nasal thermosensitive gel matrix. In the present invention, the water is preferably deionized water.

[0040] In the present invention, the antidepressant preparation preferably adopts the nasal administration method, and the dosage is in accordance with the doctor's advice.

[0041] The present invention also provides a preparation method of the antidepressant preparation according to the above technical solution, including the following steps:

[0042] 1) Mix the hydroxytyrosol-Angiopep-2 complex with water to obtain a complex solution, and the mass concentration of hydroxytyrosol in the complex solution is 5%;

[0043] 2) Mix the poloxamer, sodium hyaluronate and water to obtain a raw material for the thermosensitive gel;

[0044] The mass concentration of poloxamer in the thermosensitive gel raw material is 30%, and the mass concentration of sodium hyaluronate is 0.1%.

[0045] 4) Mix the complex solution obtained in the step 1) and the thermosensitive gel raw material obtained in the step 2) in equal volume to obtain an antidepressant preparation.

[0046] In the present invention, the poloxamer, sodium hyaluronate and water are mixed to obtain a thermosensitive gel raw material. In the present invention, the mass concentration of poloxamer in the thermosensitive gel raw material is 25%, and the mass concentration of sodium hyaluronate is 0.1%.

[0047] The present invention also provides the application of the antidepressant preparation described in the above technical solution in the preparation of a drug for treating chronic restraint stress depression. The present invention has no special limitation on the dosage form of the drug, etc., and those skilled in the art can perform conventional operations.

[0048] The present invention also provides the application of the antidepressant preparation described in the above technical solution in the preparation of a drug for treating chronic unpredictable mild stress depression. The present invention has no special limitation on the dosage form of the drug, etc., and those skilled in the art can perform conventional operations.

[0049] In order to further illustrate the present invention, the present invention will be described in detail below with reference to examples, but they should not be construed as limiting the protection scope of the present invention.

[0050] Example 1

[0051] Preparation of an antidepressant nasal administration preparation:

[0052] (1) Hydroxytyrosol (20%, w / v) and Angiopep-2 (5%, w / v) polypeptide were respectively dissolved in phosphate buffer solution (pH 7.4). After the Angiopep-2 polypeptide solution was completely dissolved, it was added to the hydroxytyrosol solution according to the molar ratio of hydroxytyrosol to Angiopep-2 polypeptide of 1:0.05. Nitrogen was filled, stirred at room temperature and protected from light for 12 h. After the reaction, it was filtered with 0.45 μm and the unreacted hydroxytyrosol was removed by using a 1 kD dialysis bag, and then freeze-dried to obtain a hydroxytyrosol-Angiopep-2 complex.

[0053] (2) The thermosensitive gel raw material contains 25% (w / v) poloxamer 188, 5% (w / v) poloxamer 407, 0.1% (w / v) sodium hyaluronate with a weight average molecular weight of 90 kDa, and the rest is deionized water.

[0054] (3) After quantifying the content of hydroxytyrosol in the hydroxytyrosol-Angiopep-2 complex in step 1 by LC-MS / MS, it was redissolved with deionized water to make the content of hydroxytyrosol in it 5% (w / v).

[0055] (4) The complex solution obtained in step (1) was mixed with the thermosensitive gel raw material obtained in step (2) in equal volume and mixed evenly to obtain an antidepressant nasal administration preparation.

[0056] Example 2

[0057] The difference from Example 1 is that the molar ratio of hydroxytyrosol to Angiopep-2 polypeptide is 1:0.1.

[0058] Example 3

[0059] The difference from Example 1 is that the molar ratio of hydroxytyrosol to Angiopep-2 polypeptide is 1:0.2.

[0060] Example 4

[0061] The difference from Example 1 is that the molar ratio of hydroxytyrosol to Angiopep-2 polypeptide is 1:0.1, and the weight-average molecular weight of sodium hyaluronate is 60 kDa.

[0062] Example 5

[0063] The difference from Example 1 is that the molar ratio of hydroxytyrosol to Angiopep-2 polypeptide is 1:0.1, and the weight-average molecular weight of sodium hyaluronate is 120 kDa.

[0064] Comparative Example 1

[0065] The difference from Example 1 is that the molar ratio of hydroxytyrosol to Angiopep-2 polypeptide is 1:0, that is, the hydroxytyrosol-Angiopep-2 complex is not prepared in step (1), and hydroxytyrosol is used instead of the hydroxytyrosol-Angiopep-2 complex in steps (3) and (4).

[0066] Comparative Example 2

[0067] The difference from Example 1 is that the molar ratio of hydroxytyrosol to Angiopep-2 polypeptide is 1:0.5.

[0068] Comparative Example 3

[0069] The difference from Example 1 is that the molar ratio of hydroxytyrosol to Angiopep-2 polypeptide is 1:0.1, and the weight-average molecular weight of sodium hyaluronate is 30 kDa.

[0070] Comparative Example 4

[0071] The difference from Example 1 is that the molar ratio of hydroxytyrosol to Angiopep-2 polypeptide is 1:0.1, and the weight-average molecular weight of sodium hyaluronate is 150 kDa.

[0072] Experiment 1: Investigation on the stability of hydroxytyrosol-Angiopep-2 thermosensitive gel

[0073] 1. Investigation on light stability

[0074] Take 6 portions each of the hydroxytyrosol raw material, each example and comparative example, corresponding to samples with 500 mg of hydroxytyrosol component respectively, and conduct strong light irradiation. The light intensity is set to 5500 Lux, 70 μW / cm 2 , and the temperature inside the chamber is 25 °C. Samples are taken before irradiation and on the 10th day of irradiation, and the remaining content of hydroxytyrosol in the samples is analyzed by HPLC. Each sample is measured in parallel three times, and the final result is the average of the three samples. As shown in Table 1, the content of hydroxytyrosol in the hydroxytyrosol raw material decreased by 8.25% after 10 days of light irradiation, and the decrease in the content of hydroxytyrosol in Examples 1-5 was not obvious, indicating that the hydroxytyrosol-Angiopep-2 thermosensitive gel has good light stability.

[0075] Table 1 Light stability experiment

[0076] Grouping 0 days 10 days Hydroxytyrosol raw material 100％ 91.75％ Example 1 100％ 99.7％ Example 2 100％ 99.8％ Example 3 100％ 99.8％ Example 4 100％ 99.8％ Example 5 100％ 99.8％ Comparative Example 1 100％ 95.8％ Comparative Example 2 100％ 99.8％ Comparative Example 3 100％ 97.6％ Comparative Example 4 100％ 99.7％

[0077] 2. Long-term storage test

[0078] Take 6 portions each of the hydroxytyrosol raw material, each example and comparative example, corresponding to samples with 500 mg of hydroxytyrosol component respectively, place them in the dark at room temperature, and sample and measure at the end of 0, 1, 3, and 6 months. The content of hydroxytyrosol in the samples is analyzed by HPLC. Each sample is measured in parallel three times, and the final result is the average of the three samples. Table 2 shows the changes in the content of hydroxytyrosol in each group. After stable storage for 6 months, the content in the hydroxytyrosol raw material group decreased by 28.55%, and the content of hydroxytyrosol in Examples 1, 2, 3, 4, and 5 decreased by only 2.82%, 2.53%, 2.35%, 2.65%, and 3.71% respectively. After 6 months of placement, the content of hydroxytyrosol in Comparative Example 1 decreased by 14.35%, and the content of hydroxytyrosol in the thermosensitive gel of Comparative Example 3 decreased by 9.19% after 6 months of placement, indicating that the hydroxytyrosol-Angiopep-2 thermosensitive gel prepared by the present invention can more significantly improve the chemical stability of hydroxytyrosol.

[0079] Table 2 Accelerated stability experiment

[0080] Grouping 0 days 1 month 3 months 6 months Hydroxytyrosol raw material 100％ 88.43％ 75.63％ 71.45％ Example 1 100％ 99.55％ 98.27％ 97.18％ Example 2 100％ 99.62％ 98.32％ 97.47％ Example 3 100％ 99.74％ 98.05％ 97.65％ Example 4 100％ 99.79％ 97.61％ 97.35％ Example 5 100％ 99.49％ 97.58％ 96.29％ Comparative Example 1 100％ 95.58％ 90.67％ 85.65％ Comparative Example 2 100％ 99.56％ 98.02％ 97.32％ Comparative Example 3 100％ 96.94％ 92.55％ 90.18％ Comparative Example 4 100％ 99.76％ 98.59％ 97.32％

[0081] Experiment 2: Investigation on the in vitro release of hydroxytyrosol-Angiopep-2 thermosensitive gel

[0082] Preparation of artificial nasal fluid: Artificial nasal fluid was prepared according to the electrolyte composition of nasal fluid. 7.91 g of sodium chloride, 2.56 g of sodium bicarbonate, 3.68 g of potassium chloride and 0.51 g of calcium chloride were accurately weighed, dissolved in deionized water, and fixed volume was made up to 1000 mL volumetric flask, and the pH was adjusted to 6.8 with 0.1 mol / L sodium hydroxide solution. The in vitro release of hydroxytyrosol-Angiopep-2 thermosensitive gel was investigated by Franz diffusion cell method. The activated 10 kD dialysis bag was cut into semipermeable membrane and fixed between the diffusion cell and the receiving cell. In the blank group, 50 mg / mL hydroxytyrosol solution was selected. In the experimental group, 1 mL of the corresponding hydroxytyrosol-Angiopep-2 thermosensitive gel was taken and added into the diffusion cell respectively, and it was evenly distributed on the surface of the semipermeable membrane. It was placed in a water bath at 35 °C for 30 min to fully gel the thermosensitive gel. 7 mL of artificial nasal fluid was added to the receiving cell, the temperature of the diffusion cell was maintained at (35 ± 0.5) °C, and the magnetic stirring speed was 200 r / min. 1 mL of samples was taken at 0.25, 0.5, 0.75, 1, 2, 4, 8, 12, 24, 48 h respectively, and 1 mL of isothermal artificial nasal fluid was supplemented at the same time. The obtained sample solution was filtered through a 0.45 μm microporous membrane, and the total amount of hydroxytyrosol in the sample was determined by LC-MS / MS method, and the cumulative release (Q n ) was calculated. Q n The calculation formula is as follows. With time (t) as the abscissa (X) and Q n as the ordinate (Y), the release curve was plotted.

[0083]

[0084] Note: Q 0 is the content of hydroxytyrosol; V 0 is the total volume of the release medium (7 mL); V is the volume at each sampling (1 mL); C n is the concentration of the receiving solution at different sampling points; C i is the concentration of the sampling solution at each sampling point

[0085] (μg / mL); i and n are the number of samplings.

[0086] Figure 1 As can be seen from Table 2 and Table 3, the release rate of hydroxytyrosol was stable in Examples 1-5, Comparative Example 2 and Comparative Example 4, and the cumulative release rates at 12 h were 89.64 ± 5.60%, 90.36 ± 9.54%, 84.85 ± 3.67%, 79.35 ± 7.51%, 72.38 ± 6.84%, 68.58 ± 3.68%, 65.38 ± 3.74% respectively, while the release rates of hydroxytyrosol solution, Comparative Example 1 and Comparative Example 3 were relatively fast. The results showed that the thermosensitive gels of Examples 1-3 had good drug release characteristics.

[0087] Table 3 In vitro release test

[0088]

[0089] Experiment 3: Pharmacokinetics study of thermosensitive gel in brain tissue:

[0090] 360 8-week-old rats were randomly divided into 10 groups. The control group was given 500 μL / kg of hydroxytyrosol solution (25 mg / mL), and the experimental groups were given Examples 1-5 and Comparative Examples 1-4 (each containing 25 mg / mL of hydroxytyrosol) at 500 μL / kg, respectively. The rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (40 mg / kg), placed supine, with their heads and limbs fixed. The neck was opened, the esophagus was exposed and ligated to prevent the loss of the liquid medicine. The thermosensitive gel was administered nasally using a microsyringe. Rats in each group were sacrificed at 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h, respectively, to obtain brain tissues and the hippocampus was isolated and stored at -80 °C for later use.

[0091] Chromatographic conditions: Chromatographic column: Accucorea Q (2.1×50 mm, 2.6 μm), mobile phase: 0.1% formic acid in water (A): acetonitrile (B), gradient elution, column temperature: 40 °C, injection volume: 5 μL.

[0092] Mass spectrometry conditions: AB SCIEX 5500 triple quadrupole tandem mass spectrometry system, ESI source, curtain gas pressure: -35 psi, collision gas: -8 psi, ionization voltage: -5500 psi, ion source temperature: 550 °C, nebulizing gas: -55 psi, auxiliary heater: -55 psi, multiple reaction monitoring (MRM) mode. The detection conditions are as follows:

[0093] Table 4 MS conditions of hydroxytyrosol

[0094]

[0095]

[0096] Treatment of brain tissue samples: The tissue was melted on ice, weighed on an analytical balance, homogenized with normal saline (2 mL / g) for 1 min at 4 °C, centrifuged at 4000 r / min for 15 min at 4 °C. 100 μL of the supernatant was taken and added to 500 μL of internal standard acetonitrile solution (TBTM 20 ng / mL), vortexed for 2 min, centrifuged at 13000 r / min for 5 min at 4 °C, and the supernatant was taken for injection of 5 μL for detection.

[0097] The main pharmacokinetic parameters were calculated using Phoenix WinNonlin 8.1, as shown in Table 5. The pharmacokinetic results showed that after intranasal administration of the thermosensitive gels of Examples 1, 2, 3, and 4, the concentration in the hippocampus reached the highest after 2 h, which was better than the peak time (t max = 0.5 h) of hydroxytyrosol solution after intranasal administration, and the half-life of hydroxytyrosol in the hippocampus was significantly prolonged. The C max of the hydroxytyrosol solution group and Comparative Example 1 group was only 30.36 ± 2.52 ng / mL and 25.32 ± 1.47 ng / mL, while the C max of the groups of Examples 1, 2, 3, 4, and 5 were 96.25 ± 5.65 ng / mL, 89.35 ± 6.52 ng / mL, 68.37 ± 7.63 ng / mL, 76.38 ± 9.24 ng / mL, and 52.87 ± 6.01 ng / mL respectively, indicating that the hydroxytyrosol-Angiopep-2 thermosensitive gel had good brain targeting.

[0098] Table 5 Main pharmacokinetic parameters (n = 6)

[0099] <![CDATA[T max (h)]]> <![CDATA[C max (ng / mL)]]> <![CDATA[t 1 / 2 (h)]]> <![CDATA[AUC (0-t) (ng / mL*h)]]> <![CDATA[MRT (0-t) (h)]]> Hydroxytyrosol solution 0.5 30.36±2.52 0.75±0.25 155.47±26.35 0.87±0.24 Example 1 2 96.25±5.65 3.71±0.87 263.54±45.25 4.65±0.36 Example 2 2 89.35±6.52 3.96±1.02 286.34±33.25 4.89±0.57 Example 3 2 68.37±7.63 4.22±0.58 242.87±36.01 4.15±0.41 Example 4 2 76.38±9.24 3.15±0.65 236.55±28.41 3.85±0.52 Example 5 2 52.87±6.01 4.59±1.04 219.33±30.11 5.54±0.94 Comparative Example 1 1 25.32±1.47 0.69±0.22 169.64±24.11 0.72±0.25 Comparative Example 2 4 49.33±9.11 5.96±0.85 183.65±24.36 6.87±0.64 Comparative Example 3 1 79.56±8.54 1.06±0.36 241.88±29.17 1.28±0.37 Comparative Example 4 4 31.36±5.96 6.84±1.27 159.64±22.97 8.07±1.39

[0100] Experiment 4: Therapeutic effect of hydroxytyrosol-Angiopep-2 thermosensitive gel on the chronic restraint stress-induced mouse model of depression:

[0101] Eighty 6-week-old male ICR mice were purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. The temperature in the animal room was controlled at 20°C - 25°C, the relative humidity was 45% - 55%, and the light-dark cycle was 12 h / 12 h. The mice had free access to water and food. After one week of environmental adaptation, the animals were randomly divided into 8 groups with 10 mice in each group: normal group, model group, fluoxetine group (10 mg / kg / d), hydroxytyrosol solution group (25 mg / mL), Comparative Example 1 group, Example 1 group, Example 2 group, and Example 3 group. Except for the normal group, the mice were restricted in a well-ventilated conical-bottom centrifuge tube (50 mL, Corning, USA) for 6 h (9:00 - 15:00) every day for 21 consecutive days. On the 22nd day, behavioral experiments such as tail suspension, forced swimming, and sucrose consumption were performed to detect the successful establishment of the chronic restraint stress-induced depression model.

[0102] Acute tail suspension experiment: About 1 cm from the tip of the mouse's tail, the mouse was suspended 50 cm above the bottom of the tail suspension experimental box using transparent tape. After 2 min of adaptation, the observer measured the stationary time of the mouse in the suspended tail state using a timer during the subsequent 4 min. The experiment was carried out in a relatively quiet environment, and the tail suspension device should be wiped after each mouse's test to eliminate the influence of odor on other mice. An increase in the immobility duration indicated depressive-like behavior.

[0103] Forced swimming test: 10 cm of pure water (24°C ± 1°C) was added to a transparent cylinder with a diameter of 10 cm and a height of 20 cm. During the swimming test, the animals were unable to touch the bottom of the tank with their feet or tails. After 2 min of adaptation, the immobility time of the mice in the water was recorded within 4 min. Immobility was defined as an animal floating in the water without any movement except for the movements necessary to keep the nose above the water surface. An increase in the immobility duration indicates depressive-like behavior.

[0104] Sucrose preference test: Mice were allowed to freely choose between two drinking solutions, water or a sucrose solution. Generally, rodents show a distinct preference for sweet water, while depressed animals show less interest. On the 1st day before the test, the animals were accustomed to a 1% (w / v) sucrose solution by placing two bottles of 1% sucrose solution in each cage. On the 2nd day, the two bottles of sucrose solution were replaced with pure water. After adaptation, on the 3rd day, each mouse was placed in a separate cage and had free access to two identical bottles, one filled with 1% sucrose solution and the other filled with pure water. On the 4th day, the consumption of sucrose and water was recorded, and the sucrose preference was calculated as follows: Sucrose preference (%) = sucrose solution consumption / (pure water consumption + sucrose solution consumption) × 100%.

[0105] After successful model establishment, drug administration was started. The fluoxetine group was intragastrically administered a fluoxetine suspension at a dose of 10 mg / kg / d, and the intragastric administration volume was 0.1 mL / 10 g body weight. The hydroxytyrosol solution group was nasally administered a hydroxytyrosol solution at a dose of 25 μL / 10 g body weight. The control group 1, example group 1, example group 2, and example group 3 were nasally administered a thermosensitive gel (containing 25 mg / mL of hydroxytyrosol) at a dose of 25 μL / 10 g body weight every day. The model group was nasally administered 25 μL / 10 g of blank thermosensitive gel (i.e., without hydroxytyrosol - Angiopep - 2) every day, and the normal group was not treated with anything. Continuous drug administration was carried out for 21 d. On the 22nd day, behavioral experiments such as tail suspension, forced swimming, and sucrose preference were performed, and data statistics were carried out to analyze the antidepressant effects of the drugs in each group.

[0106] The results of the behavioral experiments showed ( Figure 2 ), that the thermosensitive gels containing different doses of hydroxytyrosol - Angiopep - 2 could significantly improve the immobility time of the restraint - stressed mice in the tail suspension state and in water, as well as their preference for sucrose solution, indicating that the hydroxytyrosol - Angiopep - 2 thermosensitive gel of the present invention can better exert antidepressant effects.

[0107] Experiment 5: Therapeutic effect of hydroxytyrosol - Angiopep - 2 thermosensitive gel on a rat model of depression induced by chronic unpredictable mild stress:

[0108] Sixty 6-week-old male SD rats were purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. The animal breeding environment was the same as that in Example 7. After one week of environmental adaptation, except for the normal group, other groups were subjected to chronic unpredictable mild stress (CUMS) from week 0 to week 13, that is, CUMS was applied by randomly and irregularly applying seven possible stresses every day. The stimulation methods were as follows: (1) water deprivation for 12 h, (2) the cage was tilted at 45° for 12 h, (3) wet bedding (250 mL of water per cage) for 24 h, (4) physical restraint for 4 h (9:00 - 13:00), (5) cold water swimming (4 °C) for 10 min, (6) fasting for 24 h, (7) light-dark cycle inversion for 24 h. Behavioral experiments such as forced swimming and sucrose consumption were conducted at week 6, and 48 successfully modeled depressive rats were selected for grouping. The animals were randomly divided into 8 groups, with 6 rats in each group: normal group, model group, fluoxetine group (10 mg / kg / d), hydroxytyrosol solution group (25 mg / mL), Comparative Example 1 group, Example 1 group, Example 2 group, and Example 3 group. The fluoxetine group was intragastrically administered a fluoxetine suspension of 10 mg / kg / d, and the intragastric administration volume was 1 mL / 100 g body weight. The hydroxytyrosol solution group was administered 500 μL / kg body weight of hydroxytyrosol solution via nasal administration. The Comparative Example 1 group, Example 1 group, Example 2 group, and Example 3 group were administered 500 μL / kg body weight of thermosensitive gel (containing 25 mg / mL of hydroxytyrosol) via nasal administration every day for 6 consecutive weeks. Behavioral experiments were conducted at week 7.

[0109] Forced swimming experiment: A 50-cm deep transparent water bucket was selected for the rat forced swimming bucket, and pure water with a depth of 25 - 30 cm was added. Other operations were the same as those in the forced swimming experiment in Test 4. The sucrose consumption experiment had the same operation steps as those in Test 4. After the behavioral tests were completed, the rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (40 mg / kg). Blood was taken from the abdominal aorta to separate serum, the rats were sacrificed, and brain tissues were taken to detect the concentration differences of dopamine, 5-hydroxytryptamine, and norepinephrine in the serum and brain tissues of rats in each group, and to evaluate the antidepressant activities of the drugs in each group.

[0110] Treatment of serum with brain tissue homogenate: Take 100 μL of serum sample and place it in a 1.5 mL centrifuge tube. Add 500 μL of internal standard acetonitrile solution (TBTM 20 ng / mL), vortex for 2 min, centrifuge at 13000 r / min for 5 min at 4 °C. Take the supernatant and inject 5 μL for detection. For the preparation of brain tissue homogenate, add cold PBS with a volume ratio of 1:6 (w / v, mg / μL) to rat brain tissue, and homogenize the mixture twice by shaking at 30 Hz for 90 s. Centrifuge at 13000 r / min for 5 min at 4 °C. Take 900 μL of ice-cold acetonitrile and add it to 100 μL of the supernatant for deproteinization, vortex for 5 min, and then centrifuge at 13000 r / min for 10 min at 4 °C. After centrifugation, add 100 μL of the aliquot to 50 μL of acetonitrile and 50 μL of TBTM (20 ng / mL) internal standard solution, then vortex for 5 min, centrifuge at 13000 r / min for 5 min at 4 °C, and take 5 μL of the supernatant for analysis.

[0111] Chromatographic conditions: Accucore C18 chromatographic column (100 mm × 2.1 mm, 2.6 μm, Thermo, USA), column temperature 40 °C. Mobile phase A: aqueous solution containing 10% acetonitrile and 0.1% formic acid, mobile phase B: acetonitrile / water (1:1, v / v) containing 0.1% formic acid, flow rate 0.4 mL / min. Gradient elution: 0 - 1 min, 20 - 100% B; 1 - 7 min, 100% B; 7 - 7.5 min, 100 - 20% B; 7.5 - 11 min, 20% B.

[0112] Mass spectrometry conditions: SCIEX Exion LC-TQ5500 with electrospray ionization (ESI) source, positive ion mode. Ion source temperature 500 °C, ion source voltage 5000 V, collision gas 6 psi, curtain gas 30 psi, nebulizer gas and auxiliary gas 50 psi. The multiple reaction monitoring (MRM) conditions for dopamine, serotonin, and norepinephrine are shown in Table 6. Data acquisition was performed using Phoenix WinNonlin 8.1 (Certara, USA).

[0113] Table 6 Multiple ion monitoring mass spectrometry conditions

[0114]

[0115] The results of the forced swimming and sucrose consumption experiments showed ( Figure 3 ) that the antidepressant effects of the groups of Examples 1, 2, and 3 were significant and superior to those of the group of Comparative Example 1 and the hydroxytyrosol solution group. Figure 4After continuous administration for 6 weeks, the rats were sacrificed, and serum and brain tissues were collected. The levels of dopamine, serotonin, and norepinephrine in serum and brain tissues were detected by LC / MS. The results showed that the levels of the above three neurotransmitters in the groups of Examples 1, 2, and 3 were higher than those in the model group. However, there was no significant improvement in the neurotransmitter levels in serum and brain tissues in the group of Comparative Example 1 and the hydroxytyrosol solution group. The results further demonstrated that the hydroxytyrosol-Angiopep-2 thermosensitive gel exerted excellent antidepressant efficacy by virtue of its excellent brain targeting function and had good clinical application prospects.

[0116] Although the above embodiments have made a detailed description of the present invention, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antidepressant preparation for nasal mucosa administration, characterized in that, it is prepared from the following raw materials: hydroxytyrosol-Angiopep-2 complex, poloxamer, sodium hyaluronate and water; the hydroxytyrosol-Angiopep-2 complex is prepared from hydroxytyrosol and Angiopep-2 with a molar ratio of 1:0.05 - 0.2; the weight-average molecular weight of the sodium hyaluronate is 60 - 120 kDa; the poloxamer includes poloxamer 188 and poloxamer 407, and the mass ratio of poloxamer 188 to poloxamer 407 is 25:5; the preparation method of the hydroxytyrosol-Angiopep-2 complex includes: dissolving hydroxytyrosol and Angiopep-2 polypeptide in phosphate buffer respectively, and then mixing to obtain a reaction solution to be reacted; stirring and reacting the reaction solution to be reacted for 12 h under a nitrogen atmosphere at 20 - 30 °C, and then filtering and dialyzing in sequence to obtain the hydroxytyrosol-Angiopep-2 complex.

2. The antidepressant preparation according to claim 1, characterized in that, the hydroxytyrosol-Angiopep-2 complex is prepared from hydroxytyrosol and Angiopep-2 with a molar ratio of 1:0.

1.

3. The antidepressant preparation according to claim 1, characterized in that, the pH value of the phosphate buffer is 7.4; the pore size of the filter membrane used for filtration is 0.45 µm; the dialysis is carried out using a 1 kD dialysis bag, and the dialysis time is 12 h.

4. The antidepressant preparation according to claim 1, characterized in that, the weight-average molecular weight of the sodium hyaluronate is 90 kDa.

5. The antidepressant preparation according to claim 1, characterized in that, the water is deionized water.

6. A preparation method of the antidepressant preparation according to any one of claims 1 - 5, characterized in that, it includes the following steps: 1) Mix the hydroxytyrosol-Angiopep-2 complex with water to obtain a complex solution, and the mass concentration of hydroxytyrosol in the complex solution is 5%; 2) Mix the poloxamer, sodium hyaluronate and water to obtain a thermosensitive gel raw material; the mass concentration of poloxamer in the thermosensitive gel raw material is 30%, and the mass concentration of sodium hyaluronate is 0.1%; 3) Mix the complex solution obtained in step 1) and the thermosensitive gel raw material obtained in step 2) in equal volume to obtain the antidepressant preparation.

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