Preparation method for brain-targeting agomelatine nasal spray micelle and use thereof

Abstract

Disclosed in the present invention are a preparation method for a brain-targeting agomelatine nasal spray micelle and use thereof, which belong to the technical field of pharmaceutical formulations. According to the present invention, an injectable pharmaceutical excipient polyethylene glycol (15)-hydroxystearate (HS15) is taken as a carrier material, HS15 and agomelatine are dissolved in an organic solvent, and the resulting solution is subjected to rotary evaporation under reduced pressure to form a uniform film, which, upon hydration, forms an agomelatine micelle solution capable of being sprayed nasally for treating depression and insomnia. According to the present invention, the problem of low oral bioavailability of agomelatine due to poor solubility, serious first pass effect of hepar, difficulty in penetrating a blood-brain barrier, etc., is solved. The micelle structure constructed in the present invention is quickly delivered into the brain after nasal administration, and it is stable in cerebrospinal fluid and can achieve slow release of the drug, thereby improving the intracerebral bioavailability of the drug and achieving high brain targeting property and low systemic toxic and side effects. The micelle structure has good clinical application value and market prospects.

Description

Preparation method and application of brain-targeted agomelatine nasal spray micelles Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a preparation method and application of brain-targeted agomelatine nasal spray micelles. Background Art

[0002] Agomelatine is an antidepressant that can restore the disturbed biological rhythm to normal by stimulating the melatonin MT1 and MT2 receptors, thereby exerting an antidepressant effect. 2C Neutral antagonists of the receptor, with their binding sites concentrated in the hippocampus, amygdala and prefrontal cortex of the brain, can increase the release of norepinephrine and dopamine in the prefrontal cortex, and this antagonistic effect can prevent the inhibition of melatonin synthesis by light stimulation. At present, the commercially available preparation of agomelatine is an oral tablet, but due to poor solubility, severe first-pass effect in the liver and difficulty in penetrating the blood-brain barrier, its bioavailability in the brain is low, only 3%-5%, which increases the difficulty of agomelatine in treating depression and insomnia. Traditional agomelatine oral tablets show adverse reactions of nausea and vomiting in clinical use, and have potential liver toxicity, which greatly limits its application. Therefore, the research and development of a new administration method and new dosage form is of great clinical significance.

[0003] As a non-invasive drug delivery method, nasal administration facilitates self-administration and dosage adjustment by patients, and has good patient compliance. Nasal administration has also been shown to bypass the blood-brain barrier and directly enter the brain through the nasal olfactory region without damaging it. This allows for rapid onset of action, avoids enzymatic and microbial degradation of the drug in the gastrointestinal tract, and avoids first-pass effects in the liver. This helps improve brain bioavailability and reduces peripherally induced adverse reactions, opening up a promising therapeutic approach for the treatment of neurological diseases. Currently, the olfactory-brain pathway is the most direct route to the brain via the nasal mucosa. After drug absorption through the nasal mucosa and uptake by the olfactory nerves, it can be transported via axons to the olfactory bulb and further to the olfactory brain. This pathway is an effective route for the transfer of hydrophilic agents. Therefore, developing agomelatine into a nasal spray formulation that can be targeted to the brain via the olfactory-brain pathway can improve agomelatine's brain bioavailability, reduce systemic exposure, and achieve rapid onset of action after administration.

[0004] Summary of the Invention

[0005] To address the low bioavailability in the brain caused by poor solubility, severe hepatic first-pass effect, and difficulty penetrating the blood-brain barrier of currently available agomelatine oral tablets, the present invention aims to provide a method for preparing and applying brain-targeted agomelatine nasal spray micelles. Given the unique physicochemical properties of agomelatine, the present invention uses HS15, which promotes nasal absorption and solubilizes the drug, as a carrier material to develop a low-cost, large-scale, industrially produced brain-targeted agomelatine nasal spray micelle. After nasal administration, these micelles can be rapidly delivered to the brain and increase the concentration of agomelatine in the brain, achieving brain targeting and low systemic toxicity. This effectively enhances the therapeutic efficacy of agomelatine for depression and insomnia while reducing the drug's side effects, resulting in promising clinical application value and market prospects.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing brain-targeted agomelatine nasal spray micelles. The method comprises the following steps: dissolving HS15 and agomelatine in an organic solvent, removing the organic solvent by rotary evaporation until a uniform film is formed, and adding a water-soluble solvent for hydration to form agomelatine-loaded micelles.

[0008] Based on the above technical solution, the preparation method further comprises the following steps:

[0009] (1) Accurately weigh 60-100 mg of HS15 and 4-40 mg of agomelatine, add them to 4-10 ml of organic solvent, dissolve them, and stir them at 4-30°C to obtain a mixed solution;

[0010] (2) The mixed solution obtained in step (1) was transferred to an eggplant-shaped flask, and the organic solvent was removed by rotary evaporation under reduced pressure for 5 to 20 minutes in a water bath at 25 to 50° C. to form a uniform thin film, and 0.2 to 20 ml of a water-soluble solvent was added and hydrated for 3 to 30 minutes to obtain a solution of agomelatine-loaded micelles;

[0011] (3) adding 0.02% to 0.2% (w / v) of a preservative to the solution of agomelatine-loaded micelles obtained in step (2), mixing the mixture evenly, and then adding a pH regulator dropwise to adjust the pH value to 6.8 to 7.4, thereby obtaining brain-targeted agomelatine nasal spray micelles.

[0012] Based on the above technical solution, further, the organic solvent described in step (1) is one or a mixed solvent of two or more of methanol, ethanol, chloroform, and acetone, preferably ethanol.

[0013] Based on the above technical solution, further, the stirring conditions in step (1) are a stirring speed of 200 to 1200 rpm and a stirring time of 5 to 60 min.

[0014] Based on the above technical solution, further, the water-soluble solvent described in step (2) is one of physiological saline or PBS.

[0015] Based on the above technical solution, further, the preservative described in step (3) is benzalkonium bromide, hydroxyphenyl esters, benzoic acid, sorbic acid, chlorhexidine acetate, o-phenylphenol, sodium thiosulfate, preferably benzalkonium bromide.

[0016] Based on the above technical solution, further, the pH adjuster described in step (3) includes a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with a molar ratio of 1:1.3.

[0017] Based on the above technical solution, further, the composition ratio of agomelatine in the brain-targeted agomelatine nasal spray micelles is 4wt%-50wt%, and the composition ratio of HS15 is 50wt%-96wt%.

[0018] Based on the above technical solution, after preparing the agomelatine-loaded micelle solution, an isotonicity regulator and a stabilizer are further added. The isotonicity regulator is one or a combination of two or more selected from the group consisting of sodium chloride, mannitol, sorbitol, glycerol, glucose, and xylitol, with an addition amount of 0.5% to 1% (w / v). The stabilizer is one or a combination of two or more selected from the group consisting of sodium sulfite, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, glycine, citric acid, tartaric acid, ascorbic acid, and ethylenediaminetetraacetic acid, with an addition amount of 0.1% to 1% (w / v).

[0019] Another aspect of the present invention provides brain-targeted agomelatine nasal spray micelles obtained by the above preparation method.

[0020] Another aspect of the present invention provides a brain-targeted agomelatine nasal spray micelle freeze-dried powder. A freeze-dried protective agent is added to the above-mentioned brain-targeted agomelatine nasal spray micelle, and the mixture is freeze-dried to obtain the brain-targeted agomelatine nasal spray micelle freeze-dried powder.

[0021] Based on the above technical solution, further, the lyoprotectant is one or a combination of two or more of glucose, mannitol, glycine, maltose, trehalose, sucrose, polyethylene glycol 2000, polyethylene glycol 4000 or polyethylene glycol 6000, and the added amount of the lyoprotectant is 1% to 5% (w / v).

[0022] Based on the above technical solution, further, the brain-targeted agomelatine nasal spray micellar lyophilized powder is added with a diluent as needed before use and reconstituted into a micellar solution, and the diluent is one of physiological saline or buffer.

[0023] The present invention also provides use of the brain-targeted agomelatine nasal spray micelles and the brain-targeted agomelatine nasal spray micelle freeze-dried powder in preparing nasal spray medicines for treating depression and insomnia.

[0024] Compared with existing preparations, the beneficial effects of the present invention are as follows:

[0025] (1) The present invention uses HS15 as the carrier material, and the micelles formed have small particle size, large encapsulation efficiency and high stability.

[0026] (2) The micelle structure constructed by the present invention can achieve slow release of drugs and is stable and completely released in the cerebrospinal fluid. After nasal administration, it is quickly delivered to the brain and has good brain targeting, thereby improving the bioavailability of drugs and is suitable for poorly soluble drugs that exert therapeutic effects in the central nervous system.

[0027] (3) The present invention overcomes the problems of low oral bioavailability of agomelatine due to poor solubility, severe first-pass effect in the liver, and difficulty in penetrating the blood-brain barrier.

[0028] (4) The brain-targeted agomelatine nasal spray micelles prepared by the present invention are easy to use, simple to operate, and have readily available raw materials and low cost, and have great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 is a transmission electron micrograph of agomelatine-loaded micelles;

[0030] FIG2 is a characterization of the particle size distribution and surface potential of agomelatine-loaded micelles by dynamic light scattering;

[0031] FIG3 shows the in vitro release curve of agomelatine-loaded micelles in nasal fluid from 0 to 10 h;

[0032] FIG4 is an in vitro release curve of agomelatine-loaded micelles in cerebrospinal fluid from 0 to 10 h;

[0033] Figure 5 shows the stability of agomelatine-loaded micelles in PBS, normal saline, and cerebrospinal fluid;

[0034] FIG6 is a characterization of the particle size distribution of agomelatine-loaded micelles at 4° C. for 7 days using dynamic light scattering;

[0035] FIG7 is a characterization of the particle size distribution of agomelatine-loaded micelles at 4° C. for 14 days using dynamic light scattering;

[0036] FIG8 is a picture of lyophilized powder of agomelatine-loaded micelles;

[0037] FIG9 is a characterization of the particle size distribution of agomelatine-loaded micelles before and after spraying by dynamic light scattering;

[0038] FIG10 is a quantitative fluorescence imaging method for evaluating the targeting effect of HS15-carrier micelles in rat brain tissue;

[0039] FIG11 is a quantitative fluorescence imaging method for evaluating the targeting effect of HS15-based micelles in major rat tissues. DETAILED DESCRIPTION

[0040] The present invention will be further described below in conjunction with specific examples, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or replacements made to the inventive method, steps, and conditions are intended to fall within the scope of the present invention. Unless otherwise specified, the experiments and techniques, reagents, and materials used in the examples are all commercially available.

[0041] Example 1 Preparation of agomelatine-loaded micelles

[0042] 4 mg of agomelatine and 80 mg of 15-hydroxystearate polyethylene glycol ester (HS15) were weighed and dissolved in 5 ml of anhydrous ethanol. The mixture was slowly stirred at room temperature until uniformly mixed. The mixed solution was transferred to a 50 ml eggplant-shaped flask and rotary evaporated under reduced pressure. The anhydrous ethanol was removed in a 40°C water bath to form a uniform agomelatine-HS15 film. 2 ml of 0.1 M PBS was added and hydrated for 5 minutes to obtain a solution of agomelatine micelles.

[0043] Example 2 Characterization of agomelatine-loaded micelles

[0044] Transmission electron microscopy and dynamic light scattering were used to observe the structure, morphology, size, and potential of the agomelatine-loaded micelles. The results are shown in Figures 1 and 2. The results show that the agomelatine-loaded micelles prepared in Example 1 have an ultrasmall micelle size of approximately (14.22±0.73) nm, which is consistent with the nanomicelle size observed using transmission electron microscopy. The particles are uniformly dispersed, with a polydispersity coefficient of 0.153±0.042, indicating good monodispersity. The agomelatine-loaded micelles have a relatively stable zeta potential of approximately (-2.76±0.42) mV.

[0045] Example 3 Drug loading of agomelatine loaded micelles

[0046] The drug loading (DL) and encapsulation efficiency (EE) of agomelatine-loaded micelles were determined using ultracentrifugation. Accurately measure 1 ml of agomelatine micelle solution, dilute to volume with ethanol, and disrupt the drug-loaded micelles by sonication at 400W for 15 minutes to release all entrapped drug. The absorbance at 303 nm was measured using a UV spectrophotometer to calculate the total amount of agomelatine. Separately, 1 ml of the agomelatine-loaded micelle solution was placed in an ultrafiltration cup and centrifuged at 3000 × g for 20 minutes to retain the drug-loaded micelles. The absorbance of the filtrate was measured using a UV spectrophotometer to calculate the amount of unentrapped agomelatine.

[0047] The results showed that the encapsulation efficiency and drug loading of agomelatine-loaded micelles were (95.95%±2.68)% and (4.50±0.88)% (n=3), respectively, indicating that the micelles obtained by this method had a high loading capacity for agomelatine.

[0048] Example 4 In vitro release behavior of agomelatine-loaded micelles

[0049] The in vitro release of agomelatine-loaded micelles was investigated using dynamic dialysis. Two milliliters of agomelatine-loaded micelle solution were precisely measured and placed in a 3.5 kDa dialysis bag. The bag was then tied tightly at both ends and placed in 10 milliliters of artificial nasal fluid (composed of 13.6% (w / v) potassium dihydrogen phosphate and 0.1 mol / L sodium hydroxide, pH 6.8) containing 1% w / v DMSO and artificial cerebrospinal fluid (aCSF, 0.72% (w / v) sodium chloride, 0.02% (w / v) potassium chloride, 0.05% (w / v) magnesium sulfate, 0.22% (w / v) sodium bicarbonate, 0.02% (w / v) calcium chloride, 0.40% (w / v) D-glucose, pH 7.4, sterile). To eliminate factors influencing adsorption, an agomelatine solution was used as a control. The mixture was shaken at 200 rpm / min at 37°C for 10 hours. At 0, 2, 4, 6, 8, and 10 hours, 2 ml of release medium was aspirated and promptly replenished with an equal amount of the same release medium at the same temperature. The agomelatine content in the release medium at different time points was determined by UV-visible spectrophotometry, and the cumulative release rate was calculated. The release curves are shown in Figures 3 and 4. The agomelatine solution reached maximum cumulative release after 2 hours. The cumulative release rate of agomelatine-loaded micelles in the cerebrospinal fluid was 100% after 6 hours, but the cumulative release rate of agomelatine-loaded micelles in nasal fluid was only approximately 80%.

[0050] The results showed that the agomelatine-loaded micelles prepared in Example 1 had an obvious sustained-release effect without a burst-release effect, and the environment of the cerebrospinal fluid was more conducive to the release of agomelatine from the HS15 micelle structure.

[0051] Example 5 Dilution stability of agomelatine-loaded micelles

[0052] The agomelatine-loaded micelles prepared in Example 1 were diluted with 100 ml of PBS, physiological saline, and cerebrospinal fluid, respectively. Dynamic light scattering was used to analyze the particle size after 1, 2, 6, 10, 14, and 24 hours. The particle size measurement results are shown in Figure 5 and Table 1.

[0053] Table 1 Stability of agomelatine-loaded micelles in normal saline and cerebrospinal fluid

[0054] The results showed that the agomelatine-loaded micelles prepared in Example 1 remained stable after dilution in PBS, normal saline and cerebrospinal fluid, indicating that the constructed micelle system can be stable during storage and release drugs in the brain in the form of micelles, thereby improving the scope and convenience of clinical use.

[0055] Example 6 Investigation on the storage stability of agomelatine-loaded micelle solution at 4°C

[0056] The agomelatine-loaded micelle solution prepared in Example 1 was stored at 4°C for 14 days, and the particle size was analyzed by dynamic light scattering technology at 7 days and 14 days, respectively. The measurement results are shown in Figures 6-7. The particle sizes at 7 days and 14 days were 13.54±0.23 nm and 14.34±0.43 nm, respectively. The particle size remained basically unchanged, the particles were evenly dispersed, and no aggregation occurred.

[0057] The results showed that the agomelatine-loaded micelle solution prepared in Example 1 could be directly stored at 4° C., so the agomelatine-loaded micelle solution could be directly used.

[0058] Example 7 Stability of agomelatine-loaded micelle freeze-dried powder

[0059] A lyoprotectant consisting of 2% (w / v) glucose and 3% (w / v) mannitol was added to the agomelatine-loaded micelle solution prepared in Example 1. The freeze-drying procedure was as follows: pre-freezing: 1 hour at -50°C; sublimation drying: 1 hour at -40°C; 1 hour at -20°C; 1 hour at -10°C; and desorption drying: 6 hours at 0°C. After freeze-drying, the solution was allowed to stand at room temperature. The freeze-dried product exhibited a uniform color, dense pores, minimal volume change before and after freeze-drying, and essentially unchanged properties, forming a spongy, blocky structure. The freeze-dried agomelatine micelles were then reconstituted in 2 ml of PBS (the concentration of the agomelatine-loaded micelles was 42 mg / ml). The reconstituted solution was placed at 4°C and room temperature (25°C) for 24 h, and the particle size of the reconstituted lyophilized preparation was measured by dynamic light scattering technology at 0, 0.5, 1, 2, 4, 6, and 24 h, respectively. The measurement results are shown in Table 2.

[0060] Table 2 Stability of agomelatine-loaded micelle freeze-dried powder

[0061] The results showed that the agomelatine-loaded micelle freeze-dried preparation prepared in Example 1 had good stability within 24 hours after reconstitution. To facilitate product storage, the agomelatine-loaded micelles can be stored in a freeze-dried form and reconstituted before use.

[0062] Example 8 Stability of agomelatine-loaded micelle solution before and after spraying

[0063] The agomelatine-loaded polymicelle solution prepared in Example 1 was added to a nasal spray device, and the particle size after overspray was measured using dynamic light scattering technology. The measurement results are shown in Figure 9. The particle sizes before and after overspray were 14.22±0.73 nm and 17.97±0.33 nm, respectively.

[0064] The results showed that the shear force of the spray device had no effect on the formulation properties of the agomelatine-loaded micelles prepared in Example 1. The particle size of the micelles remained stable before and after spraying, and the particle size distribution was good, providing feasibility for clinical use. The solution of agomelatine-loaded micelles can be used for administration through a nasal spray device.

[0065] Example 9 Brain targeting ability of micelles with HS15 as carrier

[0066] To preliminarily investigate the brain-targeting ability of the brain-targeted agomelatine nasal spray micelles constructed in this paper, HS15 micelles were used to encapsulate Cy7, a hydrophobic fluorescent dye with similar physicochemical properties to agomelatine. The distribution of the HS15-carrying micelles in mice was observed using in vivo imaging. A Cy7-loaded micelle solution was prepared using a thin film dispersion method: 600 μg of Cy7 and 12 mg of HS15 were dissolved in 5 ml of anhydrous ethanol. The mixture was gently stirred at room temperature until uniformly mixed. The mixture was transferred to a 25 ml eggplant-shaped flask and rotary evaporated under reduced pressure. The anhydrous ethanol was removed in a 40°C water bath to form a uniform agomelatine-Cy7 thin film. The solution was then hydrated with 600 μl of 0.1 M PBS for 5 minutes to obtain the Cy7-loaded micelle solution.

[0067] Six rats were randomly divided into two groups (n=6): one group received a Cy7 solution and the other group received a Cy7 micelle-loaded solution. Each rat received 100 μl of either Cy7 solution or Cy7 micelle-loaded solution intranasally, each with the same fluorescence intensity. Four hours after administration, the rats were sacrificed, and heart, liver, spleen, lung, kidney, and brain tissues were removed. The fluorescence intensity of the isolated organs was observed and quantitatively analyzed using a small animal imaging device.

[0068] The results, as shown in Figures 10 and 11, demonstrate that the brain-targeting effect of HS15 micelles is significantly stronger than that of the simple drug solution, with the fluorescence intensity in the brain approximately double that of the simple solution. Furthermore, toxicity to the heart, liver, spleen, lung, and kidneys was lower when administered in the form of HS15 micelles, with liver and lung toxicity significantly reduced. This demonstrates that the constructed micelles, when administered via nasal spray, exhibit brain-specific targeted drug delivery in vivo.

[0069] The above description is merely an example and explanation of the concept of the present invention. Those skilled in the art may make various modifications or additions to the described specific embodiments or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they are all within the scope of protection of the present invention.

Claims

1. A preparation method of brain-targeted agomelatine nasal spray micelles, characterized in that, Dissolve HS15 and agomelatine in an organic solvent, remove the organic solvent by rotary evaporation until a uniform film is formed, add a water-soluble solvent for hydration to form agomelatine-loaded micelles.

2. The preparation method according to claim 1, wherein The preparation method described above includes the following steps: (1) Weigh accurately 60 - 100 mg of HS15 and 4 - 40 mg of agomelatine, add them to 4 - 10 ml of an organic solvent for dissolution, and stir evenly at 4 - 30 °C to obtain a mixed solution; (2) Transfer the mixed solution obtained in step (1) to a eggplant-shaped flask, remove the organic solvent by rotary evaporation under reduced pressure in a water bath at 25 - 50 °C for 5 - 20 min to form a uniform film, add 0.2 - 20 ml of a water-soluble solvent for hydration for 3 - 30 min to obtain a solution of agomelatine-loaded micelles; (3) Add 0.02% - 0.2% (w / v) of a preservative to the solution of agomelatine-loaded micelles obtained in step (2), mix evenly and then add a pH regulator to adjust the pH value to 6.8 - 7.4 to obtain brain-targeted agomelatine nasal spray micelles.

3. The preparation method according to claim 2, characterized in that, The organic solvent described in step (1) is one or a mixture of two or more of methanol, ethanol, chloroform, and acetone, preferably ethanol; the stirring conditions are a stirring speed of 200 - 1200 rpm and a stirring time of 5 - 60 min; the water-soluble solvent described in step (2) is one of normal saline or PBS.

4. The preparation method according to claim 2, characterized in that, The preservative described in step (3) is benzalkonium bromide, parabens, benzoic acid, sorbic acid, chlorhexidine acetate, o-phenylphenol, sodium thiosulfate, preferably benzalkonium bromide; the pH regulator includes a sodium dihydrogen phosphate - disodium hydrogen phosphate buffer solution with a molar ratio of 1:1.

3.

5. The preparation method according to claim 2, characterized in that, The composition ratio of agomelatine in the brain-targeted agomelatine nasal spray micelles is 4 wt% - 50 wt%, and the composition ratio of HS15 is 50 wt% - 96 wt%.

6. The preparation method according to claim 2, characterized in that, After the solution of agomelatine-loaded micelles is prepared, an isotonicity regulator and a stabilizer are also added; the isotonicity regulator is one or a combination of two or more of sodium chloride, mannitol, sorbitol, glycerol, glucose, and xylitol, and the addition amount is 0.5% - 1% (w / v); the stabilizer is one or a combination of two or more of sodium sulfite, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, glycine, citric acid, tartaric acid, ascorbic acid, and ethylenediaminetetraacetic acid, and the addition amount is 0.1% - 1% (w / v).

7. Brain-targeted agomelatine nasal spray micelles obtained by the preparation method according to any one of claims 1 - 6.

8. A brain-targeted agomelatine nasal spray micelle lyophilized powder, characterized in that, After adding a lyoprotectant to the brain-targeted agomelatine nasal spray micelles according to claim 7, freeze-dry to obtain a freeze-dried powder of brain-targeted agomelatine nasal spray micelles.

9. The brain-targeted agomelatine nasal spray micelle lyophilized powder according to claim 8, characterized in that, The lyoprotectant is one or a combination of two or more of glucose, mannitol, glycine, maltose, trehalose, sucrose, polyethylene glycol 2000, polyethylene glycol 4000, or polyethylene glycol 6000, and the addition amount of the lyoprotectant is 1% - 5% (w / v).

10. Use of the brain-targeted agomelatine nasal spray micelles according to claim 7 and the freeze-dried powder of the brain-targeted agomelatine nasal spray micelles according to claim 8 or 9 in the preparation of a nasal spray drug for treating depression and insomnia.

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