Brain-targeted nasal spray amphotericin B phospholipid complex as well as preparation method and application thereof

Abstract

The invention discloses a brain-targeted nasal-spray amphotericin B phospholipid complex and a preparation method and application thereof, and belongs to the technical field of pharmaceutical preparations, the brain-targeted nasal-spray amphotericin B phospholipid complex is prepared by adopting a film dispersion method, and the preparation method comprises the following steps: dissolving 15-hydroxystearic acid polyethylene glycol ester, phospholipid, cholesterol, amphotericin B and optional other auxiliary components in a solvent, removing the solvent until a film is formed, and drying to obtain the brain-targeted nasal-spray amphotericin B phospholipid complex. And carrying out hydration dispersion to obtain the amphotericin B-loaded phospholipid complex. According to the amphotericin B sodium deoxycholate injection, the problems that the traditional amphotericin B sodium deoxycholate injection is high in renal toxicity, the intrathecal injection is poor in solubility, and low in bioavailability and patient compliance, large in side effect and the like due to the fact that the traditional amphotericin B sodium deoxycholate injection is difficult to penetrate through a blood brain After nasal administration, the drug is quickly delivered into the brain, so that the slow release of the drug is realized, the intracerebral bioavailability of the drug is improved, high brain targeting and low systemic toxic and side effects are realized, and good clinical application value and market prospect are achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical preparations, and particularly relates to a brain-targeting nasal spray amphotericin B phospholipid complex as well as a preparation method and application thereof. BACKGROUND

[0002] As a polyene antifungal drug, amphotericin B mainly binds to ergosterol in the fungal cell membrane to form pores in the membrane structure, increase the permeability of the membrane, and cause the leakage and loss of intracellular essential substances such as potassium ions, nucleotides and amino acids. In addition, amphotericin B can also induce lipid peroxidation in the cell membrane through a series of oxidation reactions, causing oxidative damage to the fungal cell, and ultimately leading to the death of the fungal cell. This mechanism of action endows amphotericin B with broad antifungal activity, making it one of the most widely used and most potent antifungal drugs in clinical practice, and it is widely used to treat severe invasive fungal infections affecting internal organs or the whole body, especially for infections resistant to other antifungal drugs.

[0003] Amphotericin B is the first choice for treating cryptococcal meningitis / meningoencephalitis, and its clinical application is mainly intravenous administration. However, traditional intravenous administration of amphotericin B has certain safety risks, such as chills, high fever, nausea, vomiting, decreased blood pressure, dizziness and other adverse reactions during infusion. In some clinical practices, intrathecal injection is used, but the patient compliance of this administration method is very poor, and it may cause serious adverse reactions such as headache, fever, vomiting, neck stiffness, lower limb pain, urinary retention, and even lower limb paralysis in severe cases. In addition, most patients may have varying degrees of renal function impairment during treatment, manifested as the presence of red blood cells, white blood cells, protein and cast in urine, elevated blood urea nitrogen and creatinine levels, and decreased creatinine clearance rate, and some patients may also develop renal tubular acidosis.

[0004] In view of these risks, a new method of preparing amphotericin B into a phospholipid complex and directly delivering it to the lesion site through the nasal mucosa shows considerable safety and practicality. The advantage of nasal administration is its non-invasiveness, convenience, painlessness and high bioavailability. This administration route can directly deliver the drug to the brain through the olfactory region under certain conditions, bypassing the blood-brain barrier (BBB), thereby improving the bioavailability of the drug and minimizing peripheral toxicity. At the same time, nasal administration avoids the inactivation of proteins and peptides in the gastrointestinal tract and the rapid metabolism in the liver, reduces mucociliary clearance, enhances the permeability and bioavailability of the drug, actively targets the brain, and has the advantage of preventing enzymatic degradation of the drug, which is beneficial for the treatment of brain diseases. Therefore, the preparation of amphotericin B into a phospholipid complex for nasal administration shows great potential and advantage in the treatment of intracranial fungal infections and other diseases. SUMMARY

[0005] In view of the special physicochemical properties of amphotericin B, 15-hydroxystearic acid polyethylene glycol ester (HS15) and common pharmaceutical excipients are used as carrier materials to develop a brain-targeting nasal spray amphotericin B phospholipid complex with low price and large-scale industrial production. The brain-targeting nasal spray amphotericin B phospholipid complex can be quickly delivered to the brain after nasal administration and increase the concentration of amphotericin B in the brain, realize the brain targeting of amphotericin B and low systemic toxic side effects, effectively enhance the treatment effect of amphotericin B on cryptococcal meningitis, and reduce the side effects of the drug, which has good clinical application value and market prospect.

[0006] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme: The present application provides a preparation method of brain-targeting nasal spray amphotericin B phospholipid complex, which is prepared by film dispersion method, including dissolving HS15, phospholipid, cholesterol and amphotericin B, and optional other auxiliary components in a solvent, concentrating and removing the solvent to form a film, and then performing hydration dispersion to obtain amphotericin B-loaded phospholipid complex.

[0007] Based on the above technical scheme, further, the preparation method comprises the following steps: HS15, phospholipid, cholesterol and amphotericin B, and optional other auxiliary components are weighed, dissolved in an organic solvent, and then stirred uniformly at 25-80 DEG C. The obtained mixed solution is subjected to rotary evaporation under reduced pressure at 25-80 DEG C for 5-20 min to remove the organic solvent and form a uniform film. Water-soluble solvent is added for hydration for 3-30 min to obtain a solution of amphotericin B-loaded phospholipid complex.

[0008] Further, the phospholipid is selected from one or more than two combinations of soybean phospholipid (SPC), egg yolk phospholipid (EPC), hydrogenated soybean phospholipid (HSPC), hydrogenated egg yolk phospholipid (HEPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), phosphatidylglycerol (PG) and phosphatidylserine (PS). Preferably, soybean phospholipid (SPC) and egg yolk phospholipid (EPC) are used.

[0009] Further, the cholesterol is selected from one or more than two combinations of natural cholesterol, hydrogenated cholesterol and cholesteryl sulfate sodium. Preferably, natural cholesterol is used.

[0010] Further, the organic solvent is selected from one or more than two mixed solvents of methanol, ethanol, chloroform, and acetone, and preferably is methanol or ethanol.

[0011] Further, the stirring condition is that the stirring speed is 200 rpm to 1200 rpm and the stirring time is 5 min to 60 min.

[0012] Further, the water-soluble solvent is selected from one or more than two mixed solvents of physiological saline, purified water, and PBS buffer solution.

[0013] Further, in the brain-targeting nasal amphotericin B phospholipid complex, the mass fraction of each component is as follows: amphotericin B 4.5wt% to 12.5wt%, HS15 37.5wt% to 45wt%, phospholipid 37.5wt% to 45wt%, cholesterol 4.5wt% to 12.5wt%, and optionally other auxiliary components, accounting for 100%.

[0014] Further, after the solution of the amphotericin B-loaded phospholipid complex is prepared, 0.02% to 0.2% (w / v) of a preservative and a pH adjuster (adjusting the pH value to 6.8 to 7.4) can be added. The pH adjuster includes a sodium dihydrogen phosphate-sodium hydrogen phosphate buffer solution with a molar ratio of 1:1.3; and the preservative is one or more than two combinations of benzalkonium bromide, hydroxybenzoic esters (including hydroxybenzoic acid methyl ester, hydroxybenzoic acid ethyl ester, and hydroxybenzoic acid propyl ester), benzoic acid, sorbic acid, chlorhexidine acetate, o-phenylphenol, and sodium thiosulfate.

[0015] Further, after the solution of the amphotericin B-loaded phospholipid complex is prepared, an isotonicity adjusting agent and a stabilizer can be added. The isotonicity adjusting agent is one or more than two combinations of sodium chloride, mannitol, sorbitol, glycerol, glucose, and xylitol, and the addition amount is 0.5% to 1% (w / v); and the stabilizer is one or more than two combinations of sodium sulfite, sodium pyrosulfite, sodium bisulfite, sodium thiosulfate, glycine, citric acid, tartaric acid, ascorbic acid, and ethylenediaminetetraacetic acid, and the addition amount is 0.1% to 1% (w / v).

[0016] Another aspect of the present application provides a brain-targeting nasal amphotericin B phospholipid complex prepared by the above preparation method.

[0017] Another aspect of the present application provides a brain-targeting nasal amphotericin B phospholipid complex lyophilized preparation. The brain-targeting nasal amphotericin B phospholipid complex obtained by the above preparation method is added with a lyophilization protective agent, and then freeze-drying to obtain the brain-targeting nasal amphotericin B phospholipid complex lyophilized preparation.

[0018] Further, the freeze-drying protective agent is one or a combination of two or more of glucose, mannitol, glycine, maltose, trehalose, sucrose, polyethylene glycol 2000, polyethylene glycol 4000, and polyethylene glycol 6000, and the freeze-drying protective agent is added in an amount of 1% to 5% (w / v).

[0019] Further, the freeze-dried preparation of the brain-targeting nasal spray amphotericin B phospholipid complex is diluted with a diluent before use to form a phospholipid complex solution, and the diluent is one or more of water for injection, normal saline, a phosphate buffer with a pH of 6.0 to 8.0, an acetic acid-sodium acetate buffer, and 5% glucose injection.

[0020] The application also provides the use of the brain-targeting nasal spray amphotericin B phospholipid complex and / or the freeze-dried preparation thereof in the preparation of a nasal spray drug for treating cryptococcal meningitis, invasive aspergillosis, disseminated candidiasis, and local fungal infections. In the phospholipid complex of the application, the components are tightly combined through intermolecular forces, and the drug is directly delivered to the central nervous system through the nasal-brain pathway after nasal administration, thereby significantly increasing the drug concentration in the brain and reducing systemic toxicity. The freeze-dried powder of the brain-targeting nasal spray amphotericin B phospholipid complex of the application improves the stability of the preparation and facilitates storage and transportation.

[0021] Compared with the prior art, the application has the following beneficial effects: (1) The application uses HS15, phospholipids, and cholesterol as carrier materials to form a phospholipid complex with uniform particle size, good encapsulation efficiency, and high stability.

[0022] (2) The phospholipid complex structure constructed in the application can achieve slow release of the drug, and the drug is stable and completely released in the cerebrospinal fluid, is quickly delivered to the brain after nasal administration, and has good brain targeting, thereby improving the bioavailability of the drug, achieving high brain targeting and low systemic toxicity, and being suitable for poorly soluble drugs that exert a therapeutic effect in the brain.

[0023] (3) The application overcomes the problems of high renal toxicity of traditional amphotericin B sodium deoxycholate injection, poor solubility of intrathecal injection, low bioavailability and low patient compliance caused by the difficulty of penetrating the blood-brain barrier, and large side effects.

[0024] (4) The brain-targeting nasal spray amphotericin B phospholipid complex prepared in the application is convenient to use, simple to operate, and easy to obtain raw materials, and has great application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 FIG. 1 is a transmission electron micrograph of the amphotericin B-loaded phospholipid complex; Figure 2 FIG. 2 is a characterization of the particle size distribution and zeta potential of the amphotericin B-loaded phospholipid complex by dynamic light scattering. Figure 3 Figure 1 is a graph showing the in vitro release curve of amphotericin B phospholipid complex in the cerebrospinal fluid at 0 h-24 h; Figure 4 Figure 2 is a graph showing the characterization of the particle size distribution of the amphotericin B phospholipid complex lyophilized powder after reconstitution by dynamic light scattering method; Figure 5 Figure 3 is a graph showing the in vitro release curve of amphotericin B phospholipid complex in the cerebrospinal fluid at 0 h-24 h; Figure 6 Figure 4 is a graph showing the stability of amphotericin B phospholipid complex in PBS; Figure 7 Figure 5 is a graph showing the stability of amphotericin B phospholipid complex in physiological saline; Figure 8 Figure 6 is a graph showing the stability of amphotericin B phospholipid complex in artificial cerebrospinal fluid; Figure 9 Figure 7 is a graph showing the characterization of the particle size distribution of the amphotericin B phospholipid complex solution at 4°C for 7 days by dynamic light scattering method; Figure 10 Figure 8 is a graph showing the characterization of the particle size distribution of the amphotericin B phospholipid complex solution at 4°C for 14 days by dynamic light scattering method; Figure 11 Figure 9 is a graph showing the characterization of the particle size distribution of the amphotericin B phospholipid complex before and after spraying by dynamic light scattering method; Figure 12 Figure 10 is a graph showing the evaluation of the targeting effect of phospholipid complex in the mouse brain tissue by fluorescence imaging quantification method; Figure 13 Figure 11 is a graph showing the change of blood drug concentration with time after intravenous injection of amphotericin B commercial injection preparation; Figure 14 Figure 12 is a graph showing the change of blood drug concentration with time after intranasal administration of amphotericin B phospholipid complex; Figure 15 Figure 13 is a graph showing the change of brain tissue drug concentration with time after intranasal administration of amphotericin B phospholipid complex. DETAILED DESCRIPTION

[0026] To better explain the technical content of the present application, specific examples will be provided below. These examples are for illustration only and do not constitute a limitation on the scope of the present application. Simple modifications and substitutions of methods or steps based on the principles of the present application also fall within the scope of the present application. The experimental techniques, reagents and materials used in the examples, if not specifically stated, are commercially available products.

[0027] Example 1

[0028] The preparation of amphotericin B phospholipid complex is as follows: Take 80 mg of 15-hydroxystearic acid polyethylene glycol ester (HS15), 80 mg of egg yolk phospholipid (EPC), 10 mg of natural cholesterol and 20 mg of amphotericin B (MeilunBio ® ), dissolved in 5 mL of anhydrous ethanol, slowly stirred at room temperature until mixed evenly, the mixed solution was transferred to a 50 mL flask and rotary evaporated under reduced pressure, the anhydrous ethanol was removed in a 60°C water bath, a uniform phospholipid complex film was formed, 2 mL of 0.1M PBS was added and watered for 5 min, and the solution of amphotericin B-loaded phospholipid complex was obtained.

[0029] Example 2

[0030] Preparation of amphotericin B-loaded phospholipid complex, the operation steps are as follows: Take 80 mg of 15-hydroxystearic acid polyethylene glycol ester (HS15), 80 mg of egg yolk phospholipid (EPC), 10 mg of natural cholesterol and 20 mg of amphotericin B (MeilunBio ® ), dissolved in 5 mL of anhydrous ethanol, slowly stirred at room temperature until mixed evenly, the mixed solution was transferred to a 50 mL flask and rotary evaporated under reduced pressure, the anhydrous ethanol was removed in a 60°C water bath, a uniform phospholipid complex film was formed, 2 mL of 0.1M PBS was added and watered for 5 min, and the solution of amphotericin B-loaded phospholipid complex was obtained.

[0031] Example 3

[0032] Preparation of amphotericin B-loaded phospholipid complex, the operation steps are as follows: Take 80 mg of 15-hydroxystearic acid polyethylene glycol ester (HS15), 80 mg of egg yolk phospholipid (EPC), 10 mg of natural cholesterol and 20 mg of amphotericin B (MeilunBio ® ), dissolved in 5 mL of anhydrous ethanol, slowly stirred at room temperature until mixed evenly, the mixed solution was transferred to a 50 mL flask and rotary evaporated under reduced pressure, the anhydrous ethanol was removed in a 60°C water bath, a uniform phospholipid complex film was formed, 2 mL of 0.1M PBS was added and watered for 5 min, and the solution of amphotericin B-loaded phospholipid complex was obtained.

[0033] Example 4

[0034] Characterization of amphotericin B-loaded phospholipid complex: The structure and morphology, size and potential of amphotericin B-loaded phospholipid complex were observed by transmission electron microscopy and dynamic light scattering technology. The results are as follows: Figure 1 and Figure 2The results show that the amphotericin B-loaded phospholipid complex prepared in Example 1 has an ultra-small phospholipid complex size of about 180 nm, which is consistent with the result of nanophospholipid complex size observed using a transmission electron microscope; the particle size is uniformly dispersed, with a polydispersity coefficient of 0.169±0.039, indicating good monodispersity; and the amphotericin B-loaded phospholipid complex has a relatively stable zeta electric potential of about -8.05±0.86 mV.

[0035] Example 5

[0036] Determination of drug loading of amphotericin B-loaded phospholipid complex: The drug loading (DL) and encapsulation efficiency (EE) of the amphotericin B-loaded phospholipid complex were determined by ultracentrifugation. 1 mL of the amphotericin B-loaded phospholipid complex solution was precisely measured, and ethanol was added to constant volume, and the drug-loaded phospholipid complex was broken by ultrasonic treatment at 400 W for 15 min to release all the drugs encapsulated therein. The absorbance value was determined at a wavelength of 405 nm using a UV spectrophotometer, and the total drug amount of amphotericin B was calculated. Another 1 mL of the amphotericin B-loaded phospholipid complex solution was placed in an ultrafiltration cup, and the drug-loaded phospholipid complex was separated by centrifugation at 3000×g for 20 min. The absorbance value of the filtrate was determined using a UV spectrophotometer, and the amount of amphotericin B not encapsulated was calculated.

[0037] The results show that the encapsulation efficiency and drug loading of the amphotericin B-loaded phospholipid complex are 89% and 2.1%, respectively, indicating that the drug-loaded phospholipid complex prepared by the method of the present application has a high loading amount of amphotericin B.

[0038] Example 6

[0039] Stability test of the freeze-dried powder of amphotericin B-loaded phospholipid complex: The amphotericin B-loaded phospholipid complex solution prepared in Example 1 was added with a freeze-drying protective agent composed of 2% (w / v) glucose and 3% (w / v) mannitol, and the test sample was placed in a vacuum freeze dryer, and freeze-drying operation was performed according to the following procedure: Pre-freezing stage: the cold trap temperature of the equipment was reduced to -50°C, and the sample was pre-frozen at this temperature for 1 h; Sublimation drying stage: the sample was dried at a gradient temperature program: first gradient: temperature -40°C, and the sample was dried for 1 h; second gradient: the temperature was increased to -20°C, and the sample was dried for 1 h; third gradient: the temperature was increased to -10°C, and the sample was dried for 1 h; Desorption drying stage: the temperature was increased to 0°C, and the sample was dried for 6 h.

[0040] After freeze-drying, the product was placed at room temperature, and the morphology of the freeze-dried product was as shown in Figure 3As shown, the freeze-dried product is uniform in color, dense in pores, has little change in volume before and after freeze-drying, has no change in properties, and forms a sponge-like block structure. The particle size of the amphotericin B phospholipid complex after re-dissolution with 2 mL of PBS is 180.5 ± 8.8 nm (as shown in Figure 4 ), which is not significantly different from that before freeze-drying.

[0041] The results show that the amphotericin B phospholipid complex freeze-dried preparation prepared in Example 1 has good stability within 24 h after re-dissolution. In order to facilitate product storage, the amphotericin B phospholipid complex can be stored in a freeze-dried form, and re-dissolved before use.

[0042] Example 7

[0043] In vitro release behavior test of amphotericin B phospholipid complex: The in vitro release behavior of the amphotericin B phospholipid complex was investigated by dynamic dialysis. 2 mL of the amphotericin B phospholipid complex solution was precisely measured and placed in a 3.5 kDa dialysis bag, and the dialysis bag was tightly tied at both ends. The dialysis bag was placed in 10 mL of artificial nose fluid (composition: potassium dihydrogen phosphate 13.6% (w / v) and 0.1 mol / L sodium hydroxide, pH 6.8) and artificial cerebrospinal fluid (aCSF, sodium chloride 0.72% (w / v), potassium chloride 0.02% (w / v), magnesium sulfate 0.05% (w / v), sodium bicarbonate 0.22% (w / v), calcium chloride 0.02% (w / v), D-glucose 0.40% (w / v), pH 7.4, sterile) containing 1% (w / v) DMSO.

[0044] At 37°C, the dialysis bag was shaken in a shaking table at 200 rpm / min for 10 h. At 0, 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h, all the solvents were removed and the dialysis bag was rinsed to ensure accurate measurement of the release results. The content of amphotericin B after demulsification at different time points was determined by ultraviolet-visible spectrophotometry, and thus the un-released amphotericin B was obtained. The drug release curve is shown in Figure 5 As shown, the amphotericin B solution can reach the maximum cumulative release amount at 12 h, and the release rate is 49%. The results show that the amphotericin B phospholipid complex prepared in Example 1 has obvious sustained release effect and no burst release effect.

[0045] Example 8

[0046] Dilution stability test of amphotericin B phospholipid complex solution in PBS, normal saline, and artificial cerebrospinal fluid: The amphotericin B-loaded phospholipid complex prepared in Example 1 was diluted with 100 mL of PBS, normal saline and cerebrospinal fluid, respectively, and the particle size was analyzed after 0, 0.5, 1, 2, 4, 6, 12 and 24 hours using dynamic light scattering technology. The particle size distribution is shown in Figure 6 , Figure 7 , Figure 8 .

[0047] The results show that the amphotericin B-loaded phospholipid complex prepared in Example 1 can remain stable after dilution in PBS, normal saline and artificial cerebrospinal fluid, indicating that the phospholipid complex system constructed can exist stably during storage and release the drug in the brain in the form of phospholipid complex, thereby improving the range and convenience of clinical use.

[0048] Example 9

[0049] Storage stability of the amphotericin B-loaded phospholipid complex solution at 4°C: The solution of the amphotericin B-loaded phospholipid complex prepared in Example 1 was stored at 4°C for 14 days, and the particle size was analyzed at 7 days and 14 days using dynamic light scattering technology. The results are shown in Figure 9 , Figure 10 . The particle sizes at 7 days and 14 days were 227.5 ± 0.5 nm and 183.5 ± 4.9 nm, respectively, and the particle size did not change substantially, the particle size was uniform, and no aggregation occurred.

[0050] The results show that the solution of the amphotericin B-loaded phospholipid complex prepared in Example 1 can be directly stored at 4°C, and therefore the amphotericin B-loaded phospholipid complex can be directly applied in the form of a solution.

[0051] Example 10

[0052] Stability test of the amphotericin B-loaded phospholipid complex solution before and after spraying: The solution of the amphotericin B-loaded phospholipid complex prepared in Example 1 was added to a nasal spray device, and the particle size of the product after spraying was determined using dynamic light scattering technology. The results are shown in Figure 11 . The particle sizes before and after spraying were 205.3 ± 14.2 nm and 200.4 ± 0.5 nm, respectively.

[0053] The results show that the shear force of the spraying device has no effect on the properties of the amphotericin B-loaded phospholipid complex prepared in Example 1. The particle size of the phospholipid complex remains stable before and after spraying, the particle size distribution is good, and the clinical use is feasible. The solution of the amphotericin B-loaded phospholipid complex can be applied to the nasal spray device for administration.

[0054] Example 11

[0055] Test of brain targeting ability of phospholipid complex: To preliminarily investigate the brain targeting ability of the brain targeting amphotericin B phospholipid complex prepared in the application, the hydrophobic fluorescent dye Cy7 having the same physicochemical properties as amphotericin B was loaded into the HS15 phospholipid complex, and the distribution of the phospholipid complex with HS15 as the carrier in the mouse body was observed by a small animal live imaging instrument. Since both Cy7 and amphotericin B are hydrophobic drugs, the phospholipid complex solution loaded with Cy7 was prepared by the film dispersion method, and 6 Kunming rats were randomly divided into 2 groups (n=3), one group was administered with the Cy7 solution, and the other group was administered with the phospholipid complex solution loaded with Cy7. Each rat was administered with 100 μL of the Cy7 solution and the phospholipid complex solution loaded with Cy7 with the same fluorescence intensity, and 4 hours after administration, the rats were placed under the small animal imaging instrument to observe the brain fluorescence intensity and quantitatively analyze the fluorescence intensity.

[0056] The results are shown in Table 1. Figure 12 As shown in Table 1, at 1 h, the brain targeting effect of the HS15 phospholipid complex group (AmB@HS15-LC) was lower than that of the simple drug solution group (Control), but at 4 h, the brain targeting effect of the HS15 phospholipid complex group was significantly stronger than that of the simple drug solution group, indicating that the phospholipid complex could slowly release and gradually accumulate in the brain.

[0057] Example 12

[0058] Pharmacokinetics of amphotericin B phospholipid complex after nasal administration: To preliminarily investigate the rapid delivery of the amphotericin B phospholipid complex prepared in the application to the brain after nasal administration, we performed a pharmacokinetics experiment, and the drug concentration in the brain of SD rats was determined by high performance liquid chromatography (HPLC).

[0059] The animals were all SD rats (200 g-220 g) and were divided into 4 groups, 5 rats in each group: 1. Amphotericin B commercial injection preparation group (drug identification code: 86902697000113): 1.2 mg was injected into the tail vein; 2. Amphotericin B phospholipid complex group of the application: 1.2 mg was administered nasally; 3. Intravenous injection blank control group: physiological saline (0.1 mL in volume) was injected into the tail vein; 4. Nasal administration blank control group: physiological saline (0.1 mL in volume) was administered nasally.

[0060] The rats in each group were respectively subjected to orbit blood collection at 0.5 h, 0.75 h, 1 h, 1.5 h, 2 h, 6 h, 12 h and 24 h after administration, and the blood was placed in an anticoagulant tube with heparin sodium, gently shaken to prevent coagulation, and centrifuged to obtain 100 μL of supernatant for standby; the brain tissues of the rats in each group were collected at 0.25 h, 0.5 h, 0.75 h, 1 h, 2 h and 24 h after administration, and treated with normal saline for homogenization.

[0061] Chromatographic analysis was performed using an Agilent-C18 chromatographic column (250x4.6 mm) with a mobile phase consisting of acetonitrile and water (60:40) at a flow rate of 1 mL / min; the ultraviolet detection wavelength was 405 nm, the column temperature was 37℃, and the injection volume was 20 μL.

[0062] The results are shown in Table 1. Figure 13 The amphotericin B commercial injection preparation group reached a peak in the plasma at 1 h, with a blood drug concentration of 50.92 μg / mL, but no drug was detected in the brain tissue; the amphotericin B-loaded phospholipid complex group of the present application reached a peak in the plasma at 2 h, with a blood drug concentration of 8.83 μg / mL, and the drug content in the brain tissue reached a peak at 0.75 h, with a concentration of 1.04 μg / mL. No drug was detected after intravenous injection and nasal administration of normal saline. The amphotericin B commercial injection preparation group can maintain a relatively high blood drug concentration in the plasma, while the drug content in the plasma of the amphotericin B-loaded phospholipid complex group administered nasally is much lower than that of the amphotericin B commercial injection preparation group, and the drug is detected in the brain and reaches a peak at 0.75 h, proving that the amphotericin B-loaded phospholipid complex constructed in the present application can cross the blood-brain barrier and be rapidly delivered to the brain after nasal administration. Figure 14 Figure 15 The results are shown in Table 1. Figure 13 The amphotericin B commercial injection preparation group reached a peak in the plasma at 1 h, with a blood drug concentration of 50.92 μg / mL, but no drug was detected in the brain tissue; the amphotericin B-loaded phospholipid complex group of the present application reached a peak in the plasma at 2 h, with a blood drug concentration of 8.83 μg / mL, and the drug content in the brain tissue reached a peak at 0.75 h, with a concentration of 1.04 μg / mL. No drug was detected after intravenous injection and nasal administration of normal saline. The amphotericin B commercial injection preparation group can maintain a relatively high blood drug concentration in the plasma, while the drug content in the plasma of the amphotericin B-loaded phospholipid complex group administered nasally is much lower than that of the amphotericin B commercial injection preparation group, and the drug is detected in the brain and reaches a peak at 0.75 h, proving that the amphotericin B-loaded phospholipid complex constructed in the present application can cross the blood-brain barrier and be rapidly delivered to the brain after nasal administration.

Claims

1. A method for preparing a brain-targeting nasal spray amphotericin B phospholipid complex, characterized in that, The preparation method comprises the following steps: dissolving 15-hydroxypolyethylene glycol stearate, phospholipid, cholesterol and amphotericin B, and optionally other auxiliary components in an organic solvent, stirring to obtain a uniform solution, concentrating to remove the organic solvent to form a uniform film, and then adding an aqueous solvent to obtain a solution of the amphotericin B-loaded phospholipid complex.

2. The method of preparing brain targeting nasal spray amphotericin B phospholipid complex according to claim 1, wherein, Specifically comprising the following steps: The preparation method comprises the following steps: dissolving 15-hydroxypolyethylene glycol stearate, phospholipid, cholesterol and amphotericin B, and optionally other auxiliary components in an organic solvent, stirring to obtain a uniform solution, concentrating to remove the organic solvent to form a uniform film, and then adding an aqueous solvent to obtain a solution of the amphotericin B-loaded phospholipid complex.

3. The method of claim 1, wherein the brain targeting nasal spray amphotericin B phospholipid complex is prepared by, The phospholipid is selected from one or more than two combinations of soybean phospholipid, egg yolk phospholipid, hydrogenated soybean phospholipid, hydrogenated egg yolk phospholipid, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, phosphatidylglycerol and phosphatidylserine; and the cholesterol is selected from one or more than two combinations of natural cholesterol, hydrogenated cholesterol and cholesteryl sulfate sodium.

4. The method of claim 1, wherein the brain targeting nasal spray amphotericin B phospholipid complex is prepared by, The organic solvent is selected from one or more than two combinations of methanol, ethanol, chloroform and acetone; and the aqueous solvent is selected from one or more than two combinations of physiological saline, purified water and PBS buffer solution.

5. The method for preparing the brain-targeting nasal spray amphotericin B phospholipid complex according to claim 1, characterized in that, In the brain-targeting nasal spray amphotericin B phospholipid complex, the mass fractions of the components are as follows: amphotericin B 4.5wt%-12.5wt%, 15-hydroxypolyethylene glycol stearate 37.5wt%-45wt%, phospholipid 37.5wt%-45wt%, cholesterol 4.5wt%-12.5wt%, and optionally other auxiliary components, with the total being 100%.

6. A brain-targeting nasal amphotericin B phospholipid complex, characterized in that, The method is prepared by any one of claims 1-5.

7. A brain-targeting nasal spray amphotericin B phospholipid complex lyophilized formulation, characterized in that, The method is prepared by any one of claims 1-5.

8. The brain targeting nasal amphotericin B phospholipid complex lyophilized powder formulation according to claim 7, characterized in that, The freeze-drying protective agent is one or more than two combinations of glucose, mannitol, glycine, maltose, trehalose, sucrose, polyethylene glycol 2000, polyethylene glycol 4000 and polyethylene glycol 6000; and a diluent is added as needed before use to reconstitute the phospholipid complex solution.

9. The use of the brain-targeting nasal spray amphotericin B phospholipid complex of claim 6 in the preparation of a nasal spray drug for treating cryptococcal meningitis, invasive aspergillosis, disseminated candidiasis and local fungal infections.

10. The use of the freeze-dried preparation of the brain-targeting nasal spray amphotericin B phospholipid complex of claim 7 in the preparation of a nasal spray drug for treating cryptococcal meningitis, invasive aspergillosis, disseminated candidiasis and local fungal infections.

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