Amphotericin B nanocomposite and its preparation method and application

Abstract

The present invention relates to an amphotericin B nanocomposite and a preparation method and application thereof, and belongs to the technical field of pharmaceutical preparations. The present invention forms a nanocomposite in the form of a high aggregation of amphotericin B through the intermolecular force between amphotericin B and phytosphingosine. Compared with the prior art, the present invention can effectively reduce the hemolytic toxicity, renal cell toxicity and acute toxicity of amphotericin B, and has good in vitro and in vivo antifungal activity. In addition, the present invention also provides a preparation method of the nanocomposite, which has broad market prospects in the treatment of systemic deep fungal infections.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparation preparation, and specifically relates to an amphotericin B nanocomposite and a preparation method and application thereof. Background Art

[0002] Since the advent of amphotericin B, it has been the "gold standard" for the treatment of invasive fungal infections. However, during use, the drug will produce some toxic and side effects such as acute toxicity, hemolytic toxicity, liver and kidney damage, which limits its wide clinical application. In order to reduce its toxicity, researchers have developed a variety of new formulations such as liposomes, lipid complexes, and cholesterol sulfate complexes. Ambisome, as a classic amphotericin B liposome preparation, has obvious advantages in improving the hemolytic toxicity, cytotoxicity and acute toxicity of the drug since it was approved for marketing in 1990, and no similar liposome preparations have been developed so far. Ambisome uses DSPG phospholipids to combine with the trehalose amine group in the amphotericin B molecule to stably encapsulate the drug through drug-carrier interaction. Due to the difficulty of replacing DSPG phospholipids and the high price, as well as the complex preparation process, there is no generic preparation in China so far. Therefore, it is of great significance to find a carrier material to replace DSPG and develop a cost-effective and patient-friendly amphotericin B liposome preparation.

[0003] Based on the structural characteristics of DSPG, we screened out the compound phytosphingosine hydrochloride, which can not only promote the solubility of amphotericin B in organic solvents, but also form a highly aggregated form similar to Ambisome with amphotericin B molecules (maximum ultraviolet absorption wavelength is about 320nm), thereby achieving efficient drug encapsulation. However, the carrier materials reported in existing studies, such as deoxycholate, cholesterol sulfate, hydrogenated soybean phosphatidylcholine, cholesterol, lecithin, etc., cannot form highly aggregated forms similar to Ambisome with amphotericin B molecules. It is precisely based on this highly aggregated state that the acute toxicity test in mice found that when Ambisome was administered at a dose of 60mg / kg or above (up to 100mg / kg), no mice died within 2 weeks after a single dose, while other commercially available preparations could cause mouse death at a dose of 20mg / kg, and some preparations could even cause mouse death at a low dose of 1mg / kg.

[0004] Compared with common amphotericin B injection preparations, the present invention forms a nanocomposite by phytosphingosine and amphotericin B, and its highly aggregated form can slow down the release rate of the drug, effectively reduce the hemolytic toxicity, renal cell toxicity and acute toxicity of amphotericin B, prolong the drug circulation time in the blood, reduce the distribution of the drug in tissues and organs, and has good in vitro and in vivo antifungal activity. In addition, the preparation method of the amphotericin B nanocomposite is simple, and it can be prepared into a liposome preparation, which is very feasible for product development. Summary of the invention

[0005] In view of this, one of the objects of the present invention is to provide an amphotericin B nanocomplex; a second object of the present invention is to provide a method for preparing an amphotericin B nanocomplex; a third object of the present invention is to provide an application of an amphotericin B nanocomplex in the treatment of fungal infectious diseases; a fourth object of the present invention is to provide a pharmaceutical composition comprising an amphotericin B nanocomplex.

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

[0007] 1. An amphotericin B nanocomposite, wherein the molar ratio of amphotericin B to phytosphingosine hydrochloride in the nanocomposite is 1:1-3.

[0008] 2. A method for preparing the above-mentioned amphotericin B nanocomplex, the method comprising the following steps:

[0009] Amphotericin B and phytosphingosine hydrochloride were weighed and dispersed in a mixed solvent with a volume ratio of methanol: dichloromethane = 1:1, 0.1 mol / l dilute hydrochloric acid was added until the drugs were dissolved, and then a hydrogenated soybean phosphatidylcholine and cholesterol solution dissolved in a mixed solvent with a volume ratio of methanol: dichloromethane = 1:1 were added, mixed evenly, and the solvent was removed by reduced pressure evaporation at 60-65° C., and then an organic base aqueous solution at 60-65° C. was added to adjust the pH of the solution to 5-6, and a suspension was obtained after stirring and dispersing at 60-65° C. for 1.5-2.5 hours, and an amphotericin B nanocomplex solution was obtained after high-pressure homogenization.

[0010] Further preferably, the mass volume ratio of amphotericin B to methanol:dichloromethane mixed solvent and 0.1 mol / l dilute hydrochloric acid is 5:6-10:0.13-0.18, mg / ml;

[0011] The mass volume ratio of the cholesterol to the methanol:dichloromethane mixed solvent is 5:1-3, mg / ml;

[0012] The molar ratio of amphotericin B to hydrogenated soybean phosphatidylcholine and cholesterol is 1:5-6:2-3;

[0013] The organic base is any one of triethanolamine, tromethamine, and ethylenediamine;

[0014] The mass volume ratio of the amphotericin B to the organic base aqueous solution is 5:1-5, mg / ml.

[0015] 3. Use of the above-mentioned amphotericin B nanocomplex in the preparation of a pharmaceutical preparation for inhibiting fungal growth or treating fungal infectious diseases.

[0016] 4. A pharmaceutical composition comprising the above-mentioned amphotericin B nanocomplex.

[0017] Preferably, the pharmaceutical composition comprises a lyoprotectant;

[0018] Preferably, the dosage form of the pharmaceutical composition is lyophilized powder injection.

[0019] The beneficial effects of the present invention are:

[0020] 1. The present invention discloses an amphotericin B nanocomplex. The present invention adopts phytosphingosine hydrochloride as a carrier material. The obtained amphotericin B nanocomplex can effectively reduce the hemolytic toxicity, renal cell toxicity and acute toxicity of amphotericin B, prolong the drug circulation time in the blood, reduce the distribution of the drug in tissues and organs, and has good in vitro and in vivo antifungal activity, and has good application prospects in the treatment of fungal infectious diseases.

[0021] 2. The present invention also discloses a method for preparing an amphotericin B nanocomplex. The method is simple and can be prepared into a liposome preparation, which is highly feasible for product development.

[0022] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0024] Figure 1 The UV-visible absorption spectra of amphotericin B nanocomplex, amphotericin B and control preparation;

[0025] Figure 2 The in vitro drug release curves of amphotericin B nanocomplex and control preparation;

[0026] Figure 3This is the result of acute toxicity evaluation in mice;

[0027] Figure 4 The results of Cryptococcal fungal load experiments in the lungs and brains of infected mice;

[0028] Figure 5 These are the results of the Candida albicans fungal load experiment in the kidneys of infected mice. DETAILED DESCRIPTION

[0029] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0030] Example 1

[0031] The preparation of an amphotericin B nanocomposite comprises the following steps:

[0032] 100 mg of amphotericin B and 40 mg of phytosphingosine hydrochloride were weighed and dispersed in 160 ml of a mixed solvent with a volume ratio of methanol: dichloromethane = 1:1, 3 ml of 0.1 mol / l dilute hydrochloric acid was added until the drug was dissolved, and then 40 ml of a solution of hydrogenated soybean phosphatidylcholine (426 mg) and cholesterol (106 mg) dissolved in a mixed solvent with a volume ratio of methanol: dichloromethane = 1:1 was added, mixed evenly, and the solvent was removed by reduced pressure evaporation at 60°C, and then 20 ml of a 10% triethanolamine aqueous solution preheated to 60°C was added to adjust the pH of the solution to 5-6, and a suspension was obtained after stirring and dispersing at 60°C for 2 hours, and an amphotericin B nanocomposite solution was obtained after high-pressure homogenization. Then 1.8 g of trehalose was added to dissolve and clarify, and after mixing evenly, it was filtered through a 0.45 μm filter membrane, and the filtrate was collected and freeze-dried to obtain amphotericin B nanocomposite freeze-dried powder.

[0033] Example 2

[0034] The performance of the amphotericin B nanocomplex prepared in Example 1 was tested:

[0035] 1. Particle size and zeta potential determination of amphotericin B nanocomplexes

[0036] Weigh 10 mg of amphotericin B nanocomplex lyophilized powder, add pure water to reconstitute and dilute to a certain concentration, draw an appropriate amount of sample into the sample pool, and use Malvern laser particle size analyzer to measure its particle size and potential. The average particle size of the nanocomplex is 102.14 nm, and the Zeta potential is 35.53 mV.

[0037] 2. Determination of encapsulation efficiency and drug loading of amphotericin B nanocomplexes

[0038] 5 mg of amphotericin B nanocomplex lyophilized powder was weighed and placed in a 25 ml volumetric flask, 1 ml of DMSO was added for ultrasonic dispersion, and then methanol was added to dilute to the mark, and the content of amphotericin B in the solution was determined by HPLC. The drug loading (LC) and encapsulation efficiency (EE) were calculated as follows: drug loading (LC, %) = encapsulated drug mass / mass of drug-loaded liposomes × 100; encapsulation efficiency (EE, %) = encapsulated drug mass / feed drug mass × 100. The results showed that the encapsulation efficiency of the amphotericin B nanocomplex was 92.63 ± 0.65%, and the drug loading was 3.77 ± 0.42%.

[0039] 3. UV-visible absorption spectrum measurement

[0040] Weigh appropriate amounts of amphotericin B nanocomplex lyophilized powder, domestic ABCD preparation, and imported Ambisome preparation, add pure water to reconstitute and dilute to a certain concentration, place in an ultraviolet spectrophotometer for detection, and set the scanning wavelength range to 300-450nm. The measurement results are shown in the attached Figure 1 As shown, compared with Ambisome, the maximum absorption peak of ABCD shifts to the right, while the maximum absorption peak position of the amphotericin B nanocomplex prepared by us is consistent with that of Ambisome, reflecting a similar highly aggregated state.

[0041] 4. In vitro drug release study

[0042] Amphotericin B nanocomplex, ABCD domestic preparation and Ambisome imported preparation solution with a concentration of 1 μg / ml were prepared respectively, placed in a shaker at 37°C, and the UV-visible absorption curve was scanned at 0, 1, 3, 6, 12 and 24 hours, with a wavelength range of 300-450 nm. The ratio (a / b) of the maximum absorption peak intensities at 320-340 nm (a) and 400-420 nm (b) was calculated. The change in the absorption peak ratio reflects the process of amphotericin B from aggregates to monomers, that is, the release process. As can be seen from the results (see Appendix Figure 2), the release process of the above preparations was biphasic, with a faster release in the first hour and then gradually slowing down. At 24 hours, the cumulative release rates of Ambisome, amphotericin B nanocomplex and ABCD were 52.2%, 76.5% and 85.7%, respectively.

[0043] 5. In vitro hemolysis test

[0044] Weigh the amphotericin B nanocomplex lyophilized powder and ABCD preparation respectively, reconstitute with pure water and dilute to different drug concentrations with 5% glucose solution, then mix evenly with 2% red blood cell suspension at a volume ratio of 1:1, pure water plus 2% red blood cell suspension as positive control, 5% glucose solution plus 2% red blood cell suspension as negative control, the final drug concentration of the sample solution is 800, 400, 200, 100, 50, 25 and 12.5 μg / ml, the mixed sample is placed in a 37°C constant temperature incubator for 3 hours, then take out the sample and centrifuge at 2000rpm for 10 minutes, observe the hemolysis of red blood cells, and take 200 μl of supernatant to measure the absorbance value, and set the wavelength to 574nm. Hemolysis rate (%) = (A sample-A negative control) / (A positive control-A negative control).

[0045] The experimental results showed that the amphotericin B nanocomplex had no hemolytic toxicity at a drug concentration of 800 μg / ml or less, while obvious hemolysis was observed in the ABCD group. At a drug concentration of 400 μg / ml, the in vitro hemolysis rate of ABCD reached 22.6%, and when the concentration increased to 800 μg / ml, its hemolysis rate was as high as 100%. This result shows that the nanocomplex can significantly reduce the hemolytic toxicity of amphotericin B.

[0046] 6. In vitro pharmacodynamic evaluation

[0047] Amphotericin B nanocomplex lyophilized powder and ABCD preparation were weighed separately, reconstituted with pure water and diluted to different drug concentrations with 5% glucose solution. 40 μl of preparation solution was added to a 96-well plate, and then 160 μl of bacterial solution (1.25×10 3 CFU / ml), the final concentrations of the drugs in the wells were 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0.125 μg / ml, respectively. A negative control well was set up with bacteria but no drug, and a positive control well was set up with drug but no bacteria. The 96-well plate was placed in a 30°C constant temperature incubator for culture, and the fungal growth was observed after 48 hours. It can be seen from the experimental results (Table 1) that the amphotericin B nanocomplex has a good in vitro antifungal effect.

[0048] Table 1 Evaluation of in vitro antifungal activity

[0049]

[0050] 7. Evaluation of renal cell toxicity

[0051] Weigh the amphotericin B nanocomplex lyophilized powder and ABCD preparation respectively, reconstitute with pure water and dilute to different drug concentrations with 5% glucose solution. Weigh amphotericin B dissolved in DMSO and dilute to different drug concentrations with 5% glucose solution. Take HK-2 cells with good growth status, aspirate 160μl cell suspension and add it to a 96-well plate, with 3000 cells in each well. The plated cells were placed in a 37℃ incubator for 24h, and then 40μl preparation solution was added. The final concentration of the drug in the well was 200-3.125μg / ml. After the 96-well plate was placed in a 37℃ incubator for 24h, MTT (20μl) with a concentration of 5mg / ml was added. After incubation in the incubator for 4h, the solution in the well was removed, 150μl DMSO solution was added, and the OD value was measured in an ELISA reader, and the wavelength was set to 490nm.

[0052] The experimental results showed that amphotericin B had strong renal cytotoxicity with an IC50 range of 6.25 to 12.25 μg / ml, while amphotericin B nanocomplex and ABCD did not show renal cytotoxicity at concentrations as high as 200 μg / ml, indicating that the nanocomplex can significantly reduce the renal cytotoxicity of amphotericin B.

[0053] 8. Acute toxicity test

[0054] Healthy KM mice were randomly divided into groups of 6 mice each. The drug was injected into the tail vein of the mice at a dose of 40 mg / kg. The symptoms of the mice in each group and the deaths within 14 days were observed and recorded. Using the commercially available ABCD preparation as a control, one mouse died in the amphotericin B nanocomplex group within one week, and three mice died in the ABCD group (see attached). Figure 3 ). When the dosage was within the range of 5-30 mg / kg, no mice died.

[0055] 9. In vivo pharmacodynamic evaluation in mice

[0056] Cryptococcal fungal load experiment in mice: Cryptococcus neoformans H99 was injected into the tail vein to establish a mouse lung and brain fungal infection model. The specific method is as follows:

[0057] (1) Absorb the H99 Cryptococcus culture (5×10 6CFU / ml), and the model was established by injecting bacterial solution into the tail vein of KM mice (dose 100μl / 20g). 24h after infection, amphotericin B nanocomplex solution and control commercial preparation ABCD were injected into the tail vein, once a day, for 3 consecutive times, with a dose of 5mg / kg. 24h after the third administration, the mice were dissected on a sterile operating table, the lungs and brain tissues were removed, washed with sterilized PBS, and dried with filter paper. PBS was added according to the weight of the lung or brain tissue and the mass volume ratio of PBS 1:3 (g / ml), and then homogenized and diluted. 50μl was drawn and spread on YPD solid culture medium, placed in a 30℃ incubator for 48h, and the fungal growth in each culture plate was observed, and the colony count was performed.

[0058] From the attached Figure 4 It can be seen that compared with the untreated model group, the amphotericin B nanocomplex has obvious antifungal activity and significantly reduces the number of fungi in the lungs and brain of mice, and there is no significant difference in the therapeutic effect with the commercially available ABCD preparation.

[0059] In vivo Candida albicans fungal load experiment in mice: The mouse kidney fungal infection model was established by tail vein injection of Candida albicans ATCC 90028. The entire experimental method was the same as the Cryptococcal fungal load experiment. The results are shown in the attached Figure 5 Compared with the untreated model group, the amphotericin B nanocomplex had significant antifungal activity and significantly reduced the number of fungi in the kidneys of mice, and there was no significant difference in the therapeutic effect with the commercially available ABCD preparation.

[0060] The above results show that the amphotericin B nanocomplex prepared by the present invention has good in vivo antifungal activity.

[0061] In summary, the present invention uses phytosphingosine to encapsulate amphotericin B to form a nanocomposite, and its highly aggregated form can delay the release rate of the drug, effectively reduce the hemolytic toxicity, renal cell toxicity and acute toxicity of amphotericin B, and has good in vitro and in vivo antifungal activity. In addition, the present invention also provides a method for preparing the nanocomposite, and the prepared amphotericin B nanocomposite has broad market prospects in the treatment of systemic deep fungal infections.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. An amphotericin B nanocomposite, characterized in that: The molar ratio of amphotericin B to phytosphingosine hydrochloride in the nanocomposite is 1:1-3. The preparation method of the nanocomposite comprises the following steps: Amphotericin B and phytosphingosine hydrochloride were weighed and dispersed in a mixed solvent with a volume ratio of methanol: dichloromethane = 1:1, 0.1 mol / l dilute hydrochloric acid was added until the drugs were dissolved, and then a hydrogenated soybean phosphatidylcholine and cholesterol solution dissolved in a mixed solvent with a volume ratio of methanol: dichloromethane = 1:1 were added, mixed evenly, and the solvent was removed by reduced pressure evaporation at 60-65° C., and then an organic base aqueous solution at 60-65° C. was added to adjust the pH of the solution to 5-6, and a suspension was obtained after stirring and dispersing at 60-65° C. for 1.5-2.5 hours, and an amphotericin B nanocomplex solution was obtained after high-pressure homogenization.

2. The amphotericin B nanocomplex according to claim 1, characterized in that: The mass volume ratio of the amphotericin B to the methanol:dichloromethane mixed solvent and 0.1 mol / l dilute hydrochloric acid is 5:6-10:0.13-0.18, mg / ml, the mass volume ratio of the cholesterol to the methanol:dichloromethane mixed solvent is 5:1-3, mg / ml, the molar ratio of the amphotericin B to hydrogenated soybean phosphatidylcholine and cholesterol is 1:5-6:2-3, the organic base is any one of triethanolamine, tromethamine, and ethylenediamine, and the mass volume of the amphotericin B to the organic base aqueous solution is 5:1-5, mg / ml.

3. Use of the amphotericin B nanocomplex according to claim 1 in the preparation of a pharmaceutical preparation for inhibiting fungal growth or treating fungal infectious diseases.

4. A pharmaceutical composition, characterized in that Contains the amphotericin B nanocomposite according to claim 1.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition comprises a lyophilization protectant, and the dosage form of the pharmaceutical composition is a lyophilized powder injection.

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