Temozolomide spherocrystal based on nano confinement crystallization regulation, method and application

Abstract

The invention discloses a temozolomide spherocrystal based on nano confinement crystallization regulation and a method and application thereof. The preparation method of the temozolomide spherocrystal based on nano confinement crystallization regulation and control comprises the following steps: dissolving glycyrrhizic acid and temozolomide in pure water under an ultrasonic condition, adjusting the pH value to 3-4, heating until the solution is clear, and cooling to 0-5 DEG C under a stirring condition to obtain the temozolomide spherocrystal based on nano confinement crystallization regulation and control. Compared with the temozolomide raw material I crystal form, the temozolomide spherocrystal disclosed by the invention shows excellent nasal administration characteristics, including uniform particle size, high sphericity and excellent fluidity. Cell experiments show high cytotoxicity (IC50 = 51.9 [mu] g mL <-1 >) to glioblastoma, and in vivo experiments prove efficient brain targeting ability of the polypeptide. The preparation method has the characteristics of mild preparation conditions, easiness in process control, good reproducibility, high brain targeting property and the like, and is suitable for treating cancers delivered into the brain through the nose.

Description

TECHNICAL FIELD

[0001] The present application relates to a temozolomide, in particular to a kind of based on nanometer limited domain crystal regulation temozolomide spherulite and method and application, the application is as dry powder inhalation or spray inhalation through nose into brain cancer treatment drug application;Belong to the field of drug delivery system. BACKGROUND

[0002] Glioblastoma is a highly invasive malignant brain tumor, and its clinical treatment is a great challenge. Temozolomide is the main drug for treating GBM at present, and the I crystal form is the commonly used raw material. However, the solubility and dissolution rate of I crystal form temozolomide are low, which limits its in vivo absorption and distribution. Traditional administration methods have many adverse reactions: oral administration can easily cause gastrointestinal irritation and vomiting, and intravenous injection can cause systemic toxicity. In addition, the traditional administration method is difficult to target the drug to the brain tumor site, which significantly reduces the treatment effect.

[0003] Nasal delivery is a non-invasive drug delivery method that can directly deliver drugs to the brain tumor site through the nasal-brain pathway. However, in the existing temozolomide preparation, the I crystal form presents a plate-like crystal structure, has a low bulk density, an irregular particle size distribution, and poor flowability, which leads to low deposition efficiency and poor penetration during nasal delivery. Therefore, it is urgent to develop a new type of preparation to improve the clinical application effect.

[0004] Chinese patent application CN106491540A discloses a temozolomide sustained-release system and its preparation method and application. The temozolomide sustained-release system comprises PLGA sustained-release microspheres and temozolomide encapsulated in the PLGA sustained-release microspheres. The preparation method of the temozolomide sustained-release system is as follows: temozolomide crystals are dissolved in methanol, and PLGA copolymer is dissolved in dichloromethane; the above two solutions are mixed, and the obtained organic phase is emulsified in a solution containing polyvinyl alcohol until dichloromethane and methanol are completely volatilized. However, this technology has the problem of toxic solvent residue during preparation, and the spherulite particle size distribution is difficult to control, which may affect the safety and efficiency of nasal delivery. SUMMARY

[0005] The present application provides a nanometer limited domain crystallization engineering method for preparing temozolomide spherulites. The spherulites can achieve efficient brain targeting delivery through nasal delivery, significantly improving the treatment effect of glioblastoma. The present application utilizes the self-assembly characteristics of glycyrrhizic acid supramolecules to construct a limited domain environment on a nanometer scale, providing ideal heterogeneous nucleation sites for temozolomide. Based on the limited domain effect, the present application significantly improves the supersaturation of temozolomide, and realizes the selective growth of II and III crystal form spherulites

[0006] The present application is realized by the following technical solutions:

[0007] A method for preparing temozolomide spherulites based on nano-limited crystallization regulation: glycyrrhizic acid and temozolomide are dissolved in pure water under ultrasonic conditions, the pH value is adjusted to 3-4, heating to dissolve clear, and cooling to 0-5℃ under stirring to obtain temozolomide spherulites based on nano-limited crystallization regulation.

[0008] To further achieve the object of the present application, preferably, the mass ratio of glycyrrhizic acid to pure water is 0.005-0.05:1.

[0009] Preferably, 0.1-0.8 g of temozolomide is added per milliliter of pure water.

[0010] Preferably, the ultrasonic conditions are built-in ultrasonic probe, and the specific conditions are: ultrasonic time is 10-30 min, ultrasonic power is 400-600 W, and ultrasonic duty cycle is 33%-66%.

[0011] Preferably, the pH value is adjusted to 3-4 by adding sodium hydroxide, and the molar ratio of sodium hydroxide to glycyrrhizic acid is 1:1-3:1.

[0012] Preferably, the stirring rate is 50 rpm-200 rpm, the cooling rate is 10-40℃ per minute, and the temperature under stirring is 1-4℃.

[0013] Preferably, the heating temperature for dissolving clear is 60-80℃.

[0014] A temozolomide spherulite based on nano-limited crystallization regulation is prepared by the above method, which is temozolomide II crystal spherulite or temozolomide III crystal spherulite.

[0015] Preferably, the sphericity of the temozolomide spherulite is 0.6-0.95, the bulk density is 0.4-0.6 g / cm -3 , the rest angle is 22-40°, the particle size distribution is 15-25 μm, and the polydispersity coefficient is 0.1-0.5.

[0016] The temozolomide spherulite based on nano-limited crystallization regulation is used as a cancer treatment drug for nasal delivery into the brain in the form of dry powder inhalation or spray inhalation.

[0017] Compared with the prior art, the present application has the following advantages:

[0018] 1) The nano-limited environment is constructed by self-assembly of glycyrrhizic acid to regulate the nucleation and growth process of temozolomide, and pure II crystal spherulite and III crystal spherulite are obtained.

[0019] 2) The temozolomide spherulite has uniform particle size distribution, high sphericity and excellent flowability, and is suitable for nasal administration.

[0020] 3) In vivo studies show that the said nanocrystals have the highest brain targeting ability through nasal delivery, which is 15.8 times higher than that of traditional preparations, and significantly reduces systemic toxicity.

[0021] 4) The method for preparing temozolomide nanocrystals based on nano-limited crystallization has the advantages of mild conditions, simple process, good reproducibility, and good scale-up production advantage. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 PXRD patterns of temozolomide standard samples of each crystal form.

[0023] Figure 2 PXRD patterns of temozolomide nanocrystals obtained in Examples 1-4.

[0024] Figure 3 Microscope images of temozolomide Form II and Form III nanocrystals obtained in Examples 2 and 4.

[0025] Figure 4 Scanning electron microscope (SEM) images of temozolomide Form II nanocrystals obtained in Example 2.

[0026] Figure 5 Particle size distribution analysis of temozolomide Form II nanocrystals obtained in Example 2.

[0027] Figure 6 Dissolution behavior curves of temozolomide nanocrystals obtained in Examples 1-4.

[0028] Figure 7 Comparison results of cytotoxicity of temozolomide nanocrystals obtained in Examples 1-4.

[0029] Figure 8 Distribution of temozolomide Form II nanocrystals obtained in Example 2 in the brain when delivered through the nose into the brain. DETAILED DESCRIPTION

[0030] In order to better understand the present application, the following examples are used to further illustrate the present application, but it should be noted that the scope of protection required by the present application is not limited to the scope expressed in the following examples.

[0031] Temozolomide is a chemotherapy drug for treating malignant brain tumors (such as glioblastoma and astrocytoma). Temozolomide (C6H6N6O2, molecular weight 194.15, CAS number 85622-93-1, structure as shown in formula 1) is a model drug.

[0032]

[0033] However, temozolomide has some limitations in clinical application, such as gastrointestinal adverse reactions caused by oral administration, systemic toxicity of injection administration, and low distribution efficiency of the drug in brain tissue. In order to overcome these challenges, developing an efficient drug delivery system and improving the method of temozolomide brain delivery efficiency is of great significance to promote the further development of temozolomide and other drug research and application fields.

[0034] The present application finds that the problems of temozolomide can be effectively overcome by using spherulites and nano-confinement effect. Spherulites, as a kind of polycrystalline aggregate, have excellent stability, fluidity, processability and biochemical properties, and are particularly suitable for nasal administration. Moreover, as the advantage metastable crystal forms, II and III crystal forms have higher solubility and dissolution rate, which can significantly improve the bioavailability and delivery efficiency of the drug. Therefore, for temozolomide, it is urgent to develop a new method for preparing temozolomide spherulites of advantage crystal forms under mild conditions, in order to further improve the targeting and clinical efficacy of the drug. Nano-confinement effect refers to the changes in the physical and chemical properties of materials due to spatial confinement at the nanoscale. When the size of the material is reduced to the nanoscale, the movement of electrons, holes or other particles is limited in a very small space, which changes their aggregation form and further changes the optical, electrical and other physical properties of the material. The present application further finds that by self-assembly of glycyrrhizic acid to form nanomicelles, temozolomide is enriched in the micelles to achieve high supersaturation and change its nucleation and growth pathway. Based on this nano-confinement effect, the crystallization process is precisely controlled to induce the secondary nucleation of temozolomide, thereby realizing the selective growth of temozolomide II and III crystal spherulites.

[0035] Glycyrrhizic acid (GA, C 42 H 62 O 16 , molecular weight 822.94, CAS number 1405-86-3, as shown in formula 2) is a natural compound extracted from the roots of licorice, which has anti-inflammatory, antiviral, antioxidant and immunomodulatory biological activities, and is widely used in the fields of medicine and health products, and has high safety.

[0036]

[0037] The present application finds that glycyrrhizic acid can self-assemble to form nanoscale micellar confined environment in water due to the presence of both hydrophilic and hydrophobic ends in its chemical structure, which makes it an ideal nanometer confined template and provides ideal heterogeneous nucleation sites for the nucleation and growth of temozolomide. Therefore, the glycyrrhizic acid nanometer confined environment can provide ideal heterogeneous nucleation sites for temozolomide, increase the supersaturation of temozolomide based on the confinement effect, and realize the enrichment and molecular diffusion limitation of temozolomide inside the glycyrrhizic acid. The confined crystallization regulates the nucleation and growth process of temozolomide, and realizes the selective growth of temozolomide II crystal and III crystal.

[0038] For intranasal administration, the suitable drug particle size is 10 to 50 microns. The present application prepares temozolomide II crystal and III crystal spherulites by regulating the crystallization process of temozolomide, which exhibit a narrow and concentrated particle size distribution, high bulk density and sphericity, and excellent flowability. The excellent performance highlights the great potential of temozolomide spherulites as an inhalation preparation for intranasal administration; cell experiments show high cytotoxicity to glioblastoma; in vivo studies further verify its high brain targeting ability.

[0039] Therefore, the present application provides a method for preparing temozolomide spherulites based on nanometer confined crystallization regulation, which adopts the following measures: dissolving glycyrrhizic acid and temozolomide in pure water, adjusting the pH value to 3-4, heating to dissolution under ultrasonic conditions, and cooling to a set temperature of 0-5°C under stirring to obtain glycyrrhizic acid confined crystallization regulated temozolomide spherulites. In the technical solution, the amount of raw materials can be obtained by testing in combination with the purpose of the present application under the above measures. Preferably, the mass ratio of glycyrrhizic acid to pure water is 0.005-0.05:1. More preferably, the mass ratio of glycyrrhizic acid to pure water is 0.005-0.01:1. Preferably, 0.2-0.8 g of temozolomide is added per milliliter of pure water. More preferably, 0.4-0.6 g of temozolomide is added per milliliter of pure water.

[0040] In the technical measures of the present application, the ultrasonic is to heat the product obtained by adjusting the pH value to 3-4 to dissolution. According to the purpose, the ultrasonic conditions can be preferably obtained as follows: built-in ultrasonic probe, ultrasonic time 10-30 min, ultrasonic power 400-600 W, and ultrasonic duty cycle 33%-66%. In the technical measures, the pH value of 3-4 is preferably adjusted by adding sodium hydroxide, and the molar ratio of sodium hydroxide to glycyrrhizic acid can be calculated to be 1:1-3:1. The heating temperature for heating to dissolution is preferably 60-80°C.

[0041] In the technical measures of the present application, the stirring rate can be preferably 50 rpm to 200 rpm when the temperature is lowered to the set temperature of 0 ℃ to 5 ℃; the temperature lowering rate is preferably controlled to be 10 to 40 ℃ per minute; and the temperature is further lowered to 1 ℃ to 4 ℃ under stirring.

[0042] The obtained temozolomide spherocrystal based on nano-limited crystallization regulation is a temozolomide II crystal spherocrystal or a temozolomide III crystal spherocrystal; the sphericity of the temozolomide spherocrystal is 0.6 to 0.95; the bulk density is 0.4 to 0.6 g / cm -3 ; the rest angle is 22° to 40°; the particle size distribution is 15 to 25 μm; and the polydispersity coefficient is 0.1 to 0.5.

[0043] The present application also provides the use of the temozolomide spherocrystal based on nano-limited crystallization regulation as a cancer treatment drug for nasal delivery into the brain by dry powder inhalation or spray inhalation.

[0044] Example 1

[0045] The step of preparing the temozolomide spherocrystal based on nano-limited crystallization regulation of glycyrrhizic acid is as follows: 0.05 g of glycyrrhizic acid and 0.35 g of temozolomide are dissolved in 10 mL of pure water, 4.8 mg of sodium hydroxide is added to adjust the pH to 3, and then the solution is ultrasonically treated for 20 min (ultrasonic power is 400 W, and ultrasonic duty cycle is 33%), and the solution is heated to dissolve at 80 ℃, and then the temperature is lowered to 4 ℃ under stirring at a speed of 100 rpm to obtain the crystal. The PXRD pattern of the crystal is shown in Figure 1, and the characteristic peaks of the crystal are consistent with those of the temozolomide II crystal shown in Figure 2. The SEM pattern (Figure 3) of the crystal proves the successful preparation of the spherocrystal. The results show that the temozolomide II crystal spherocrystal is obtained in Example 1. Figure 2 Figure 1 The step of preparing the temozolomide spherocrystal based on nano-limited crystallization regulation of glycyrrhizic acid is as follows: 0.05 g of glycyrrhizic acid and 0.35 g of temozolomide are dissolved in 10 mL of pure water, 4.8 mg of sodium hydroxide is added to adjust the pH to 3, and then the solution is ultrasonically treated for 20 min (ultrasonic power is 400 W, and ultrasonic duty cycle is 33%), and the solution is heated to dissolve at 80 ℃, and then the temperature is lowered to 4 ℃ under stirring at a speed of 100 rpm to obtain the crystal. The PXRD pattern of the crystal is shown in Figure 1, and the characteristic peaks of the crystal are consistent with those of the temozolomide II crystal shown in Figure 2. The SEM pattern (Figure 3) of the crystal proves the successful preparation of the spherocrystal. The results show that the temozolomide II crystal spherocrystal is obtained in Example 1.

[0046] Example 2

[0047] The step of preparing the temozolomide spherocrystal based on nano-limited crystallization regulation of glycyrrhizic acid is as follows: 0.05 g of glycyrrhizic acid and 0.35 g of temozolomide are dissolved in 10 mL of pure water, 4.8 mg of sodium hydroxide is added to adjust the pH to 3, and then the solution is ultrasonically treated for 20 min (ultrasonic power is 400 W, and ultrasonic duty cycle is 33%), and the solution is heated to dissolve at 80 ℃, and then the temperature is lowered to 4 ℃ under stirring at a speed of 100 rpm to obtain the crystal. The PXRD pattern of the crystal is shown in Figure 1, and the characteristic peaks of the crystal are consistent with those of the temozolomide II crystal shown in Figure 2. The SEM pattern (Figure 3) of the crystal proves the successful preparation of the spherocrystal. The results show that the temozolomide II crystal spherocrystal is obtained in Example 1. Figure 2 Figure 1 The step of preparing the temozolomide spherocrystal based on nano-limited crystallization regulation of glycyrrhizic acid is as follows: 0.05 g of glycyrrhizic acid and 0.35 g of temozolomide are dissolved in 10 mL of pure water, 4.8 mg of sodium hydroxide is added to adjust the pH to 3, and then the solution is ultrasonically treated for 20 min (ultrasonic power is 400 W, and ultrasonic duty cycle is 33%), and the solution is heated to dissolve at 80 ℃, and then the temperature is lowered to 4 ℃ under stirring at a speed of 100 rpm to obtain the crystal. The PXRD pattern of the crystal is shown in Figure 1, and the characteristic peaks of the crystal are consistent with those of the temozolomide II crystal shown in Figure 2. The SEM pattern (Figure 3) of the crystal proves the successful preparation of the spherocrystal. The results show that the temozolomide II crystal spherocrystal is obtained in Example 1. Figure 5

[0048] Example 3

[0049] ​​​Dissolve 0.2 g glycyrrhizic acid and 0.6 g temozolomide in 10 mL of pure water, add 19.2 mg sodium hydroxide to adjust the pH to 3.5, sonicate for 30 min (ultrasonic power 600 W, ultrasonic duty cycle 66%), heat at 60 °C until dissolved, and then cool to 2 °C at 100 rpm to obtain crystals. The PXRD pattern of the crystals is attached. Figure 2 Crystal characteristic peaks and attached Figure 1 The temozolomide II crystals were consistent. The results showed that the temozolomide II spherulites obtained in Example 3 were indeed crystalline.

[0050] Example 4

[0051] Dissolve 0.1 g glycyrrhizic acid and 0.8 g temozolomide in 10 mL of pure water, add 9.6 mg sodium hydroxide to adjust the pH to 3.5, sonicate for 10 min (ultrasonic power 500 W, ultrasonic duty cycle 50%), heat at 80 °C until dissolved, and then cool to 4 °C at 150 rpm to obtain crystals. The PXRD pattern of the crystals is attached. Figure 2 Crystal characteristic peaks and attached Figure 1 The temozolomide III crystal form was consistent. The results showed that the temozolomide III spherulites obtained in Example 4 were indeed temozolomide III spherulites.

[0052] Comparative Example 1

[0053] 0.4 g of temozolomide was dissolved in 10 mL of pure water. The pH of the solution was adjusted to 3 using 0.1 mol / L hydrochloric acid solution. After sonication for 20 min (ultrasonic power 500 W, ultrasonic duty cycle 50%), the solution was heated to 80 °C until dissolved. The solution was then cooled to 4 °C at 100 rpm to obtain crystals. The PXRD pattern of the crystals is attached. Figure 2 Crystal characteristic peaks and attached Figure 1 The crystal form of temozolomide I was consistent with that of the sample. The results showed that the crystals obtained in Comparative Example 1 were of the temozolomide I crystal form.

[0054] The characterization test methods and test results of the embodiments and comparative examples of this invention are as follows:

[0055] This invention uses X-ray powder diffraction (PXRD) to characterize the polymorphic standard of temozolomide, the temozolomide spherulites prepared in Examples 1-4 and Comparative Example 1, respectively, as shown in the appendix. Figure 1 and attached Figure 2The characteristic peak values ​​(2θ±0.2°) of the temozolomide raw material I crystal form standard are: 10.8, 13.4, 14.2, 18.2, 19.3, 26.8, and 27.1. The characteristic peak values ​​(2θ±0.2°) of the temozolomide II crystal form standard are: 11.4, 13.1, 13.7, 16.4, 18.0, and 23.5. The characteristic peak values ​​(2θ±0.2°) of the temozolomide III crystal form standard are: 8.6, 12.0, 13.7, 17.4, 19.2, and 27.2. In comparison, the spherulites of the temozolomide II crystal form exhibit enhanced PXRD peaks at 15.0° and 18.0°, indicating directional growth along the (101) and (-101) crystal planes of the II crystal form. The enhanced characteristic peaks at 9.8° and 15.1° of temozolomide III spherulites indicate growth along the (001) and (011) crystal planes of the III form. This directional growth phenomenon is characteristic of spherulite formation, and the corresponding growth pattern determines the density of spherulite branches, thus affecting the spherulite roundness and formulation performance. (Comparison) Figure 2 and Figure 1 It can be seen that the products obtained in Examples 1-3 are temozolomide II crystalline spherulites, the product obtained in Example 4 is temozolomide III crystalline spherulites, and the product obtained in Comparative Example 1 is temozolomide I crystalline form.

[0056] The morphology and sphericity of the prepared temozolomide spherulites were characterized using a hot-stage microscope and field emission scanning electron microscopy (SEM), as shown in the appendix. Figure 3 and attached Figure 4 . Figure 3 The growth processes of temozolomide spherulites of crystal form II and III obtained in Examples 2 and 4 were characterized, respectively. Temozolomide crystal form II spherulites were obtained from needle-like crystal form II through secondary nucleation, achieving high sphericity and controllable particle size distribution through radioactive growth. Temozolomide crystal form III spherulites were obtained from elongated plate-like crystal form III through secondary nucleation, exhibiting relatively high sphericity and controllable particle size distribution. Furthermore, the present invention used scanning electron microscopy (SEM) to characterize the temozolomide crystal form II spherulites prepared in Example 2. Figure 4 The image shows a scanning electron microscope (SEM) image of the temozolomide II spherulites obtained in Example 2. The morphology clearly shows needle-like branching accumulation forming the temozolomide II spherulites, resulting in an uneven, recessed surface. These spherulites exhibit high sphericity, exceeding 0.9. In contrast, the temozolomide raw material I spherulite is plate-like with a sphericity below 0.6. The temozolomide III spherulites have a sphericity exceeding 0.8. The temozolomide II spherulites prepared in Example 2 have a sphericity of 0.95. The temozolomide III spherulites prepared in Example 4 have a sphericity of 0.83. In comparison, the temozolomide raw material I spherulite prepared in Comparative Example 1 has a sphericity of 0.6.

[0057] The particle size and polydispersity index of the temozolomide II crystal form spherulites prepared in Example 2 were characterized by dynamic light scattering (DLS) method, as shown in Figure 2. Figure 5 The particle size distribution of the temozolomide II crystal form spherulites was concentrated, with a particle size of 21.1 ± 3.2 pm and a polydispersity index of 0.107. The particle size of the temozolomide III crystal form spherulites prepared in Example 4 was 25.2 ± 8.2 pm, and the polydispersity index was 0.421. For comparison, the particle size of the temozolomide raw material I crystal form prepared in Comparative Example 1 was 33.2 ± 11.2 pm, and the polydispersity index was 0.965. The highly concentrated particle size distribution of the spherulites in the present application ensured the targeted deposition of the nasal olfactory region during the nasal delivery process, thereby improving the efficiency of the nasal brain delivery. Therefore, in Examples 1-4, the performance of the temozolomide spherulites met the requirements for nasal administration, compared with Comparative Example 1.

[0058] The in vitro dissolution performance of the temozolomide spherulites prepared in Examples 1-4 was characterized by a semi-permeable membrane dialysis method, as shown in Figure 3. Figure 6 The cumulative release amount of the raw temozolomide I crystal form within 60 minutes was 40%. Notably, the cumulative release amount within 15 minutes was less than 10%, indicating that the efficiency of the inhalation preparation was extremely low. In Example 4, the cumulative release amount of the temozolomide III crystal form spherulites within 15 minutes was 35%, and the increase in the specific surface area from bulk to spherulites achieved an increase in the dissolution rate. In the temozolomide II crystal form spherulite system, the cumulative release amount within 15 minutes was increased to 73.6%. The release rate of the temozolomide II crystal form spherulites in Example 2 was the fastest, and the nasal drug penetration efficiency during the nasal administration process was maximized. In Examples 1-4, the performance of the temozolomide spherulites met the requirements for nasal administration, compared with Comparative Example 1.

[0059] The cytotoxicity of the temozolomide spherulites prepared in Examples 1-4 was characterized by a 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) method, as shown in Figure 4. Figure 7 The cellular uptake of all crystal forms by U87 glioma cells was compared by fluorescence microscopy and flow cytometry. After 8 hours of incubation, the temozolomide II crystal form spherulites exhibited the highest cytotoxicity, with a cell survival rate of 4.7% (200 pg mL -1 ) and the lowest IC 50 value (51.9 pg mL -1 ). Notably, the temozolomide II crystal form spherulites were significantly superior to the temozolomide I crystal form (IC50 = 79.1 pg mL -1 ) and the temozolomide III crystal form spherulites (IC50 = 71.6 pg mL -1). The high cytotoxicity of temozolomide II crystal spherulites on U87 cancer cells reflects its superior anticancer effect. Therefore, the delivery efficiency of temozolomide II crystal spherulites through the nose into the brain was characterized next.

[0060] The in vivo fluorescence tracking method was used to characterize the in vivo distribution of the temozolomide II crystal spherulites prepared in Example 2, as shown in FIG. 6. Figure 8 In male BALB / c mice, the effects of different administration routes were evaluated by intragastric administration, intravenous administration, and intranasal administration of temozolomide II crystal spherulites. In vivo fluorescence imaging showed that the brain accumulation of the intranasal administration group was significantly higher than that of the intragastric administration group and the intravenous administration group, with fluorescence intensity in the brain region being 15.8 times and 7.1 times higher, respectively. This high-efficiency brain-targeted delivery effect reflects the advantages of temozolomide spherulites in the field of nasal delivery, significantly improving the therapeutic effect of temozolomide preparations.

[0061] The preparation conditions and results of each example and comparative example are compared in Table 1. All raw materials are commercially available, and the temozolomide raw material is stable I crystal form.

[0062] Table 1 Comparison of preparation conditions and results of each example and comparative example

[0063]

[0064] Table 2 Comparison of spherulite properties of each example and comparative example

[0065]

[0066] The temozolomide II crystal and III crystal spherulites prepared in the present application have higher sphericity and more concentrated particle size distribution, significantly improving the problems of low bulk density, irregular particle size distribution, and poor flowability of temozolomide, which helps to improve the deposition efficiency of the drug in the nasal cavity. At the same time, the dissolution rate of temozolomide II crystal and III crystal spherulites is significantly improved, proving the enhanced permeability of spherulites in the nasal cavity, which indicates that spherulites have higher bioavailability and are more suitable for nasal delivery. Cell experiments show that the cytotoxicity of temozolomide II crystal and III crystal spherulites is higher than that of the raw material I crystal, indicating that they have more advantages in anticancer effect. In particular, temozolomide II crystal, after nasal delivery, has higher fluorescence effect in the brain than conventional administration, proving its high efficiency in brain-targeted delivery.

[0067] The present application optimizes the performance of temozolomide preparations, improving their clinical application value. The prepared II crystal and III crystal spherulites have better physical properties, higher dissolution rate and targeting, and stronger anticancer effect. These advantages make them exhibit extremely high clinical application value in the treatment of glioblastoma through nasal delivery of temozolomide.

[0068] From the above, the temozolomide II crystal spherulite obtained by regulating the nanometer limited crystallization of glycyrrhizin provides an important basis for promoting the development of a drug delivery system for nasal brain, and provides a feasible scheme for brain-targeted cancer treatment.

[0069] The embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods, and are included in the protection scope of the present application.

Claims

1. A method for controlling temozolomide spherulites based on nano-confined crystallization, characterized in that: Glycyrrhizic acid and temozolomide were dissolved in pure water under ultrasonic conditions, the pH was adjusted to 3-4, heated until dissolved, and then cooled to 0-5℃ under stirring to obtain temozolomide spherulites based on nano-confined crystallization control.

2. The method for producing temozolomide spherulites based on nano-confined crystallization control according to claim 1, characterized in that, The mass ratio of glycyrrhizic acid to pure water is 0.005 to 0.05:

1.

3. The method for temozolomide spherulites based on nano-confined crystallization control according to claim 1, characterized in that, Add 0.1–0.8 g of temozolomide per milliliter of pure water.

4. The method for producing temozolomide spherulites based on nano-confined crystallization control according to claim 1, characterized in that, The ultrasound conditions are as follows: built-in ultrasound probe, ultrasound time of 10-30 minutes, ultrasound power of 400-600W, and ultrasound duty cycle of 33%-66%.

5. The method for producing temozolomide spherulites based on nano-confined crystallization control according to claim 1, characterized in that, The pH value is adjusted to 3-4 by adding sodium hydroxide, and the molar ratio of sodium hydroxide to glycyrrhizic acid is 1:1 to 3:

1.

6. The method for producing temozolomide spherulites based on nano-confined crystallization control according to claim 1, characterized in that, The stirring rate is 50 rpm to 200 rpm; the cooling rate is 10 to 40°C per minute; and the temperature is reduced to 1°C to 4°C under stirring conditions.

7. The method for producing temozolomide spherulites based on nano-confined crystallization control according to claim 1, characterized in that, The heating temperature for heating until the solution is clear is 60℃~80℃.

8. A temozolomide spherulite based on nano-confined crystallization regulation, characterized in that, It is prepared by the method according to any one of claims 1-7; and is temozolomide II crystalline spherulite or temozolomide III crystalline spherulite.

9. The temozolomide spherulites based on nano-confined crystallization control according to claim 8, characterized in that, The sphericity of the temozolomide spheroids is 0.6–0.95; the bulk density is 0.4–0.6 g / cm³. -3 The angle of repose is 22° to 40°; the particle size distribution is 15 to 25 μm; and the polydispersity index is 0.1 to 0.

5.

10. The use of the temozolomide spheroids based on nanoconfined crystallization as described in claim 8 or 9 as a cancer treatment drug delivered to the brain via nasal inhalation or spray inhalation.

Images