Clofazimine suspension and application thereof

Abstract

The invention discloses a medicine suspension taking clofazimine as a core and application of the medicine suspension, and the medicine suspension comprises near-infrared fluorescence and photoacoustic imaging effect for in-vivo tracing and near-infrared photothermal effect for tumor treatment, or combines near-infrared photothermal with the anti-tumor effect of a medicine, so that the medicine suspension can be applied to tumor treatment. The phototherapy and the traditional chemotherapy are combined, so that the effective inhibition effect on the occurrence and development of tumors is realized. Besides, the drug delivery system can realize long-acting retention of the drug at the tumor site, the treatment compliance is improved, and the drug delivery system with'five-in-one 'treatment imaging integrated characteristics of near-infrared photothermal treatment, near-infrared fluorescence imaging, photoacoustic imaging, traditional drug chemotherapy and long-acting retention is formed.

Description

Technical Field

[0001] The present invention relates to a clofazimine suspension and applications thereof, and in particular to a clofazimine suspension having excellent near-infrared photothermal, photoacoustic and fluorescence multiple effects and applications thereof. Background Art

[0002] Phototherapy mainly includes photodynamic therapy and photothermal therapy, which can achieve timed and localized treatment of tumors, making tumor treatment more controllable and individualized. Phototherapy can also accurately kill a variety of tumors, including subcutaneous tumors, minimizing damage to normal human cell tissues. Among them, thermal therapy destroys cell membranes, inhibits DNA synthesis, and kills cells through high temperature. The hypoxic and acidic environment in the tumor area makes tumor cells more sensitive to thermal stimulation, and because of the abnormal growth of blood vessels in the tumor site and poor heat dissipation, the temperature is 3 to 5 degrees higher than that of adjacent normal tissues, so tumor cells are more likely to die at the same high temperature.

[0003] The medium releases energy through the following three steps after being irradiated with light: (1) the incident light can be absorbed by the medium and transfer the energy to the molecules and atoms in the medium; (2) the molecules and atoms that absorb the energy quickly transition from the ground state (S0) to the excited state (Sn); (3) since the excited state is unstable, the molecules or atoms in the excited state will release energy directly back to the ground state through radiative decay and non-radiative decay, or first transition to the triplet state and then back to the ground state. In this process, light energy is converted into thermal energy (photothermal therapy, PTT) or acoustic energy (photoacoustic imaging, PAI). The above non-radiative decay forms are respectively suitable for photothermal treatment of diseases and in vivo tracing; while the fluorescent form of radiative decay is used for fluorescence imaging. Among them, near-infrared fluorescence imaging has the advantages of high sensitivity, while photoacoustic imaging has a tissue imaging depth that exceeds the penetration limit of traditional optical imaging. Combining near-infrared fluorescence imaging with photoacoustic imaging to achieve multimodal imaging can achieve ideal imaging depth while having high sensitivity, providing more reliable and comprehensive information for drug fate research in vivo and disease diagnosis.

[0004] Among the existing near-infrared responsive materials, near-infrared responsive materials such as metals, metal oxide semiconductors and carbon-based materials have problems with biocompatibility and biosafety, while small molecule dyes currently used in clinical angiography, such as indocyanine green (ICG) and methylene blue (MB), have problems such as insufficient stability, easy quenching and rapid metabolism, which have limitations in the clinical diagnostic application of the invention.

[0005] Clofazimine is an aminophenazine drug that can induce the production of ROS in tumor cells and specifically inhibit the carcinogenic Wnt signaling pathway. Oral soft capsules are currently commonly used to treat patients in clinical practice, but the poor water solubility of clofazimine and the existence of non-enzymatic metabolic pathways make its bioavailability low, requiring multiple doses, resulting in poor patient compliance. At the same time, the particle size of the capsule dosage form is too large and the specific surface area is small, which slows down the release of the drug from the entire drug delivery system to the gastrointestinal mucosa, thereby making the drug concentration in the gastrointestinal mucosa relatively low, resulting in a slow absorption rate of the drug into the blood, a slow absorption rate, and limited bioavailability.

[0006] Xueding Wang et al. respectively disclosed the application of clofazimine as a macrophage-targeted photoacoustic contrast agent (SciRep. 2016Mar 22, 6: 23528) and the photoacoustic imaging application of clofazimine hydrochloride nanoparticles accumulated in cancerous prostate and normal prostate (PLoS One. 2019Jul 15, 14(7): e0219655). Both studies used the photoacoustic responsiveness of clofazimine and its corresponding hydrochloride solvate at 450-540nm to achieve the diagnosis of pathological sections of lesions. However, the wavelength of 450-540nm has very limited tissue penetration ability and can only be limited to in vitro tissue sections. In addition, the sampling process of pathological sections is complicated and has poor compliance, which brings unignorable pain to patients. In addition, the visible light band has been plagued by low sensitivity and strong interference, which can lead to off-target and misjudgment. Summary of the invention

[0007] Purpose of the invention: The first purpose of the present invention is to provide a drug suspension with clofazimine as the core, the second purpose is to provide an application of the drug suspension in the field of tracers, and the third purpose is to provide an application of the drug suspension in the field of anti-tumor.

[0008] Technical solution: The clofazimine suspension of the present invention has an average particle size of 0.05 to 5 μm and is prepared by any of the following methods:

[0009] (1) Anti-solvent method: an organic solution containing clofazimine is stirred and reacted with a stabilizer aqueous solution or water to obtain the product;

[0010] (2) Grinding method: Grind chlorfaxamine in a stabilizer and water to obtain the product.

[0011] The present invention designs a drug delivery system for clofazimine injection suspension based on near-infrared photothermal, photoacoustic and fluorescence effects regulated by solid-state form. The drug delivery system utilizes the anti-tumor effects of near-infrared photothermal and the drug itself, and combines phototherapy with traditional chemotherapy to achieve a drug delivery system that integrates near-infrared phototherapy, near-infrared fluorescence and photoacoustic imaging, traditional drug chemotherapy and long-acting retention "five-in-one" therapeutic imaging, which is used in the field of multimodal tracers and combating the occurrence and development of cancer.

[0012] Preferably, the solid form of the clofazimine suspension is selected from Type I, Type II, Type III or amorphous.

[0013] Further preferably, when the solid form of the clofazimine suspension is Type I, it has characteristic diffraction peaks at 9.24°, 11.44°, 12.24°, 16.10°, 19.82°, 21.86°, 23.56°, and 24.70°, measured at 2θ±0.2°.

[0014] Further preferably, when the solid form of the clofazimine suspension is Type II, it has characteristic diffraction peaks at 10.18°, 13.92°, 15.4°, 16.76°, 17.7°, 19.3°, 20.12°, and 20.42°, measured at 2θ±0.2°.

[0015] Further preferably, when the solid form of the clofazimine suspension is Type III, it has characteristic diffraction peaks at 6.9°, 7.88°, 10.26°, 13.34°, 14.34°, 19.22°, 20.80°, and 22.16°, measured at 2θ±0.2°.

[0016] The suspensions corresponding to the crystal form I, crystal form II or crystal form III described in the present invention all have good near-infrared fluorescence AIE effect.

[0017] Preferably, when the solid form of the clofazimine suspension is Type I, Type II or Type III, its near-infrared fluorescence intensity is clofazimine concentration-dependent within a concentration range of 0 to 0.5 mM.

[0018] More preferably, when the solid form of the clofazimine suspension is type I, the correlation equation between its near-infrared fluorescence effect FL and the suspension concentration C is FL=32.24C+189.2, R 2 =0.9968.

[0019] More preferably, the concentration of the clofazimine suspension is 0.75-400 μM.

[0020] Preferably, when the solid form of the clofazimine suspension is form I, form II, form III or amorphous, its near-infrared photothermal conversion efficiency is 1% to 35%.

[0021] Further preferably, the photothermal conversion efficiency of Form I reaches 25.88%, the photothermal conversion efficiency of Form II is 1.36%, the photothermal conversion efficiency of Form III is 2.34%, and the amorphous form is as high as 32.01%.

[0022] The suspension corresponding to the crystal form I and the amorphous form described in the present invention has a good near-infrared photothermal effect, and can flexibly control the near-infrared photothermal conversion efficiency of the clofazimine suspension within the range of 1% to 35% by selecting different solid forms.

[0023] When the solid form of the suspension of the present invention is type I or amorphous, it has an obvious photoacoustic signal in the range of 680 to 970 nm; and regardless of whether it is crystal form I or amorphous, its photoacoustic signal has a good linear relationship related to the clofazimine concentration in the strong signal band including 720 nm.

[0024] Preferably, when the solid form of the clofazimine suspension is Type I, it has a photoacoustic signal intensity peak at 720 nm.

[0025] Preferably, when the solid form of the clofazimine suspension is amorphous, the photoacoustic signal intensity thereof gradually decreases with increasing wavelength within the range of 680 to 970 nm.

[0026] Preferably, when the solid form of the clofazimine suspension is Type I, the photoacoustic signal intensity is clofazimine concentration-dependent within a concentration range of 0 to 5 mM.

[0027] More preferably, when the solid form of the clofazimine suspension is Type I, at a wavelength of 720 nm, the correlation equation between the photoacoustic signal intensity PA and the concentration C is PA=0.4691C+0.037.

[0028] More preferably, the concentration of the clofazimine suspension is 5 mM.

[0029] Preferably, when the solid form of the clofazimine suspension is amorphous, its near-infrared fluorescence intensity is clofazimine concentration-dependent within a concentration range of 0 to 5 mM.

[0030] More preferably, when the solid form of the clofazimine suspension is amorphous, at a wavelength of 720 nm, the correlation equation between the photoacoustic signal intensity PA and the concentration C is PA=0.647C-0.112.

[0031] More preferably, the concentration of the clofazimine suspension is 5 mM.

[0032] Preferably, in the method (1), the organic solvent is dimethyl sulfoxide, the concentration of clofazimine in the organic solution is greater than 0.5 mg / mL, more preferably 0.5-5 mg / mL, and further preferably 2-5 mg / mL; the volume ratio of the organic solution of clofazimine to the aqueous stabilizer solution or water is 1:5-1:40, and further preferably 1:5-1:20; the stabilizer is selected from one or more of polysorbate, polyethylene glycol, polyethylene glycol 1000 vitamin E succinate, poloxamer, povidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose, gelatin, and soluplus; the mass volume ratio of the stabilizer to water is 0.1-100 mg / mL, and further preferably 0.1-10 mg / mL.

[0033] Preferably, the stirring speed is faster than 50 rpm and the stirring reaction time is not shorter than 5 min.

[0034] Preferably, in the method (2), the stabilizer is selected from one or more of polysorbate, polyethylene glycol, polyethylene glycol 1000 vitamin E succinate, poloxamer, povidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose, gelatin, and soluplus.

[0035] More preferably, the mass volume ratio of the stabilizer to water is 0.1 to 100 mg / mL, more preferably 2.5:1 to 100:1, and even more preferably 2.5 to 50 mg / mL.

[0036] Preferably, in the method (2), the grinding rotation speed is 200 to 1000 rpm.

[0037] More preferably, the grinding speed is 500-1000 rpm.

[0038] The drug suspension of the invention is prepared by a reverse solvent method or a wet ball milling method.

[0039] Reverse solvent method:

[0040] Under stirring and ultrasonic conditions, an organic solution containing clofazimine is added into water for reaction, and clofazimine crystals or amorphous forms with an average particle size of 0.05 to 5 μm are collected;

[0041] Wet ball milling method:

[0042] Under the condition of adding a certain stabilizer, grinding media and water are added, reacting for a period of time at a certain rotation speed, and collecting chlorfazimine crystals or amorphous with an average particle size of 0.05 to 5 μm.

[0043] Preferably, in the process of preparing the suspension by wet ball milling, the main functions of the stabilizer include preventing particle aggregation, improving dispersibility, adjusting particle size, extending the milling time, etc., and will not affect the near-infrared light responsiveness of the clofazimine system. Therefore, the stabilizer is not limited to one or more selected from polysorbate, polyethylene glycol, polyethylene glycol 1000 vitamin E succinate, poloxamer, povidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose, gelatin, and soluplus.

[0044] The clofazimine suspension of the present invention is used in preparing tracers for near-infrared photoacoustic and fluorescence imaging.

[0045] The clofazimine suspension of the present invention is used in the preparation of anti-tumor drugs.

[0046] Preferably, the drug is a drug for treating breast cancer.

[0047] Preferably, the drug is a drug used for anti-tumor near-infrared photothermal therapy, near-infrared fluorescence imaging or near-infrared photoacoustic imaging.

[0048] Preferably, the pharmaceutical concentration of the drug is 0.5-10 mM.

[0049] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0050] The suspension drug delivery system designed by the present invention can regulate near-infrared photothermal, photoacoustic and fluorescence effects based on solid-state form, and utilizes its near-infrared photothermal and anti-tumor effects of the drug itself, and combines phototherapy with traditional chemotherapy to achieve effective inhibition of tumor occurrence and development. At the same time, the good near-infrared fluorescence and photoacoustic properties give the drug delivery system the advantage of achieving multimodal imaging. In addition, the drug delivery system can achieve long-term retention of drugs at the tumor site, increase the compliance of the treatment, and form a drug delivery system with the integrated characteristics of near-infrared phototherapy, near-infrared fluorescence imaging, photoacoustic imaging, traditional drug chemotherapy and long-term retention "five-in-one" treatment imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The results of near-infrared photothermal conversion efficiency of different solid forms of clofazimine prepared in Example 14;

[0052] Figure 2 The full-band photoacoustic signal spectra at 680-970 nm of different solid forms of clofazimine prepared in Example 14;

[0053] Figure 3 The results of the photoacoustic signal intensity at 720 nm of different solid forms of clofazimine prepared in Example 14 as a function of concentration;

[0054] Figure 4a The near-infrared fluorescence AIE effect of the clofazimine crystal form I suspension prepared in Example 14;

[0055] Figure 4b The near-infrared fluorescence AIE effect of the clofazimine crystal form II suspension prepared in Example 14;

[0056] Figure 4c The near-infrared fluorescence AIE effect of the clofazimine crystal form III suspension prepared in Example 14;

[0057] Figure 4d The near-infrared fluorescence AIE effect of the clofazimine amorphous suspension prepared in Example 14;

[0058] Figure 5 The PXRD patterns of different solid forms (form I, II, III or amorphous) of clofazimine prepared in Example 14;

[0059] Figure 6 The PXRD patterns of different solid forms of clofazimine (form I, II, III or amorphous) predicted by mercury;

[0060] Figure 7 The near infrared imaging and bright field imaging results of the clofazimine mixture system with the same concentration and different water phase ratios of the clofazimine suspension prepared in Example 14;

[0061] Figure 8 Near infrared imaging of different concentration gradients of the clofazimine suspension prepared in Example 14;

[0062] Fig. 9 The near infrared spectra of different concentration gradients of the clofazimine suspension prepared in Example 14;

[0063] Fig.10 The fluorescence intensity trend line of the clofazimine suspension prepared in Example 14 at different concentrations at 824 nm;

[0064] Fig.11a The illustration shows the results of near-infrared fluorescence in vivo imaging 14 days after in situ injection.

[0065] Fig.11b The quantitative results of near-infrared fluorescence in vivo imaging within 14 days after in situ injection;

[0066] Fig.12a The illustrations show the results of the isolated tumor and major organs 14 days after orthotopic injection;

[0067] Figure 12bThe quantitative results of isolated tumors and major organs 14 days after in situ injection;

[0068] Fig.13a The illustration shows the results of near-infrared photoacoustic imaging 14 days after in situ injection.

[0069] Fig.13b The quantitative results of near-infrared photoacoustic imaging within 14 days after in situ injection;

[0070] Fig.14 The cytotoxicity results of clofazimine solution dissolved in DMSO on 4T1 cells at different concentrations;

[0071] Fig.15 The results of the cytotoxicity of the clofazimine suspension prepared in Example 14 to 4T1 cells under different treatment interventions;

[0072] Fig.16 Schematic diagram of the treatment for in vivo anti-tumor experiments;

[0073] Fig.17 The results of tumor volume changes within 14 days after the clofazimine suspension prepared in Example 14 was treated with different treatment strategies;

[0074] Fig.18 The tumor weight results after 14 days of treatment intervention with different treatment strategies for the clofazimine suspension prepared in Example 14. DETAILED DESCRIPTION

[0075] The technical solution of the present invention is further described below in conjunction with embodiments.

[0076] Example 1

[0077] 1 mL of a 5 mg / mL clofazimine solution dissolved in dimethyl sulfoxide was added to 20 mL of deionized water under stirring at 50 rpm and ultrasonic conditions for 5 minutes, and the clofazimine suspension was collected by suction filtration.

[0078] Example 2

[0079] 1 mL of 0.5 mg / mL clofazimine solution dissolved in dimethyl sulfoxide was added to 20 mL of deionized water under stirring at 50 rpm and ultrasonic conditions for 5 minutes, and the clofazimine suspension was obtained by collecting by suction filtration.

[0080] Example 3

[0081] 1 mL of a 5 mg / mL clofazimine solution dissolved in dimethyl sulfoxide was added to 20 mL of deionized water under stirring at 500 rpm and ultrasonic conditions for 5 min, and the clofazimine suspension was collected by suction filtration.

[0082] Example 4

[0083] 1 mL of 2 mg / mL clofazimine solution dissolved in dimethyl sulfoxide was added to 5 mL of deionized water under stirring at 50 rpm and ultrasonic conditions for 5 min, and the clofazimine suspension was collected by suction filtration.

[0084] Example 5

[0085] 1 mL of a 5 mg / mL clofazimine solution dissolved in dimethyl sulfoxide was added to 40 mL of deionized water under stirring at 50 rpm and ultrasonic conditions for 30 minutes, and the clofazimine suspension was obtained by collecting by suction filtration.

[0086] Example 6

[0087] 1 mL of a 5 mg / mL clofazimine solution dissolved in dimethyl sulfoxide was added to 10 mL of deionized water under stirring at 50 rpm and ultrasonic conditions for 5 minutes, and the clofazimine suspension was collected by suction filtration.

[0088] Example 7

[0089] 1 mL of 5 mg / mL clofazimine solution dissolved in dimethyl sulfoxide was added to 20 mL of deionized water containing 0.1 mg / mL soluplus under stirring at 50 rpm and ultrasonic conditions for 5 minutes, and the mixture was collected and concentrated by ultrafiltration to obtain a clofazimine suspension.

[0090] Example 8

[0091] 1 mL of 5 mg / mL clofazimine solution dissolved in dimethyl sulfoxide was added to 20 mL of deionized water containing 1 mg / mL soluplus under stirring at 50 rpm and ultrasonic conditions for 5 minutes, and the clofazimine suspension was obtained by collecting and concentrating by ultrafiltration.

[0092] Example 9

[0093] 1 mL of 5 mg / mL clofazimine solution dissolved in dimethyl sulfoxide was added to 20 mL of deionized water containing 10 mg / mL Tween 80 under stirring at 50 rpm and ultrasonic conditions for 5 minutes, and the mixture was collected and concentrated by ultrafiltration to obtain a clofazimine suspension.

[0094] Example 10

[0095] 50 mg of clofazimine (crystalline form I) was placed in a ball mill, and 6 g of grinding media and 4 mL of deionized water dissolved with poloxamer 407 (200 mg) were added, and the mixture was reacted at 800 rpm for 120 min, and a clofazimine suspension was obtained.

[0096] Embodiment 11

[0097] 50 mg of clofazimine (crystalline form I) was placed in a ball mill, and 6 g of grinding media and 4 mL of deionized water dissolved with poloxamer 407 (400 mg) were added, and the mixture was reacted at 800 rpm for 120 min, and a clofazimine suspension was obtained.

[0098] Example 12

[0099] 50 mg of clofazimine (crystal form I) was placed in a ball mill, and 6 g of grinding media and 4 mL of deionized water dissolved with soluplus (10 mg) were added. The mixture was reacted at 1000 rpm for 240 min, and a clofazimine suspension was obtained.

[0100] Embodiment 13

[0101] 50 mg of clofazimine (crystal form I) was placed in a ball mill, and 6 g of grinding media and 4 mL of deionized water dissolved with soluplus (10 mg) were added. The mixture was reacted at 500 rpm for 120 min, and a clofazimine suspension was obtained.

[0102] Comparative Example 1

[0103] 1 mL of 0.1 mg / mL clofazimine solution dissolved in dimethyl sulfoxide was added to 20 mL of deionized water under stirring at 50 rpm and ultrasonic conditions for 5 minutes, and the clofazimine suspension was obtained by collecting by suction filtration.

[0104] Comparative Example 2

[0105] 1 mL of a 5 mg / mL clofazimine solution dissolved in dimethyl sulfoxide was added to 20 mL of deionized water under stirring at 10 rpm and ultrasonic conditions for 5 minutes, and the clofazimine suspension was collected by suction filtration.

[0106] Comparative Example 3

[0107] 1 mL of a 5 mg / mL clofazimine solution dissolved in dimethyl sulfoxide was added to 1 mL of deionized water under stirring at 50 rpm and ultrasonic conditions for 5 minutes, and the clofazimine suspension was collected by suction filtration.

[0108] Comparative Example 4

[0109] 1 mL of a 5 mg / mL clofazimine solution dissolved in dimethyl sulfoxide was added to 20 mL of deionized water under stirring at 50 rpm and ultrasonic conditions for 1 min, and the clofazimine suspension was collected by suction filtration.

[0110] Comparative Example 5

[0111] 1 mL of a 5 mg / mL clofazimine solution dissolved in dimethyl sulfoxide was added to 50 mL of deionized water under stirring at 50 rpm and ultrasonic conditions for 5 min, and the clofazimine suspension was collected by suction filtration.

[0112] Compared with Example 1, the parameters of Comparative Examples 1 to Comparative Examples 5 are changed as shown in Table 1.

[0113] Table 1 Parameter comparison table

[0114]

[0115] Compared with Example 1, in Comparative Example 1, in step S1, the concentration of clofazimine is 0.1 mg / mL, and after the stirring is completed, the system is orange-yellow and there are a small number of crystals visible to the naked eye in the system. During the particle size analysis using a Malvern particle size analyzer, it was found that the final particle size was too large and the average particle size exceeded the range of the particle size analyzer.

[0116] Compared with Example 1, in Comparative Example 2, the rotation speed in step S1 is 10 rpm. After the stirring is completed, the particles are collected and analyzed by using a Malvern particle size analyzer. It is found that the particle size PDI value exceeds 0.5, indicating that the particle size uniformity is poor.

[0117] Compared with Example 1, in Comparative Example 3, in step S1, the ratio of the organic phase to the aqueous phase is 1:1, and after the stirring is completed, the crystals are collected and analyzed using a Malvern particle size analyzer, and it is found that the particle size is too large and exceeds the range.

[0118] Compared with Example 1, in Comparative Example 4, the reaction time in step S1 is 1 min. After the stirring is completed, the particles are collected immediately and the particle size is analyzed using a Malvern particle size analyzer. It is found that the particle size of the particles gradually increases with time, indicating that the system is not balanced.

[0119] Compared with Example 1, in Comparative Example 5, in step S1, the ratio of the organic phase to the aqueous phase is 1:50. After the stirring is completed, the crystals are collected and analyzed using a Malvern particle size analyzer. It is found that the particle size distribution is uneven and the PDI is greater than 0.3.

[0120] As shown in Table 1, in the process of preparing clofazimine, due to the good solubility of clofazimine in dimethyl sulfoxide (up to 5 mg / mL), dimethyl sulfoxide is selected as the organic phase of the present invention; when the concentration of the clofazimine solution is too low (less than 0.5 mg / mL), the system cannot quickly generate a large number of crystal nuclei due to the low concentration and the supersaturation condition is not reached, and the clofazimine molecules dissolved in dimethyl sulfoxide that have not yet precipitated the crystal nuclei will be gradually absorbed by the crystal nuclei that are precipitated first, and eventually grow into large-sized crystals (Comparative Example 1). It takes time for the system to reach equilibrium from the generation of crystal nuclei to the absorption of solutes. If the reaction time is too short (less than 5 min), the system does not reach equilibrium, and the crystal nuclei in the system will continue to absorb solutes until equilibrium is obtained to obtain clofazimine microparticles with uniform particle size that meet the experimental requirements (Comparative Example 4).

[0121] When the ratio of organic phase to aqueous phase is greater than 1:5 or less than 1:20, due to the inability to ensure that the many problems such as fully dispersed uniformly, the particle size will also be too large. When the volume difference between organic phase and aqueous phase is too large, the organic phase cannot achieve rapid and uniform dispersion in aqueous phase, and the local supersaturated system that cannot be quickly dispersed uniformly is extremely unstable, so the whole system first locally precipitates a small amount of crystal nuclei, resulting in the future being able to precipitate crystal nuclei dissolved in dimethyl sulfoxide. The chlorfazimine that will be gradually absorbed by the crystal nuclei first precipitated grows into large-sized crystals (Comparative Example 5). And as the volume ratio of subsequent organic phase and aqueous phase decreases, the organic phase can achieve rapid dispersion in aqueous phase and gradually reach the supersaturated conditions necessary for generating crystals, so that the whole system can achieve rapid generation of a large number of crystal nuclei, so that each crystal nucleus absorbs relatively average and controllable chlorfazimine molecules. When the volume difference between organic phase and aqueous phase is too small, it is difficult to obtain microparticles within the target range (Comparative Example 3) because the system is difficult to make the organic phase and aqueous phase quickly distributed uniformly by stirring and ultrasound.

[0122] The process of preparing the clofazimine suspension drug delivery system of the present invention requires a certain stirring speed (greater than 50 rpm) to help the system achieve rapid and uniform mixing of the organic phase and the aqueous phase. When the stirring speed is too low (less than 50 rpm), the organic phase and the aqueous phase cannot be quickly and uniformly mixed, resulting in the local generation of a large number of crystal nuclei and the beginning of absorption of solute molecules, and ultimately the entire system obtains a crystal drug delivery system with a larger particle size and poor uniformity (Comparative Example 2).

[0123] The clofazimine suspension (Examples 10 to 13) was prepared by wet ball milling. The particle size of this type of example varied depending on the stabilizer and gradually decreased with the increase in rotation speed and the extension of grinding time. The overall particle size was in the range of 0.05 to 5 μm.

[0124] Example 14: Preparation of suspensions in different solid forms

[0125] 1. Preparation of different polymorphic or amorphous clofazimine

[0126] 0.5 g of clofazimine (Form I purchased from the market) was dispersed in 5 mL of anhydrous ether, stirred evenly on a magnetic stirrer (800 rpm) for reaction for 24 h, and filtered and dried after the reaction to obtain clofazimine Form II (Form II); 1.0 g of clofazimine was dissolved in 5 mL of toluene, reacted on a heating and stirring device for 24 h (40° C., 800 rpm), and filtered and dried after the reaction to obtain clofazimine Form III (Form III); amorphous clofazimine was prepared by melt cooling, and the clofazimine powder was heated to 220° C., and then quenched to make it rapidly solidified after it was fully melted, and the obtained amorphous solid (Amorphous) was ground and sieved to obtain an amorphous powder of clofazimine.

[0127] 2. Preparation of suspension

[0128] The suspension of clofazimine in different crystal forms or amorphous forms is obtained by ball milling as follows:

[0129] 50 mg of clofazimine (Form I, Form II and Form III or amorphous), 4 mL of deionized water dissolved with 200 mg of poloxamer 407 and 6 g of oxide beads (average particle size 0.5-1 mm) were added to a ball mill and reacted at 800 rpm for 2 h. After the reaction, the mixture was filtered, washed and resuspended to obtain a clofazimine suspension (Form I, Form II, Form III or amorphous).

[0130] Example 15: Photothermal effect evaluation

[0131] Use 3W / cm 2 The clofazimine suspension (FI, F II, F III or amorphous form) prepared according to Example 14 with a concentration of 3 mg / mL was irradiated with an 808 nm laser for 10 min. During this process, the temperature change was recorded with an infrared camera every 0.5 min, and then the photothermal conversion efficiency was obtained by calculation and fitting. Figure 1 As shown, the photothermal conversion efficiency of Form I reached 25.88%, the photothermal conversion efficiency of Form II was 1.36%, the photothermal conversion efficiency of Form III was 2.34%, and the amorphous form was as high as 32.01%.

[0132] Example 16: Photoacoustic effect

[0133] The different suspensions prepared according to Example 14 with a clofazimine concentration of 5 mM were subjected to full-band (680-970 nm) photoacoustic signal scanning. Figure 2As shown, Form I of the same concentration exhibits obvious photoacoustic responsiveness and reaches the peak of the photoacoustic signal at 720nm. Form II and Form III have certain photoacoustic responsiveness compared with deionized water, but their signal intensity is weaker. Under the same conditions, the amorphous form exhibits a stronger photoacoustic signal than Form I and does not show a signal peak similar to the polymorphic form. The overall signal shows a trend of gradually weakening with increasing wavelength.

[0134] On this basis, the single wavelength (720 nm) photoacoustic signal detection and analysis were performed on the clofazimine suspensions of different concentrations prepared according to Example 14. Figure 3 As shown, the photoacoustic signals of Form I and amorphous at 720 nm have good concentration dependence, and the amorphous has a stronger photoacoustic signal than Form I. In comparison, Form II and Form III have a certain concentration dependence but their signal intensity is weaker and the linear relationship is not ideal.

[0135] Example 17: Near-infrared fluorescence AIE

[0136] The clofazimine suspension (Form I, Form II, Form III or amorphous) prepared according to Example 14 with a concentration of 400 μM and the clofazimine solution (DMSO) were mixed in different volume ratios (10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10) to obtain a 400 μM clofazimine mixed system with different non-good solvent ratios, and the fluorescence spectrum was scanned (Ex = 640 nm; Em = 800-900 nm). The results are as follows Figure 4a to Figure 4d As shown, a 400 μM clofazimine suspension (deionized water) and a clofazimine solution (DMSO) were mixed in different volume ratios to obtain a 400 μM clofazimine mixed system with different non-good solvent ratios, and the fluorescence spectrum was scanned (Ex = 640nm; Em = 800-900nm). The results show that Form I, Form II and Form III all have near-infrared AIE effects and the fluorescence intensity has no obvious difference, while the amorphous suspension shows an extremely limited fluorescence effect.

[0137] Example 18: Particle Size and Characterization

[0138] The clofazimine suspensions in Examples 1 to 13 were respectively prepared with deionized water to a suspension concentration of 0.3 mg / mL, and then the particle size and uniformity thereof were measured at 37° C. The results are shown in Table 2.

[0139] Table 2 Particle size and PDI results of suspensions

[0140] name Average particle size (nm) PDI Example 1 1532.4 0.210 Example 2 1027.6 0.256 Example 3 1126.8 0.227 Example 4 1986.5 0.279 Example 5 2645.8 0.246 Example 6 4354.1 0.271 Example 7 180.7 0.124 Example 8 68.50 0.087 Example 9 61.4 0.94 Example 10 726.7 0.247 Embodiment 11 1187.4 0.265 Example 12 486.5 0.213 Embodiment 13 4934.8 0.297

[0141] As shown in Table 2, the average particle size of the clofazimine suspension in deionized water is 0.05-5 μm and the PDI is below 0.3, indicating that the clofazimine suspension prepared by the present invention has an average particle size of 0.05-5 μm and good uniformity.

[0142] Example 19: PXRD Characterization

[0143] The different solid forms of clofazimine prepared according to Example 14 were analyzed by X-ray diffraction. The results are as follows: Figure 5 and as shown in Table 3. Comparing this result with the result predicted by mercury ( Figure 6 ), indicating that the present invention has prepared the corresponding clofazimine polymorphs and amorphous forms respectively.

[0144] Table 3 PXRD suspension characteristic peaks

[0145]

[0146] Example 20: In vivo and in vitro near-infrared fluorescence imaging

[0147] The mixed systems of clofazimine (Form I) with the same concentration and different water phase ratios in Example 17 were added to a 96-well plate for near infrared imaging. Figure 7 As shown, Form I exhibits good near-infrared AIE properties in an in vitro environment.

[0148] The clofazimine suspension (Form I) was diluted with deionized water in a concentration gradient, and the clofazimine suspension (Form I) of different concentrations was scanned by near-infrared fluorescence spectrum (Ex=640nm; Em=800-900nm). In addition, the clofazimine suspension (Form I) of different concentration gradients was added to a 96-well plate for near-infrared imaging. Figure 8 to Figure 10 As shown, the near-infrared fluorescence effect of clofazimine suspension (Form I) is concentration-dependent.

[0149] BALB / c female mice aged 6 to 8 weeks were marked and depilated, and 50 μL of 4T1 cells with a density of 1×10 7 The cell suspension was injected subcutaneously into the flank of the mouse until the tumor volume reached 100-200 mm 3At the same time, the clofazimine suspension (Form I) prepared according to Example 14 was injected into the mouse tumor at a dose of 10 mg / kg. After drug intervention, near-infrared in vivo imaging was performed on the tumor-bearing mice injected intratumorally on days 0, 1, 3, 7 and 14, and the mice were euthanized after near-infrared in vivo imaging on day 14, and the tumors and major organs (heart, liver, spleen, lungs and kidneys) were taken for near-infrared imaging. Figure 11a to Figure 11b As shown in the figure, within 0 to 14 days, the fluorescence signal intensity of the mouse tumor site gradually weakened, but relatively strong fluorescence signals were detected. In addition, on the 14th day, in addition to the tumor tissue, strong fluorescence signals were detected in the liver, spleen and kidney of the mouse ( Figure 12a to Figure 12b ). This is because clofazimine is a highly lipophilic small molecule compound that can accumulate in mammalian resident tissue macrophages as inclusion bodies and eventually bioaccumulate and crystallize in large quantities in the liver and spleen. In addition, clofazimine is mainly metabolized by the liver and kidneys, which is one of the reasons for its accumulation in the liver and kidneys.

[0150] Example 21: In vivo photoacoustic imaging

[0151] BALB / c female mice aged 6 to 8 weeks were marked and depilated, and 50 μL of 4T1 cells with a density of 1×10 7 The cell suspension was injected subcutaneously into the flank of the mouse until the tumor volume reached 100-200 mm 3 At 10 mg / kg, the clofazimine suspension (Form I) prepared according to Example 14 was injected into the mouse tumor. After drug intervention, photoacoustic imaging (720 nm) was performed on the tumor-bearing mice injected intratumorally on days 0, 7, and 14. Figure 13a to Figure 13b As shown, a relatively high photoacoustic signal was maintained for 14 days after intratumoral injection, indicating that clofazimine suspension (Form I) can achieve a long-lasting retention effect after intratumoral injection.

[0152] Example 22: In vitro anti-tumor experiment

[0153] 4T1 cells were cultured at 5 × 10 3 Cells were seeded at a density of 100 cells / well on a 96-well plate and cultured in DMEM complete medium containing 10% fetal bovine serum, 120 mg / L penicillin and 200 mg / L streptomycin. After incubation for 24 h with different doses of clofazimine (molecular state) dissolved in DMSO, the cell viability of the cells in different treatment groups was determined at 450 nm using the CCK-8 method. Fig.14As shown in the figure, as the final concentration of clofazimine gradually increases, the cell activity gradually decreases. Since the photothermal effect of clofazimine suspension (Form I) is concentration-dependent, it exhibits a significant photothermal effect at higher concentrations (above 0.25 mg / mL). However, at this concentration, the cytotoxic effect of clofazimine molecules on 4T1 is close to 100%. In order to reflect the cytotoxicity and photothermal effect of clofazimine suspension (Form I) at the cellular level, 2W / cm 2 After 808 nm laser irradiation of cells (to prevent interference from laser heat generation), the photothermal effect of high-dose clofazimine suspension (Form I) was replaced by a 50°C water bath for 10 min. Blank control group, Laser control group, CFZ group and CFZ+50°C+Laser (CFZ+PTT) group were set up. Each group was cultured for 24 hours after treatment, and the CCK-8 method was used to analyze the viability of cells in different treatment groups. Fig.15 As shown. Compared with the Blank control group, the Laser control group did not show obvious cytotoxicity, while the CFZ group significantly reduced cell viability. In addition, the CFZ+PTT group showed more significant cytotoxicity.

[0154] Example 23: In vivo anti-tumor experiment

[0155] BALB / c female mice aged 6 to 8 weeks were marked and depilated, and 50 μL of 4T1 cells with a density of 1×10 7 The cell suspension was injected subcutaneously into the flank of the mouse until the tumor volume reached 100-200 mm 3 At 10 mg / kg, the clofazimine suspension (Form I) prepared according to Example 14 was injected into the mouse tumor. Fig.16 The 4T1 tumor-bearing mice were treated with the method shown in the figure, the tumor volume was recorded every day, and the mice were killed on the 14th day and the tumor was weighed. Figure 17-18 As shown, there was no significant difference between the PBS group and the PBS+Laser group. The CFZ and CFZ+Laser groups had significant tumor inhibitory effects compared with the PBS group. The CFZ+Laser group had a more significant tumor inhibitory effect than the CFZ group. The experimental results were significantly different.

Claims

1. A clofazimine suspension, characterized in that: The average particle size is 0.05 to 5 μm, and it is prepared by any of the following methods: (1) Anti-solvent method: an organic solution containing clofazimine is stirred and reacted with a stabilizer aqueous solution or water to obtain the product; (2) Grinding method: Grind chlorfaxamine in a stabilizer and water to obtain the product.

2. The clofazimine suspension according to claim 1, characterized in that Its solid state form is selected from Form I, Form II, Form III or amorphous form.

3. The clofazimine suspension according to claim 1, characterized in that When the solid form is type I, type II or type III, the near-infrared fluorescence intensity is clofazimine concentration-dependent within the concentration range of 0 to 0.5 mM.

4. The clofazimine suspension according to claim 1, characterized in that When the solid form is type I, type II, type III or amorphous, the near-infrared photothermal conversion efficiency is 1% to 35%.

5. The clofazimine suspension according to claim 1, characterized in that When the solid form is type I or amorphous, the photoacoustic signal intensity is clofazimine concentration-dependent within the concentration range of 0 to 5 mM.

6. The clofazimine suspension according to claim 1, characterized in that In the organic solution containing clofazimine, the concentration of clofazimine is greater than 0.5 mg / mL; the volume ratio of the organic solution containing clofazimine to the stabilizer aqueous solution or water is 1:5 to 1:

40.

7. The clofazimine suspension according to claim 1, characterized in that The stabilizer is selected from one or more of polysorbate, polyethylene glycol, polyethylene glycol 1000 vitamin E succinate, poloxamer, povidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose, gelatin, and soluplus.

8. The clofazimine suspension according to claim 7, characterized in that In the method (2), the mass volume ratio of the stabilizer to water is 0.1 to 100 mg / mL.

9. Use of the clofazimine suspension according to claim 1 in preparing a tracer for near-infrared photoacoustic and fluorescence imaging.

10. Use of the clofazimine suspension according to claim 1 in the preparation of anti-tumor drugs.

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