A clofazimine suspension and application thereof

Abstract

A clofazimine-based pharmaceutical suspension and an application thereof, wherein a drug delivery system has near-infrared fluorescence, photoacoustic imaging effect and red dye characteristics for in vivo tracing, as well as a near-infrared photothermal effect for tumor treatment. The drug delivery system combines the near-infrared photothermal effect with the anti-tumor effect of the drug itself, and adopts a combination of phototherapy and traditional chemotherapy to achieve effective inhibition of tumor occurrence and development; in addition, by combining the near-infrared fluorescence effect and the photoacoustic effect, a multimodal combined tracing technique can be used to assist intraoperative lymph node dissection, and the drug can achieve long-term retention at the tumor site, overcoming the limitations of traditional imaging in "black box" environments of strong light or deep tissues, making it more suitable for intraoperative navigation; the system possesses good biosafety, high sensitivity, high tissue penetration and photostability, which can reduce the inconvenience of multiple clinical administrations and increase compliance.

Description

A Chlorfenapyridine Suspension and Its Application Technical Field

[0001] This invention relates to a chlorfenapyridine suspension and its application, and more particularly to a chlorfenapyridine suspension with excellent near-infrared photothermal effect, photoacoustic imaging, fluorescence imaging and dye staining tracing properties and its application. Background Technology

[0002] Phototherapy mainly includes photodynamic therapy and photothermal therapy, enabling timed and targeted treatment of tumors, making tumor treatment more controllable and personalized. Phototherapy can also precisely kill various tumors, including subcutaneous tumors, minimizing damage to normal human cells and tissues. Specifically, photothermal therapy destroys cell membranes and inhibits DNA synthesis through high temperatures, killing cells. The hypoxic and acidic environment of the tumor area makes tumor cells more sensitive to heat stimulation. Furthermore, due to abnormal blood vessel growth and poor heat dissipation in the tumor site, its temperature is 3-5°C higher than that of adjacent normal tissues, making tumor cells more likely to die under the same high temperature.

[0003] The release of energy by a medium upon illumination occurs through three steps: (1) the incident light is absorbed by the medium, transferring energy to molecules and atoms within it; (2) the absorbed molecules and atoms rapidly transition from the ground state (S0) to the excited state (Sn); (3) due to the instability of the excited state, molecules or atoms in the excited state release energy through radiative decay and non-radiative decay, returning directly to the ground state or first transitioning to the triplet state before returning to the ground state. During this process, light energy is converted into heat energy (photothermal therapy, PTT) or acoustic energy (photoacoustic imaging, PAI). These non-radiative decay forms are applicable to photothermal therapy and in vivo tracing of diseases, respectively; while radiative decay in the form of fluorescence is applied to fluorescence imaging. Near-infrared fluorescence imaging has advantages such as high sensitivity, while photoacoustic imaging has tissue imaging depth exceeding the penetration limit of traditional optical imaging. Combining near-infrared fluorescence imaging with photoacoustic imaging to achieve multimodal imaging can obtain ideal imaging depth while maintaining high sensitivity, providing more reliable and comprehensive information for drug fate studies and disease diagnosis.

[0004] Compared to visible light fluorescence imaging, near-infrared I (NIR-I, 700–900 nm) fluorescence imaging offers superior background noise reduction, millimeter-level tissue penetration, and higher imaging resolution, making it a promising candidate for assisting in vivo diagnosis and treatment. Compared to NIR-I fluorescence imaging, near-infrared II (NIR-II, 1000–1700 nm) fluorescence imaging, with its further emission, boasts deeper tissue penetration (up to centimeter-level), high signal-to-noise ratio, and micrometer-level spatial resolution, and has already been applied to early tumor diagnosis. In surgical navigation, the use of NIR-II fluorescent materials enables real-time, high-resolution imaging of vascular and neural structures during surgery, advancing the clinical translation of integrated diagnosis and treatment. The long wavelength of NIR-II fluorescence emission significantly reduces tissue scattering, achieving a penetration depth 2–3 times that of NIR-I (e.g., penetrating 15 mm thick tissue), and a vascular imaging resolution of up to 47.6 μm. Secondly, the NIR-II region exhibits extremely low autofluorescence background, and combining it with high-absorption-coefficient probes (such as semiconductor polymers) can improve the signal-to-noise ratio by more than 1.46 times. Furthermore, NIR-II fluorescent probes (such as rare-earth nanoparticles) possess superior biocompatibility and metabolic stability.

[0005] Among existing near-infrared responsive materials, near-infrared responsive materials such as metals, metal oxide semiconductors, and carbon-based materials have biocompatibility and biosafety issues. Meanwhile, small molecule dyes such as indocyanine green (ICG) and methylene blue (MB), which are currently used in clinical imaging, have problems such as insufficient stability, easy quenching, and rapid metabolism, which limit their clinical diagnostic applications.

[0006] In cancer treatment, lymph node metastasis significantly reduces patient survival rates. To improve surgical prognosis, lymph node tracers are often used clinically to assist lymph node dissection. However, existing methods such as indigo staining, radioactive tracing, and fluorescence tracing have the following limitations: Clinically, the most commonly used lymph node tracing method is a combination of radioactive tracer methods. Radioactive tracer methods involve injecting a radioactive isotope, allowing the tracer to spread through the lymphatic system and visualize lymph node activity on imaging. Although this method effectively locates lymph nodes and provides clear images, the use of radioactive isotopes may pose radiation exposure risks, the distribution of radioactive tracers exhibits individual variability, and the limited half-life restricts the image capture window, thus limiting its clinical application to some extent. Secondly, while injecting blue dyes (such as methylene blue) to visualize lymph node drainage is simple and economical, the rapid diffusion of the dye can reduce sensitivity, and in patients with a history of allergies, indigo staining may cause allergic reactions.

[0007] Existing NIR-II fluorescent materials still face multiple limitations in practical applications: The fabrication process for high-performance probes is complex. For example, rare-earth-doped nanoparticles (such as CeF3:Yb / Er) require high-temperature hydrothermal synthesis and precise shell coating to improve quantum yield, resulting in poor batch reproducibility and difficulty in large-scale production. Secondly, the trade-off between quantum yield and biosafety limits clinical imaging applications. While most inorganic materials (such as PbS quantum dots) exhibit high luminescence efficiency in the NIR-IIb (1500–1700 nm) band, their heavy metal components may cause long-term toxicity. Furthermore, insufficient functional integration means that existing materials often focus on a single imaging function, failing to meet the multiple requirements of therapeutic integration, such as targeted modification, photothermal conversion, and drug loading, thus hindering the leapfrog development of NIR-II fluorescence technology from the laboratory to the clinical setting. Therefore, finding safer and more photostable NIR-II fluorescent tracer materials for clinical diagnosis, including lymph node tracing, is of great significance. This will improve cancer treatment outcomes, patient prognosis and quality of life, and provide more powerful diagnostic and treatment options for the early diagnosis and intervention of lymph node metastasis.

[0008] Clofazimine is an aminophenazine antibiotic that induces the production of reactive oxygen species (ROS) in tumor cells and specifically inhibits the oncogenic Wnt signaling pathway. Currently, oral soft capsules are commonly used clinically for treatment. However, clofazimine's poor water solubility and non-enzymatic metabolism result in low bioavailability, requiring multiple doses and leading to poor patient compliance. Furthermore, the large particle size and small surface area of ​​the capsule formulation slow down the release of the drug into the gastrointestinal mucosa, resulting in relatively low drug concentrations and consequently, slower absorption into the bloodstream, thus limiting bioavailability.

[0009] Xueding Wang et al. published their research on the application of clofazimine as a macrophage-targeting photoacoustic contrast agent (Sci Rep. 2016 Mar 22, 6:23528) and the photoacoustic imaging application of clofazimine hydrochloride nanoparticles accumulating in cancerous and normal prostates (PLoS One. 2019 Jul 15, 14(7):e0219655). Both studies utilized the photoacoustic responsiveness of clofazimine and its corresponding hydrochloric acid solvates in the 450–540 nm range to diagnose pathological sections of lesions. However, the 450–540 nm wavelength has very limited tissue penetration, limiting its application to ex vivo tissue sections. Furthermore, the sampling process for pathological sections is complex and has poor compliance, causing significant discomfort to patients. In addition, the visible light band has been hampered by low sensitivity and strong interference, leading to off-target effects and misdiagnosis. In addition, CN102397561A discloses the application of mitoxantrone as a lymphatic tracer. Due to the high toxicity of mitoxantrone, its application in special populations such as pregnant women and infants is very limited. Summary of the Invention

[0010] Purpose of the invention: In view of the problems of existing tracers having limited function and poor safety, the first purpose of this 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 therapy.

[0011] Technical solution: The chloramphenicol suspension of the present invention has an average particle size of 10 nm to 50 μm, and is prepared by any of the following methods:

[0012] (1) Antisolvent method: The organic solution containing chlorazimine is reacted with an aqueous solution of stabilizer or water by stirring;

[0013] (2) Grinding method: Chloride is ground in stabilizer and water to obtain the product.

[0014] Preferably, the mass-to-volume ratio of the stabilizer to water is 0.1 to 100 mg / mL, more preferably 2.5:1 to 100:1 mg / mL, and even more preferably 2.5 to 50 mg / mL.

[0015] The chloramphenicol suspension of the present invention has an average particle size of 0.05–5 μm and is prepared by any of the following methods:

[0016] (1) Antisolvent method: The organic solution containing chlorazimine is reacted with an aqueous solution of stabilizer or water by stirring;

[0017] (2) Grinding method: Chloride is ground in stabilizer and water to obtain the product.

[0018] This invention designs a drug delivery system for clofazimine injection suspension based on near-infrared photothermal, photoacoustic, and fluorescence effects regulated by solid-state form. This drug delivery system utilizes the near-infrared photothermal effect of the drug and its own anti-tumor effect, and combines phototherapy with traditional chemotherapy to achieve a "five-in-one" integrated therapeutic imaging drug delivery system that combines near-infrared phototherapy, near-infrared fluorescence and photoacoustic imaging, traditional drug chemotherapy, and long-acting retention. It can be applied in the fields of multimodal tracers and combating the occurrence and development of cancer.

[0019] Preferably, the solid form of the chlorfenapyridine suspension is selected from type I, type II, type III or amorphous.

[0020] Further preferably, when the solid form of the chlorfenapyridine suspension is type I, it has diffraction characteristic peaks at 9.24°, 11.44°, 12.24°, 16.10°, 19.82°, 21.86°, 23.56°, and 24.70°, calculated at 2θ±0.2°.

[0021] Further preferably, when the solid form of the chlorfenapyridine suspension is type II, it has diffraction characteristic peaks at 10.18°, 13.92°, 15.4°, 16.76°, 17.7°, 19.3°, 20.12°, and 20.42°, calculated at 2θ±0.2°.

[0022] Further preferably, when the solid form of the chlorfenapyridine suspension is type III, it has diffraction characteristic peaks at 6.9°, 7.88°, 10.26°, 13.34°, 14.34°, 19.22°, 20.80°, and 22.16°, calculated at 2θ±0.2°.

[0023] Preferably, the active ingredient in the chlorfazimine suspension is chlorfazimine.

[0024] Preferably, the chlorfenapyridine suspension exhibits NIR-I fluorescence emission signal in the wavelength range of 800–900 nm.

[0025] The suspensions corresponding to crystal form I, crystal form II, or crystal form III described in this invention all exhibit good near-infrared fluorescence (AIE) effect.

[0026] Preferably, when the chlorfazimine suspension is in solid form as type I, type II or type III, its near-infrared fluorescence intensity is concentration-dependent on chlorfazimine within the concentration range of 0 to 0.5 mM.

[0027] Further preferably, when the solid form of the chlorpheniramine 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, R2 =0.9968.

[0028] More preferably, the concentration of the chlorfenapyridine suspension is 0.75–400 μM.

[0029] Preferably, when the solid form of the chlorfenapyr suspension is crystal form I, crystal form II, crystal form III or amorphous, its near-infrared photothermal conversion efficiency is 1% to 35%.

[0030] Further optimization revealed that the photothermal conversion efficiency of Form I reached 25.88%, that of Form II was 1.36%, that of Form III was 2.34%, and that of the amorphous form was as high as 32.01%.

[0031] The crystalline form I and amorphous suspensions described in this invention have good near-infrared photothermal effects, and the near-infrared photothermal conversion efficiency of the chloramine suspension can be flexibly controlled within the range of 1% to 35% by selecting different solid forms.

[0032] When the solid form of the suspension of the present invention is type I or amorphous, it has obvious photoacoustic signal in the range of 680 to 970 nm; and regardless of whether it is crystal type I or amorphous, its photoacoustic signal has a good concentration-dependent linear relationship with chlorpromazine in the strong signal band including 720 nm.

[0033] Preferably, when the chlorfenapyridine suspension is in solid form type I, it has a photoacoustic signal intensity peak at 720 nm.

[0034] Preferably, when the chlorfenapyridine suspension is in amorphous solid form, its photoacoustic signal intensity gradually decreases with increasing wavelength in the range of 680–970 nm.

[0035] Preferably, when the chlorfazimine suspension is in solid form (Type I), its photoacoustic signal intensity exhibits a chlorfazimine concentration-dependent relationship within the concentration range of 0–5 mM.

[0036] Further preferred, when the chlorfenapyridine suspension is in solid form type I, at a wavelength of 720 nm, the correlation equation between its photoacoustic signal intensity PA and concentration C is PA = 0.4691C + 0.037.

[0037] More preferably, the concentration of the chlorfenapyridine suspension is 5 mM.

[0038] Preferably, when the chlorfazimine suspension is in amorphous solid form, its near-infrared fluorescence intensity is concentration-dependent on chlorfazimine within a concentration range of 0–5 mM.

[0039] Further preferred, when the chlorfenapyridine suspension is in amorphous solid form, the correlation equation between its photoacoustic signal intensity PA and concentration C at a wavelength of 720 nm is PA = 0.647C - 0.112.

[0040] More preferably, the concentration of the chlorfenapyridine suspension is 5 mM.

[0041] Preferably, in the method (1), the organic solvent is dimethyl sulfoxide, and the concentration of chlorfazimine in the organic solution is greater than 0.5 mg / mL, more preferably 0.5-5 mg / mL, and even more preferably 2-5 mg / mL; the volume ratio of the organic solution of chlorfazimine to the aqueous solution of the stabilizer or water is 1:5-1:40, more 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, more preferably 0.1-10 mg / mL.

[0042] Preferably, the stirring speed is faster than 50 rpm and the stirring reaction time is not less than 5 min.

[0043] 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.

[0044] More preferably, the mass-to-volume ratio of the stabilizer to water is 0.1 to 100 mg / mL, even more preferably 2.5:1 to 100:1 mg / mL, and even more preferably 2.5 to 50 mg / mL.

[0045] Preferably, in the method (2), the grinding speed is 200 to 1000 rpm.

[0046] More preferably, the grinding speed is 500-1000 rpm.

[0047] The drug suspension of the present invention is prepared by reverse solvent method or wet ball milling method.

[0048] Reverse solvent method:

[0049] Under stirring and ultrasonic conditions, an organic solution containing chlorpromazine was added to water for reaction, and chlorpromazine crystals or amorphous particles with an average particle size of 0.05–5 μm were collected.

[0050] Wet ball milling method:

[0051] With the addition of a certain stabilizer, grinding media and water are added, and the mixture is reacted at a certain speed for a period of time. Crystals or amorphous materials with an average particle size of 0.05–5 μm are then collected.

[0052] Preferably, in the process of preparing suspensions by wet ball milling, the main functions of the stabilizer include preventing particle aggregation, improving dispersibility, adjusting particle size, and extending milling time, and it does not affect the near-infrared light responsiveness of the chloramine 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.

[0053] The chlorfenapyridine suspension described in this invention is used in the preparation of tracers for near-infrared photoacoustic and fluorescence imaging.

[0054] The chlorfazimine suspension described in this invention is used in the preparation of photothermal therapy reagents.

[0055] The chlorfazimine suspension described in this invention is used in the preparation of antitumor drugs.

[0056] Preferably, the drug is a drug for treating breast cancer.

[0057] Preferably, the drug is a drug used for anti-tumor near-infrared photothermal therapy, near-infrared fluorescence imaging, or near-infrared photoacoustic imaging.

[0058] Preferably, the pharmaceutical concentration of the drug is 0.5 to 10 mM.

[0059] The clofazimine suspension described in this invention is used in the preparation of drugs for treating bacterial infections and for anti-inflammatory and analgesic purposes.

[0060] The chlorfenapyridine suspension of the present invention is used in the preparation of NIR-II fluorescent tracers, wherein the average particle size of the suspension is 10 nm to 50 μm.

[0061] Preferably, the NIR-II fluorescent tracer is an NIR-II tumor fluorescent tracer or an NIR-II lymphocyte fluorescent tracer.

[0062] Preferably, the NIR-II fluorescent tracer has a fluorescent emission signal in the wavelength range of 1100–1300 nm.

[0063] Further preferably, the fluorescence emission signal of the NIR-II fluorescent tracer is concentration-dependent.

[0064] More preferably, the fluorescence emission signal of the NIR-II fluorescent tracer is concentration-dependent in the concentration range of 0 to 1 mM.

[0065] Preferably, in the suspension used as an NIR-II fluorescent tracer, the aggregation morphology of chlorofazimine is selected from crystal form I, crystal form II, and crystal form III.

[0066] Further preferably, when the aggregated morphology of the chlorfenapyridine suspension is crystal form I, it has diffraction characteristic peaks at 9.24°, 11.44°, 12.24°, 16.10°, 19.82°, 21.86°, 23.56°, and 24.70°, calculated at 2θ±0.2°.

[0067] Further preferably, when the aggregated morphology of the chlorfenapyridine suspension is crystal form II, it has diffraction characteristic peaks at 10.18°, 13.92°, 15.4°, 16.76°, 17.7°, 19.3°, 20.12°, and 20.42°, calculated at 2θ±0.2°.

[0068] Further preferably, when the aggregated morphology of the chlorfenapyridine suspension is crystal form III, it has diffraction characteristic peaks at 6.9°, 7.88°, 10.26°, 13.34°, 14.34°, 19.22°, 20.80°, and 22.16°, calculated at 2θ±0.2°.

[0069] Further preferably, when the aggregation morphology of chlorofazimine is crystal form I, the fluorescence emission signal of the NIR-II fluorescent tracer is concentration-dependent.

[0070] More preferably, when the aggregation morphology of chlorofazimine is crystal form I, the fluorescence emission signal of the NIR-II fluorescent tracer is concentration-dependent in the concentration range of 0 to 1 mM.

[0071] More preferably, when the aggregation morphology of chlorofazimine is crystal form I, the linear equation of the fluorescence emission signal and concentration of the NIR-II fluorescent tracer is Y = 332.36X - 1622.39.

[0072] Preferably, the PDI of the chlorfenapyridine suspension is less than 0.3.

[0073] Preferably, the active ingredient in the suspension used as an NIR-II fluorescent tracer is chlorofazimine.

[0074] The NIR-II fluorescent phototherapy agent provided by this invention, through a drug delivery system based on clofazimine, has become a tracer with high sensitivity, high tissue penetration, good photostability, and better surgical navigation operability.

[0075] The clofazimine suspension used in this invention exhibits centimeter-level tissue penetration and a higher signal-to-background ratio (SBR) in NIR-II fluorescence. Simultaneously, the clofazimine suspension combines excellent biocompatibility, high sensitivity, high tissue penetration, and photostability, reducing the inconvenience of multiple clinical administrations and improving patient compliance, thus showing promising application prospects in clinical NIR-II fluorescence imaging. Compared to clofazimine's NIR-I fluorescence imaging, clofazimine's NIR-II fluorescence imaging capability overcomes the limitations of traditional imaging in strong light or deep tissue "black box" environments, making it more suitable for intraoperative navigation and possessing broader application scope and greater application potential. Furthermore, compared to clinical tracers (ICGs) with fluorescent signals at the molecular level, specific clofazimine crystals exhibit NIR-II fluorescence responsiveness, effectively avoiding interference from drug molecules prematurely released from the administration system for imaging and diagnosis.

[0076] The chlorfenapyridine suspension of the present invention is used in the preparation of lymphatic tracers, wherein the average particle size of the suspension is 10 nm to 500 nm.

[0077] The clofazimine suspension used in this invention exhibits good staining ability on mouse lymph nodes without staining surrounding tissues. Therefore, clofazimine can be developed into an effective lymph node tracing diagnostic reagent. Combined with its inhibitory effects on cancer development, antibacterial and anti-inflammatory properties, and its biocompatibility for mothers and infants, it can combat lymphedema while inhibiting tumor development. Furthermore, its staining properties can be used for lymph node dissection during cancer surgery. Simultaneously, clofazimine's excellent NIR-II fluorescence and NIR-I photoacoustic imaging properties hold promise for replacing isotope tracing, achieving combined fluorescence-photoacoustic-dye tracing, reducing the inconvenience of injecting multiple tracers in clinical practice, and improving patient compliance. Moreover, unlike traditional free small molecules with weak lymph node targeting ability and leakage, clofazimine, after micro / nano-processing, exhibits superior lymph node targeting effects and shows promising application prospects in lymph node tracing.

[0078] Preferably, in the suspension used as a lymphatic tracer, the active ingredient is selected from chlorofazimine, chlorofazimine hydrochloride, chlorofazimine methanesulfonate, chlorofazimine oxalate, chlorofazimine sulfate, chlorofazimine sulfite, chlorofazimine phosphate, chlorofazimine acetate, chlorofazimine citrate, chlorofazimine isonicotinic acid, chlorofazimine nicotinic acid, chlorofazimine salicylate, chlorofazimine maleate, chlorofazimine hydrated methanesulfonate, chlorofazimine malonic acid, chlorofazimine dodecyl sulfate, chlorofazimine p-aminobenzoate, and chlorofazimine ethyl acetate solvate.

[0079] Preferably, the application modes of the lymphatic tracer are fluorescence tracer, photoacoustic tracer, dye tracer, and the above-mentioned multimodal combined tracer.

[0080] Preferably, the lymphatic tracer is an NIR-II fluorescent lymphatic tracer or an NIR-I photoacoustic lymphatic tracer.

[0081] Preferably, the lymphatic tracer is a tracer for popliteal lymph nodes, iliac lymph nodes, and abdominal aortic lymph nodes.

[0082] Preferably, the aggregation morphology of chlorofazimine in the suspension is selected from crystal form I, crystal form II, crystal form III or amorphous.

[0083] The chloramine suspension of the present invention is prepared by any of the following methods:

[0084] (1) Antisolvent method: The organic solution containing chlorazimine is reacted with an aqueous solution of stabilizer or water by stirring;

[0085] (2) Grinding method: Chloride is ground in stabilizer and water to obtain the product.

[0086] Preferably, in the method (1), the organic solvent is dimethyl sulfoxide, and the concentration of chlorfazimine in the organic solution is greater than 0.5 mg / mL, more preferably 0.5-5 mg / mL, and even more preferably 2-5 mg / mL; the volume ratio of the organic solution of chlorfazimine to the aqueous solution of the stabilizer or water is 1:5-1:40, more 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, more preferably 0.1-10 mg / mL.

[0087] Preferably, the stirring speed is faster than 50 rpm and the stirring reaction time is not less than 5 min.

[0088] Preferably, in the method (2), the main functions of the stabilizer include preventing particle aggregation, improving dispersibility, adjusting particle size, and extending grinding time, and it does not affect the near-infrared light responsiveness of the chloramphenicol 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.

[0089] More preferably, the mass-to-volume ratio of the stabilizer to water is 0.1 to 100 mg / mL, even more preferably 2.5:1 to 100:1 mg / mL, and even more preferably 2.5 to 50 mg / mL.

[0090] Preferably, in the method (2), the grinding speed is 200 to 1000 rpm.

[0091] More preferably, the grinding speed is 500-1000 rpm.

[0092] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0093] The suspension delivery system designed in this invention, by micro- and nano-processing chlorofazimine suspension, can regulate near-infrared photothermal, photoacoustic, and fluorescence effects based on solid-state form. Utilizing its near-infrared photothermal effect and the anti-tumor effect of the drug itself, it combines phototherapy with traditional chemotherapy to achieve effective inhibition of tumor development and progression.

[0094] Meanwhile, the excellent near-infrared fluorescence and photoacoustic properties give this drug delivery system the advantage of enabling multimodal imaging. Clofazimine suspension exhibits NIR-II fluorescence responsiveness only in specific aggregate states, effectively avoiding imaging and diagnostic interference caused by premature release from the drug delivery system. Its NIR-II fluorescence imaging capability overcomes the limitations of traditional imaging in strong light or deep tissue "black box" environments, making it more suitable for intraoperative navigation and assisting in the diagnosis and treatment of diseases in pregnant women and infants. It also boasts advantages such as high sensitivity, low interference, high tissue penetration, long-term photostability, good biocompatibility, and high cost-effectiveness.

[0095] Furthermore, in anti-tumor applications, this drug delivery system enables long-term drug retention at the tumor site, increasing treatment compliance and forming a comprehensive drug delivery system integrating near-infrared phototherapy, near-infrared fluorescence imaging, photoacoustic imaging, traditional chemotherapy, and long-term retention—a truly integrated therapeutic imaging system. In lymph node tracing, it exhibits superior lymph node targeting and more durable lymph node staining, combining fluorescence-photoacoustic-dye three-in-one tracing functions. This reduces the inconvenience of requiring injections of two or more tracers in clinical combination tracing, improving patient compliance. Attached Figure Description

[0096] Figure 1 shows the near-infrared photothermal conversion efficiency of different solid forms of chloramphenicol prepared in Example 23.

[0097] Figure 2 shows the full-band photoacoustic signal spectrum of chlorazimine prepared in different solid forms in Example 23 at 680–970 nm.

[0098] Figure 3 shows the photoacoustic signal intensity at 720 nm as a function of concentration for different solid forms of chlorazimine prepared in Example 23.

[0099] Figure 4a shows the near-infrared fluorescence AIE effect of the chloromethacin crystal form I suspension prepared in Example 23;

[0100] Figure 4b shows the near-infrared fluorescence AIE effect of the chloromethacin crystal form II suspension prepared in Example 23;

[0101] Figure 4c shows the near-infrared fluorescence AIE effect of the chloromethacin crystal form III suspension prepared in Example 23.

[0102] Figure 4d shows the near-infrared fluorescence AIE effect of the chloramphenicol amorphous suspension prepared in Example 23.

[0103] Figure 5 shows the homogenized NIR-II fluorescence emission spectra of the chlorfenapyridine suspensions (crystal form I, crystal form II, and crystal form III) prepared in Example 16.

[0104] Figure 6 shows the concentration-dependent NIR-II fluorescence emission signal results of the chlorfazimine suspension (crystal form I) prepared in Example 16;

[0105] Figure 7 shows the aggregation-state induced emission effect of NIR-II fluorescence of the chlorfenapyridine suspension (crystal form I) prepared in Example 16;

[0106] Figure 8 shows the fluorescence stability results of the NIR-II chlorpromazine suspension (crystal form I) prepared in Example 16;

[0107] Figure 9 shows the fluorescence stability results of indocyanine green solution NIR-II;

[0108] Figure 10 shows the PXRD patterns of different solid forms (crystal forms I, II, III or amorphous) of chloromethacin prepared by Example 23;

[0109] Figure 11 shows the PXRD patterns of different solid morphologies (crystal forms I, II, III or amorphous) of chlorinated zimine predicted by mercury.

[0110] Figure 12 shows the near-infrared and bright-field imaging results of the chloramphenicol suspension prepared in Example 23 with different water phase ratios and the same concentration of chloramphenicol suspension.

[0111] Figure 13 shows near-infrared images of the chlorpromazine suspension prepared in Example 23 at different concentration gradients;

[0112] Figure 14 shows the near-infrared spectra of the chloramphenicol suspension prepared in Example 23 at different concentration gradients;

[0113] Figure 15 shows the fluorescence intensity trend line at 824 nm for different concentrations of the chloramphenicol suspension prepared in Example 23.

[0114] Figure 16a shows the results of near-infrared fluorescence in vivo imaging 14 days after in situ injection.

[0115] Figure 16b shows the quantitative results of near-infrared fluorescence in vivo imaging within 14 days after in situ injection.

[0116] Figure 17a shows the results of the isolated tumor and major organs 14 days after in situ injection;

[0117] Figure 17b shows the quantitative results of the ex vivo tumor and major organs 14 days after in situ injection;

[0118] Figure 18 shows the NIR-I / II images of the pre-filled capillary glass tube and the SBR analysis results;

[0119] Figure 19a shows the results of near-infrared photoacoustic imaging within 14 days after in situ injection.

[0120] Figure 19b shows the quantitative results of near-infrared photoacoustic imaging within 14 days after in situ injection.

[0121] Figure 20 shows the photoacoustic signal intensity results of the chlorfenapyridine suspension (crystal form I) prepared in Example 16 compared with deionized water;

[0122] Figure 21 shows the cytotoxicity results of chlorfazimine solution dissolved in DMSO on 4T1 cells at different concentrations;

[0123] Figure 22 shows the results of the cytotoxicity of clofazimine suspension prepared in Example 23 on 4T1 cells under different treatment interventions;

[0124] Figure 23 is a schematic diagram of the treatment in an in vivo anti-tumor experiment;

[0125] Figure 24 shows the tumor volume changes within 14 days after treatment with different treatment strategies using the clofazimine suspension prepared in Example 23.

[0126] Figure 25 shows the tumor weight results after 14 days of treatment intervention with different treatment strategies using the clofazimine suspension prepared in Example 23.

[0127] Figure 26 shows the effect of methylene blue staining on mouse lymph nodes;

[0128] Figure 27 shows the staining effect of the chlorfazimine suspension prepared in Example 14 on the lymph nodes after injection into the paw of a mouse.

[0129] Figure 28 shows the staining effect of the salicylic acid chlorofazimine suspension prepared in Example 21 on the primary lymph nodes (popliteal lymph nodes) after injection into the paw of a mouse;

[0130] Figure 29a shows the intratumoral NIR-II fluorescence imaging effect of the chlorfazimine suspension (crystal form I) prepared in Example 16 two weeks after intratumoral injection in mice;

[0131] Figure 29b shows the quantitative results of intratumoral NIR-II fluorescence imaging of the chlorfazimine suspension (crystal form I) prepared in Example 16 two weeks after intratumoral injection in mice;

[0132] Figure 30 shows the NIR-II fluorescence imaging-assisted labeling effect of the chlorfazimine suspension (crystal form I) prepared in Example 16 on the primary lymph nodes (popliteal lymph nodes) after injection into the paw of a mouse.

[0133] Figure 31 shows the NIR-I photoacoustic lymphatic tracing signal analysis results of the primary lymph nodes (popliteal lymph nodes) after injection of the chlorfazimine suspension (crystal form I) prepared in Example 16 into the paw of a mouse. Detailed Implementation

[0134] The technical solution of the present invention will be further described below with reference to the embodiments.

[0135] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials and reagents used are all commercially available.

[0136] Chlorfenapyridine suspensions in crystal form I, crystal form II, crystal form III, and amorphous aggregates were prepared according to the following examples.

[0137] Example 1

[0138] 1 mL of 5 mg / mL chlorfenapyridine solution dissolved in dimethyl sulfoxide was added to 20 mL of deionized water and reacted for 5 min under stirring and sonication at 50 rpm. The chlorfenapyridine suspension was then collected by filtration.

[0139] Example 2

[0140] 1 mL of 0.5 mg / mL chlorfenapyridine solution dissolved in dimethyl sulfoxide was added to 20 mL of deionized water and reacted for 5 min under stirring and sonication at 50 rpm. The chlorfenapyridine suspension was then collected by filtration.

[0141] Example 3

[0142] 1 mL of 5 mg / mL chlorfenapyridine solution dissolved in dimethyl sulfoxide was added to 20 mL of deionized water and reacted for 5 min under stirring and sonication at 500 rpm. The chlorfenapyridine suspension was then collected by filtration.

[0143] Example 4

[0144] 1 mL of 2 mg / mL chlorfenapyridine solution dissolved in dimethyl sulfoxide was added to 5 mL of deionized water and reacted for 5 min under stirring and sonication at 50 rpm. The chlorfenapyridine suspension was then collected by filtration.

[0145] Example 5

[0146] 1 mL of 5 mg / mL chlorfenapyridine solution dissolved in dimethyl sulfoxide was added to 40 mL of deionized water and reacted for 30 min under stirring and sonication at 50 rpm. The chlorfenapyridine suspension was then collected by filtration.

[0147] Example 6

[0148] 1 mL of 5 mg / mL chlorfenapyridine solution dissolved in dimethyl sulfoxide was added to 10 mL of deionized water and reacted for 5 min under stirring and sonication at 50 rpm. The chlorfenapyridine suspension was then collected by filtration.

[0149] Example 7

[0150] 1 mL of chlorfazimine solution (5 mg / mL) dissolved in dimethyl sulfoxide was added to 20 mL of deionized water containing 0.1 mg / mL soluplus under stirring and sonication at 50 rpm for 5 min. The solution was then collected and concentrated by ultrafiltration to obtain chlorfazimine suspension.

[0151] Example 8

[0152] 1 mL of chlorfazimine solution (5 mg / mL) dissolved in dimethyl sulfoxide was added to 20 mL of deionized water containing 1 mg / mL soluplus under stirring and sonication at 50 rpm for 5 min. The solution was then collected and concentrated by ultrafiltration to obtain chlorfazimine suspension.

[0153] Example 9

[0154] 1 mL of clofazimine solution (5 mg / mL) dissolved in dimethyl sulfoxide was added to 20 mL of deionized water containing 10 mg / mL Tween 80 under stirring and sonication at 50 rpm for 5 min. The solution was then collected and concentrated by ultrafiltration to obtain clofazimine suspension.

[0155] Example 10

[0156] 50 mg of clofazimine (crystal form I) was placed in a ball mill jar, along with 6 g of grinding media and 4 mL of deionized water containing 200 mg of beroxam 407. The mixture was reacted at 800 rpm for 120 min, and the clofazimine suspension was collected.

[0157] Example 11

[0158] 50 mg of clofazimine (crystal form I) was placed in a ball mill jar, and 6 g of grinding media and 4 mL of deionized water containing 400 mg of beroxam 407 were added. The mixture was reacted at 800 rpm for 120 min, and the clofazimine suspension was collected.

[0159] Example 12

[0160] 50 mg of clofazimine (crystal form I) was placed in a ball mill jar, along with 6 g of grinding media and 4 mL of deionized water containing 10 mg of soluplus. The mixture was reacted at 1000 rpm for 240 min, and the clofazimine suspension was collected.

[0161] Example 13

[0162] 50 mg of clofazimine (crystal form I) was placed in a ball mill jar, along with 6 g of grinding media and 4 mL of deionized water containing 10 mg of soluplus. The mixture was reacted at 500 rpm for 120 min, and the clofazimine suspension was collected.

[0163] Comparative Example 1

[0164] 1 mL of 0.1 mg / mL chlorfenapyridine solution dissolved in dimethyl sulfoxide was added to 20 mL of deionized water and reacted for 5 min under stirring and sonication at 50 rpm. The chlorfenapyridine suspension was then collected by filtration.

[0165] Comparative Example 2

[0166] 1 mL of 5 mg / mL chlorfenapyridine solution dissolved in dimethyl sulfoxide was added to 20 mL of deionized water and reacted for 5 min under stirring and sonication at 10 rpm. The chlorfenapyridine suspension was then collected by filtration.

[0167] Comparative Example 3

[0168] 1 mL of 5 mg / mL chlorfenapyridine solution dissolved in dimethyl sulfoxide was added to 1 mL of deionized water and reacted for 5 min under stirring and sonication at 50 rpm. The chlorfenapyridine suspension was then collected by filtration.

[0169] Comparative Example 4

[0170] 1 mL of 5 mg / mL chlorfenapyridine solution dissolved in dimethyl sulfoxide was added to 20 mL of deionized water and reacted for 1 min under stirring at 50 rpm and sonication. The chlorfenapyridine suspension was then collected by filtration.

[0171] Comparative Example 5

[0172] 1 mL of 5 mg / mL chlorfenapyridine solution dissolved in dimethyl sulfoxide was added to 50 mL of deionized water and reacted for 5 min under stirring and sonication at 50 rpm. The chlorfenapyridine suspension was then collected by filtration.

[0173] Compared with Example 1, the parameters of Comparative Examples 1 to 5 were changed as shown in Table 1.

[0174] Table 1 Parameter Comparison Table

[0175] Compared with Example 1, in Comparative Example 1, the concentration of chlorfazimine was 0.1 mg / mL in step S1. After stirring, the system was orange-yellow and contained a small number of crystals visible to the naked eye. During the analysis of the particle size 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.

[0176] Compared with Example 1, in Comparative Example 2, the rotation speed was 10 rpm in step S1. After stirring, the particles were collected and analyzed using a Malvern particle size analyzer. It was found that the particle size PDI value exceeded 0.5, indicating poor particle size uniformity.

[0177] Compared with Example 1, in Comparative Example 3, the ratio of organic phase to water phase in step S1 was 1:1. After stirring, the crystals were collected and their particle size was analyzed using a Malvern particle size analyzer. It was found that the particle size was too large and exceeded the measurement range.

[0178] Compared with Example 1, in Comparative Example 4, the reaction time in step S1 was 1 min. After stirring, the particles were collected immediately and their particle size was analyzed using a Malvern particle size analyzer. It was found that the particle size gradually increased over time, indicating that the system was not in equilibrium.

[0179] Compared with Example 1, in Comparative Example 5, the ratio of organic phase to aqueous phase in step S1 was 1:50. After stirring, the crystals were collected and analyzed using a Malvern particle size analyzer. It was found that the particle size distribution was uneven and the PDI was greater than 0.3.

[0180] As shown in Table 1, during the preparation of chlorozimine, dimethyl sulfoxide (DMSO) was chosen as the organic phase in this invention due to its good solubility (up to 5 mg / mL). When the concentration of the chlorozimine solution was too low (less than 0.5 mg / mL), the system did not reach supersaturation and thus could not rapidly generate a large number of crystal nuclei. The chlorozimine molecules dissolved in DMSO that had not yet precipitated were gradually absorbed by the precipitated nuclei, eventually growing into large-sized crystals (Comparative Example 1). The process from nucleus formation to solute absorption reaching equilibrium requires time. If the reaction time is too short (less than 5 min), the system will not reach equilibrium, and the nuclei will continue to absorb solute until equilibrium is reached, resulting in chlorozimine microparticles with uniform particle size meeting experimental requirements (Comparative Example 4).

[0181] When the ratio of organic phase to aqueous phase is greater than 1:5 or less than 1:20, excessively large particle sizes can result due to various problems, such as the inability to ensure sufficient and uniform dispersion. When the volume difference between the organic and aqueous phases is too large, the organic phase cannot achieve rapid and uniform dispersion in the aqueous phase. Since a locally supersaturated system that cannot be rapidly and uniformly dispersed is extremely unstable, a small number of crystal nuclei initially precipitate in the entire system. This causes the chlorofazimine dissolved in dimethyl sulfoxide before the nuclei precipitate to be gradually absorbed by the precipitated nuclei and grow into large crystals (Comparative Example 5). However, as the volume ratio of organic to aqueous phase decreases, the organic phase can achieve rapid dispersion in the aqueous phase and gradually reach the supersaturation conditions necessary for crystal formation. This allows the entire system to rapidly generate a large number of crystal nuclei, resulting in each nucleus absorbing a relatively even and controllable number of chlorofazimine molecules. When the volume difference between the organic and aqueous phases is too small, it is difficult to achieve rapid and uniform distribution of the organic and aqueous phases through stirring and sonication, making it difficult to obtain particles within the target range (Comparative Example 3).

[0182] The process of preparing the chlorfenapyridine 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 mixed quickly and uniformly, resulting in the formation of a large number of crystal nuclei in some areas and the absorption of solute molecules. Ultimately, the entire system becomes a crystal drug delivery system with large particle size and poor uniformity (Comparative Example 2).

[0183] Chlorinated chloramine suspension was prepared by wet ball milling (Examples 10-13). The particle size of these examples varies depending on the stabilizer and gradually decreases with increasing rotation speed and milling time, with the overall particle size in the range of 0.05-5 μm.

[0184] Example 14: Antisolvent method

[0185] Add 1 mL of 5 mg / mL chlorfenapyridine dimethyl sulfoxide solution to 20 mL of 0.1% (m / m) Soluplus deionized water solution and stir for 5 min to obtain chlorfenapyridine suspension.

[0186] Example 15: Antisolvent Method

[0187] Add 1 mL of 5 mg / mL chlorozimine dimethyl sulfoxide solution to 40 mL of 10% (m / m) Tween 80 deionized water solution and stir for 5 min to obtain chlorozimine suspension.

[0188] Example 16: Grinding method

[0189] Add 50 mg of chlorfazimine (crystal form I, crystal form II, crystal form III) to a grinding jar, along with 6 g of grinding media and 4 mL of 10% (m / m) Tween 80 deionized water. Grind at 800 rpm for 120 min and collect the chlorfazimine suspension.

[0190] Example 17: Grinding method

[0191] Add 50 mg of clofazimine (crystal form I, crystal form II, crystal form III) to a grinding jar, along with 6 g of grinding media and 4 mL of 0.1 mg / mL beroxam 488 deionized water. Grind at 800 rpm for 10 min and collect the clofazimine suspension.

[0192] Example 18: Preparation of Chlorpheniramine Phosphate Suspension

[0193] Add 1 mL of 5 mg / mL clofazimine dimethyl sulfoxide solution to 40 mL of 10% (m / m) Tween 80 0.1 M PBS (pH 4.0) buffer solution and stir for 5 min to obtain clofazimine phosphate suspension.

[0194] Example 19: Preparation of Chlorfenapyridine Hydrochloride Suspension

[0195] Add 1 mL of 5 mg / mL chlorozimine dimethyl sulfoxide solution to 20 mL of 1% (m / m) Tween 80 0.1 M hydrochloric acid solution and stir for 5 min to obtain chlorozimine hydrochloride suspension.

[0196] Example 20: Preparation of a suspension of pharmaceutically acceptable salts and solvates of chlorfazimine

[0197] 50 mg each of chlorofazimine hydrochloride, chlorofazimine methanesulfonate, chlorofazimine oxalate, chlorofazimine sulfate, chlorofazimine sulfite, chlorofazimine phosphate, chlorofazimine acetate, chlorofazimine citrate, chlorofazimine isonicotinic acid, chlorofazimine nicotinic acid, chlorofazimine salicylate, chlorofazimine maleate, chlorofazimine hydrated methanesulfonate, chlorofazimine malonic acid, chlorofazimine dodecyl sulfate, chlorofazimine p-aminobenzoate, and chlorofazimine ethyl acetate solvate were added to a grinding jar, along with 4 g of grinding media and 4 mL of 3% (m / m) Tween 80 deionized water. The mixture was ground at 800 rpm for 12 h, filtered, and washed to obtain the final product.

[0198] Example 21: Preparation of a suspension of pharmaceutically acceptable salts and solvates of chlorfazimine

[0199] The difference from Example 20 is that a 10% (m / m) deionized aqueous solution of Tween 80 was used for preparation.

[0200] Example 22: Preparation of suspensions of pharmaceutically acceptable salts and solvates of chlorfazimine

[0201] The difference from Example 20 is that a 10% (m / m) deionized aqueous solution of beroxam 407 was used for preparation.

[0202] Example 23: Preparation of suspensions in different solid forms

[0203] 1. Preparation of different polymorphs or amorphous chlorine esters

[0204] 0.5 g of chlorfenapyridine (commercially available Form I) was dispersed in 5 mL of anhydrous diethyl ether and stirred uniformly on a magnetic stirrer (800 rpm) for 24 h. After the reaction was completed, the product was filtered and dried to obtain chlorfenapyridine crystal form II (Form II). 1.0 g of chlorfenapyridine was dissolved in 5 mL of toluene and reacted on a heated stirring apparatus for 24 h (40 °C, 800 rpm). After the reaction was completed, the product was filtered and dried to obtain chlorfenapyridine crystal form III (Form III). Amorphous chlorfenapyridine was obtained by melt cooling. Chlorfenapyridine powder was heated to 220 °C until it was fully melted, then rapidly cooled to solidify it. The resulting amorphous solid was ground and sieved to obtain amorphous chlorfenapyridine powder.

[0205] 2. Preparation of suspensions

[0206] Suspensions of different crystalline or amorphous forms of chloramphenicol were obtained by ball milling, as follows:

[0207] 50 mg of clofazimine (Form I, Form II, and Form III or amorphous), 4 mL of deionized water containing 200 mg of beroxam 407, and 6 g of oxidized 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 was completed, the mixture was filtered, washed, and resuspended to obtain clofazimine suspension (Form I, Form II, Form III or amorphous).

[0208] Example 24: Evaluation of Photothermal Effect

[0209] Use 3W / cm 2 An 808 nm laser was used to irradiate a chlorfenapyridine suspension (FI, FII, FIII, or amorphous) prepared according to Example 23 at a concentration of 3 mg / mL for 10 min. During this process, the temperature change was recorded every 0.5 min using an infrared camera, and the photothermal conversion efficiency was subsequently calculated and fitted to obtain the efficiency. As shown in Figure 1, the photothermal conversion efficiency of Form I reached 25.88%, that of Form II was 1.36%, that of Form III was 2.34%, while that of the amorphous form was as high as 32.01%.

[0210] Example 25: Photoacoustic Effect

[0211] Photoacoustic signals were scanned across the entire wavelength range (680–970 nm) using different suspensions prepared according to Example 23 at a concentration of 5 mM chlorfenapyridine. As shown in Figure 2, Form I at the same concentration exhibited significant photoacoustic reactivity and reached a peak photoacoustic signal at 720 nm. Form II and Form III showed some photoacoustic reactivity compared to deionized water, but their signal intensity was weaker. Amorphous suspensions, under the same conditions, exhibited stronger photoacoustic signals than Form I and did not show a signal peak similar to that of polymorphic suspensions. Overall, the signal gradually weakened with increasing wavelength.

[0212] Based on this, single-wavelength (720 nm) photoacoustic signals of different concentrations of chlorfenapyridine suspension prepared according to Example 23 were detected and analyzed. As shown in Figure 3, the photoacoustic signals of Form I and the amorphous form at 720 nm showed good concentration dependence, and the amorphous form had a stronger photoacoustic signal than Form I. In contrast, Form II and Form III showed some concentration dependence, but their signal intensity was weaker and the linear relationship was not ideal.

[0213] Example 26: Near-infrared fluorescence AIE

[0214] A 400 μM chlorpromazine suspension (Form I, Form II, Form III, or amorphous) prepared according to Example 23 and a chlorpromazine solution (DMSO) were mixed at 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 chlorpromazine mixture systems with different non-good solvent ratios at 400 μM concentrations, and their fluorescence spectra were scanned (Ex = 640 nm; Em = 800–900 nm). The results are shown in Figures 4a–4d. The results showed that Form I, Form II and Form III all exhibited near-infrared AIE effects and their fluorescence intensities did not differ significantly, while the amorphous suspension showed extremely limited fluorescence effects.

[0215] Example 27: Evaluation of NIR-II fluorescence emission effect of chlorfenapyridine suspension

[0216] NIR-II fluorescence signal quantification and homogenization were performed on chlorofazamine suspensions (CFZ NC, crystal form I, crystal form II, and crystal form III) of the same concentration but different aggregation states prepared according to Example 16. The results are shown in Figure 5. Different chlorofazamine crystal forms all exhibited NIR-II fluorescence emission signals, and crystal form I showed the most obvious fluorescence signal. Therefore, the chlorofazamine suspension corresponding to crystal form I was selected for subsequent NIR-II fluorescence emission characteristic testing. NIR-II fluorescence spectra of chlorofazamine suspensions (CFZ NC) of different concentrations of crystal form I prepared according to Example 16 were tested. The results are shown in Figure 6. The NIR-II fluorescence emission signal of the chlorofazamine suspension (CFZ NC) showed good concentration dependence (Y = 332.36X - 1622.39, 0–1.0 mM, R...). 2 =1).

[0217] The above tests were further performed on the 600 μM crystalline form I chlorozimine suspension (CFZ NC) prepared according to Example 16 and the DMSO solution of chlorozimine. The results are shown in Figure 7. The NIR-II fluorescence signal of the chlorozimine suspension (CFZ NC) originated from the aggregation-induced emission effect (AIE).

[0218] In addition, the stability of the NIR-II fluorescence signal of the crystalline form I chloromethamine suspension (CFZ NC) prepared according to Example 16 was tested overall. The exposure time was 5 h, and the signal value was recorded every 10 min. The results are shown in Figures 8 and 9. The chloromethamine suspension (CFZ NC) has a long-term stable NIR-II fluorescence emission signal (Figure 8); while the NIR-II fluorescence signal of indocyanine green (ICG) was bleached by 70% within a 5 min exposure time, showing obvious photobleaching (Figure 9).

[0219] Example 28: Particle size and characterization

[0220] The chlorfenapyridine suspensions from Examples 1 to 13 were prepared with deionized water to form suspensions with a concentration of 0.3 mg / mL. The particle size and uniformity were then measured at 37°C. The results are shown in Table 2.

[0221] Table 2. Particle size and PDI results of the suspension.

[0222] As shown in Table 2, the average particle size of the chlorpheniramine suspension in deionized water is 0.05–5 μm and the PDI is below 0.3, indicating that the chlorpheniramine suspension prepared by this invention has an average particle size of 0.05–5 μm and good uniformity.

[0223] Example 29: Particle size and potential

[0224] The chlorfazimine and its pharmaceutically acceptable salts and solvates obtained in Examples 14 to 22 were prepared into 0.3 mg / mL suspensions with deionized water, and their particle size and potential were measured at 37°C. The results are shown in Table 3.

[0225] Table 3. Particle size and uniformity (PDI) of clofazimine and its pharmaceutically acceptable salts and solvates suspensions.

[0226] As shown in Table 3, the average particle size of the chlorfenapyr suspension is 10 nm to 50 μm, and the PDI is less than 0.3, indicating good uniformity.

[0227] Example 30: PXRD Characterization

[0228] The different solid forms of chloramine prepared according to Example 23 were analyzed by X-ray diffraction, and the results are shown in Figure 10 and Table 4. Comparing these results with the results predicted by Mercury (Figure 11) demonstrates that the present invention has prepared the corresponding polymorphs and amorphous forms of chloramine.

[0229] Table 4 Characteristic peaks of PXRD suspensions

[0230] Example 31: Characterization of different aggregation states

[0231] Qualitative analysis of the aggregation state of the chlorpheniramine suspensions obtained in Examples 14 to 17 was performed by PXRD. The results confirmed that the grinding method was used to prepare each suspension formulation with the corresponding dosage aggregation state, while the bottom-up method could be used to prepare chlorpheniramine nano-amorphous particles with a particle size distribution between 10 and 100 nm (as shown in Table 5).

[0232] Table 5 Aggregation state of chlorfenapyridine suspension

[0233] Example 32: In vivo and in vitro near-infrared fluorescence imaging

[0234] The same concentration of chlorfazimine (Form I) with different water phase ratios as described in Example 26 was added to 96-well plates for near-infrared imaging. As shown in Figure 12, Form I exhibited good near-infrared AIE properties in vitro.

[0235] Chlorfenapyridine suspension (Form I) was serially diluted with deionized water, and near-infrared fluorescence spectroscopy (Ex = 640 nm; Em = 800–900 nm) was performed on different concentrations of chlorfenapyridine suspension (Form I). Furthermore, chlorfenapyridine suspension (Form I) at different concentration gradients was added to 96-well plates for near-infrared imaging. As shown in Figures 13–15, the near-infrared fluorescence effect of chlorfenapyridine suspension (Form I) is concentration-dependent.

[0236] Female BALB / c mice aged 6–8 weeks were marked and shaved. 50 μL of 4T1 cells were then injected at a density of 1 × 10⁻⁶. 7 A cell suspension of 100 cells / mL was subcutaneously injected into the lateral abdomen of mice. The tumor volume was increased to 100–200 mm². 3At that time, clofazimine suspension (Form I) prepared according to Example 23 was injected into mouse tumors at a dose of 10 mg / kg. After drug intervention, near-infrared in vivo imaging was performed on tumor-bearing mice injected intratumorally on days 0, 1, 3, 7, and 14. Mice were euthanized after near-infrared in vivo imaging on day 14, and tumors and major organs (heart, liver, spleen, lungs, and kidneys) were harvested for near-infrared imaging. As shown in Figures 16a-16b, the fluorescence signal intensity at the mouse tumor sites gradually decreased from day 0 to day 14, but relatively strong fluorescence signals were still detected in all areas. Furthermore, on day 14, strong fluorescence signals were detected in the liver, spleen, and kidneys of mice, in addition to the tumor tissue (Figures 17a-17b). This is because clofazimine is a highly lipophilic small molecule compound that can accumulate as inclusion bodies in mammalian resident tissue macrophages, eventually leading to large-scale bioaccumulation and crystallization in the liver and spleen. In addition, clofazimine is mainly metabolized by the liver and kidneys, which is one of the reasons why it accumulates in the liver and kidneys.

[0237] Example 33: Evaluation of the in vitro imaging performance of chlorfazimine suspension

[0238] The in vitro imaging performance of clofazimine suspensions (CFZ NC, crystal form I, crystal form II, and crystal form III) of the same concentration but different aggregation states prepared according to Example 16 was evaluated. The signal-to-background ratio (SBR) of different crystal forms of clofazimine suspensions was mainly quantitatively analyzed. The results are shown in Table 6. All different polymorphs had SBR values ​​significantly higher than the blank control (PBS) group, indicating that clofazimine suspensions (crystal form I, crystal form II, and crystal form III) all have NIR-II fluorescence imaging performance. The clofazimine suspension corresponding to crystal form I has the most significant SBR and the most ideal imaging performance. This is consistent with the NIR-II fluorescence spectroscopy results in Example 27. Therefore, the clofazimine suspension corresponding to crystal form I prepared according to Example 16 was selected for subsequent imaging performance evaluation.

[0239] Table 6. Chlorfenapyridine suspensions in different aggregation states

[0240] Because the clofazimine suspension (CFZ NC) exhibits both NIR-I and NIR-II fluorescence emission signals, the effects of NIR-I and NIR-II fluorescence imaging can be compared under identical environmental conditions. Specifically, 600 μM of crystalline form I clofazimine suspension (CFZ NC) prepared according to Example 16 was injected into a capillary, and NIR-I and NIR-II fluorescence imaging were performed under conditions of no coverage and chicken breast coverage thicknesses of 1 mm, 4 mm, and 8 mm, respectively.

[0241] As shown in Figure 18, in the phantom experiment, both NIR-I and NIR-II fluorescence imaging could clearly distinguish the CFZ fluorescence signal of clofazimine suspension (CFZ NC) within the capillary glass tube. Even when these samples were covered by a 1 mm thick layer of chicken breast tissue, the signal remained clearly imaged and distinguishable. However, when covered by 4.0 mm of chicken breast tissue, NIR-I imaging could not accurately distinguish the signal, while NIR-II fluorescence imaging could still clearly distinguish the CFZ fluorescence signal, indicating that NIR-II fluorescence has better optical resolution and tissue penetration than NIR-I. Furthermore, NIR-I fluorescence imaging lost its resolving power under 8 mm of chicken breast tissue coverage, but NIR-II fluorescence imaging maintained its ability to distinguish the CFZ signal source. Further quantitative analysis showed that the signal-to-background ratio (SBR) of both NIR-I and NIR-II fluorescence decreased with increasing tissue thickness. However, the SBR of NIR-II fluorescence was consistently significantly higher than that of NIR-I (p < 0.01), indicating that NIR-II fluorescence imaging has better overall imaging resolution and depth.

[0242] Example 34: In vivo photoacoustic imaging

[0243] Female BALB / c mice aged 6–8 weeks were marked and shaved. 50 μL of 4T1 cells were then injected at a density of 1 × 10⁻⁶. 7 A cell suspension of 100 cells / mL was subcutaneously injected into the lateral abdomen of mice. The tumor volume was increased to 100–200 mm². 3 At that time, clofazimine suspension (Form I) prepared according to Example 23 was injected into mouse tumors at a dose of 10 mg / kg. After drug intervention, photoacoustic imaging (720 nm) was performed on tumor-bearing mice that had been injected intratumorally on days 0, 7, and 14. As shown in Figures 19a and 19b, a high photoacoustic signal was maintained for 14 days after intratumoral injection, indicating that clofazimine suspension (Form I) can achieve a long-term retention effect after intratumoral injection.

[0244] Example 35: Evaluation of the photoacoustic effect of chloramphenicol suspension

[0245] The crystalline form I chloromethacin suspension (CFZ NC) prepared according to Example 16 was subjected to single-wavelength (720 nm) photoacoustic signal detection and analysis. The results are shown in Figure 20. Compared with the aqueous phase, the chloromethacin suspension (CFZ NC) has a significant photoacoustic signal at 720 nm, showing potential for application in photoacoustic imaging.

[0246] Example 36: In vitro antitumor experiment

[0247] 4T1 cells were loaded at 5 × 10 3Cells were seeded at a density of 10 cells / well in 96-well plates and cultured in DMEM complete medium containing 10% fetal bovine serum, 120 mg / L penicillin, and 200 mg / L streptomycin. After incubation with different doses of clofazimine (molecular form) dissolved in DMSO for 24 h, cell viability at 450 nm was determined using the CCK-8 assay. As shown in Figure 21, cell viability gradually decreased with increasing final concentration of clofazimine. Since the photothermal effect of clofazimine suspension (Form I) is concentration-dependent, a significant photothermal effect was observed at higher concentrations (above 0.25 mg / mL). However, at this concentration, the cytotoxic effect of clofazimine molecules on 4T1 cells approached 100%. To reflect the cytotoxicity and photothermal effect of clofazimine suspension (Form I) at the cellular level, a 2 W / cm² incubation was performed under ice bath conditions. 2 Cells were irradiated with an 808 nm laser (to prevent interference from laser heat generation) and then incubated in a 50 °C water bath for 10 min to replace the photothermal effect observed with a high dose of clofazimine suspension (Form I). Blank control, Laser control, CFZ, and CFZ+50 °C+Laser (CFZ+PTT) groups were established. After each treatment, cells were cultured for 24 h, and cell viability was analyzed using the CCK-8 assay. (See Figure 22). Compared to the Blank control group, the Laser control group did not show significant cytotoxicity, while the CFZ group significantly reduced cell viability. Furthermore, the CFZ+PTT group showed more significant cytotoxicity.

[0248] Example 37: In vivo anti-tumor experiment

[0249] Female BALB / c mice aged 6–8 weeks were marked and shaved. 50 μL of 4T1 cells were then injected at a density of 1 × 10⁻⁶. 7 A cell suspension of 100 cells / mL was subcutaneously injected into the lateral abdomen of mice. The tumor volume was increased to 100–200 mm². 3 At that time, clofazimine suspension (Form I) prepared according to Example 23 was injected into mouse tumors at a dose of 10 mg / kg. 4T1 tumor-bearing mice were treated as shown in Figure 23. Tumor volume was recorded daily, and mice were sacrificed on day 14 for tumor weighing. As shown in Figures 24 and 25, there was no significant difference between the PBS group and the PBS+Laser group. Both the CFZ and CFZ+Laser groups showed significant tumor inhibition compared to the PBS group, with the CFZ+Laser group exhibiting a more significant tumor inhibition effect than the CFZ group. The experimental results showed significant differences.

[0250] Example 38: Evaluation of the dye-lymphatic tracing performance of suspensions of chlorfazimine and pharmaceutically acceptable salts and solvates.

[0251] A score of 0-10 represents the staining condition of lymph nodes, and the score is determined according to the following criteria:

[0252] 0 represents no color change;

[0253] 5 represents staining of the sinus region of a lymph node;

[0254] 10 represents staining of the entire lymph node or marginal sinus.

[0255] According to pharmacodynamic experiments, a staining score of 4 or higher is sufficient for lymphatic tracing. Ten 6-8 week old male Balb / c mice were used, with five mice at each time point. The amorphous chlorofazimine suspension prepared in Example 14 and a 1% methylene blue aqueous solution (self-made, control) were subcutaneously injected into the right hind paw of each mouse. The mice were euthanized 10 and 30 minutes after administration, and the first, second, and third-order lymph nodes (popliteal, iliac, and abdominal aortic lymph nodes) were immediately dissected. The staining was observed, and the degree of lymph node staining was scored. The results showed that the 1% methylene blue injection had a problem with excessively rapid fading. At 10 minutes, it had a good staining effect on the first and second-order lymph nodes, but a certain off-target effect on the third-order lymph nodes. At 30 minutes, staining faded in all three order lymph nodes (Figure 26 and Tables 7-8).

[0256] Sixty male Balb / c mice aged 6-8 weeks were used, with 5 mice in each group. The amorphous clofazimine suspension prepared in Example 14 and the salicylate clofazimine suspension prepared in Example 21 were injected subcutaneously into the right hind paw of each mouse. The mice were euthanized at 10, 30, 60, 120, 180, and 240 minutes after administration, and the primary, secondary, and tertiary lymph nodes (popliteal, iliac, and abdominal aortic lymph nodes) were immediately dissected. The staining was observed, and the degree of lymph node staining was scored.

[0257] The experimental results showed that 180 minutes after administration, the staining scores of the primary, secondary, and tertiary lymph nodes of mice in Example 14 group all reached 4 or higher (Figure 27 and Table 7); and 240 minutes after administration, the staining effect on the primary lymph nodes was still good. The primary lymph node staining score of mice in Example 21 group reached 4 or higher, and the effect on sentinel lymph node tracing remained stable for 240 minutes (Figure 28 and Table 8).

[0258] Table 7. Lymphocyte staining scores 30 minutes after administration of clofazimine suspension and methylene blue aqueous solution.

[0259] Table 8. Lymphocyte staining scores of chlorpromazine suspension and methylene blue aqueous solution within 240 min.

[0260] Experimental results show that, compared with methylene blue, various clofazimine-related preparations have the advantages of rapid staining and accurate localization, and the color fades more slowly, making them suitable for the needs of clinical surgery. They are excellent lymphatic tracers and are suitable for widespread application.

[0261] Example 39: Evaluation of the in vivo NIR-II fluorescence imaging performance of chlorfazimine suspension

[0262] Because chlorofazimine polymorph I exhibited the strongest NIR-II fluorescence emission characteristics among the three room-temperature stable polymorphs, chlorofazimine polymorph I prepared according to Example 16 was subsequently selected for in vivo NIR-II fluorescence imaging testing. Six- to eight-week-old female BALB / c mice were labeled and shaved, and 50 μL of 4T1 cells at a density of 1 × 10⁻⁶ were added. 7 A cell suspension of 100 cells / mL was subcutaneously injected into the lateral abdomen of mice. The tumor volume was increased to 100–200 mm². 3 At that time, clofazimine suspension (crystal form I) prepared according to Example 16 was injected into mouse tumors at a dose of 10 mg / kg. After drug intervention, NIR-II in vivo fluorescence imaging was performed on tumor-bearing mice injected into the tumor at days 0, 1, 3, 7 and 14. The results are shown in Figures 29a and 29b. From day 0 to day 14, the fluorescence signal intensity at the mouse tumor site gradually decreased, but a relatively strong fluorescence signal was detected at all times, indicating that clofazimine suspension has good and stable intratumoral imaging effect.

[0263] Since primary lymph nodes (sentinel lymph nodes) are the first site of cancer cell metastasis, real-time monitoring of primary lymph nodes is crucial for clinical cancer treatment. The intervention group treated with the above-mentioned crystalline form I clofazimine suspension (CFZ NC) underwent NIR-II fluorescence imaging of primary lymph nodes after subcutaneous injection in the hindlimb. The results are shown in Figure 30, demonstrating that clofazimine suspension (CFZ NC) has a good labeling effect on primary lymph nodes.

[0264] Example 40: Evaluation of the in vivo NIR-I photoacoustic imaging performance of chlorfenapyridine suspension

[0265] Since the chlorozimine crystalline form I prepared according to Example 16 exhibits the strongest NIR-I photoacoustic properties among the three room-temperature stable polymorphs, chlorozimine crystalline form I was selected for in vivo NIR-I photoacoustic lymph node tracing tests. The chlorozimine crystalline form I suspension (CFZ NC) intervention group from Example 39 was subjected to NIR-I in vivo photoacoustic lymph node tracing (720 nm), and the in vivo signal was quantitatively evaluated 3 hours after intervention. The results are shown in Figure 31. The chlorozimine crystalline form I suspension (CFZ NC) prepared according to Example 16 has a good NIR-I photoacoustic tracing effect on first-order lymph nodes, and its photoacoustic signal results show significant differences from surrounding tissues.

Claims

1. A suspension of clofazimine characterized in that, The average particle size is 10 nm to 50 μm, and it is prepared by any of the following methods: (1) Antisolvent method: The organic solution containing chlorazimine is reacted with an aqueous solution of stabilizer or water by stirring; (2) Grinding method: Chloride is ground in stabilizer and water to obtain the product.

2. A suspension of clofazimine characterized in that, The average particle size is 0.05–5 μm, and it is prepared by any of the following methods: (1) Antisolvent method: The organic solution containing chlorazimine is reacted with an aqueous solution of stabilizer or water by stirring; (2) Grinding method: Chloride is ground in stabilizer and water to obtain the product.

3. The clarithromycin suspension according to claim 1 or 2, characterized in that, Its solid form is selected from type I, type II, type III or amorphous.

4. The clarithromycin suspension according to claim 1 or 2, characterized in that, The active ingredient in the suspension is chlorfazimine.

5. The clarithromycin suspension according to claim 1 or 2, characterized in that, It exhibits NIR-I fluorescence emission signals in the wavelength range of 800–900 nm.

6. The clarithromycin suspension according to claim 1 or 2, characterized in that, When its solid form is type I, type II or type III, its near-infrared fluorescence intensity is concentration-dependent on chlorofazimine in the concentration range of 0 to 0.5 mM.

7. The clarithromycin suspension according to claim 1 or 2, characterized in that, When its solid form is type I, type II, type III or amorphous, its near-infrared photothermal conversion efficiency is 1% to 35%.

8. The clarithromycin suspension according to claim 1 or 2, characterized in that, When its solid form is type I or amorphous, its photoacoustic signal intensity exhibits a concentration dependence of chlorfazimine within the concentration range of 0–5 mM.

9. The clarithromycin suspension according to claim 1 or 2, characterized in that, In the organic solution containing chlorfazimine, the concentration of chlorfazimine is greater than 0.5 mg / mL; the volume ratio of the organic solution containing chlorfazimine to the aqueous solution of the stabilizer or water is 1:5 to 1:

40.

10. The clarithromycin suspension according to claim 1 or 2, 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, sodium carboxymethyl cellulose, gelatin, and soluplus.

11. The clarithromycin suspension of any one of claims 1, 2, or 10, wherein, In the method (2), the mass-to-volume ratio of stabilizer to water is 0.1 to 100 mg / mL.

12. The use of the chlorfenapyr suspension according to claim 1 or 2 in the preparation of tracers for near-infrared photoacoustic and fluorescence imaging.

13. The use of the chlorfazimine suspension according to claim 1 or 2 in the preparation of photothermal therapy reagents.

14. The use of the clofazimine suspension according to claim 1 or 2 in the preparation of an antitumor drug.

15. The use of the clofazimine suspension according to claim 1 or 2 in the preparation of a medicament for treating bacterial infections and for anti-inflammatory and analgesic purposes.

16. The application of a chlorfenapyridine suspension in the preparation of NIR-II fluorescent tracers, wherein the average particle size of the suspension is 10 nm to 50 μm.

17. Use according to claim 16, characterized in that, The aforementioned NIR-II fluorescent tracer is used for tumor or lymph node tracing.

18. The use according to claim 16, characterized in that, The NIR-II fluorescent tracer exhibits fluorescence emission signals in the wavelength range of 1100–1300 nm.

19. Use according to claim 18, characterized in that, The fluorescence emission signal of the NIR-II fluorescent tracer is concentration-dependent in the concentration range of 0–1 mM.

20. The use according to claim 16, characterized in that, The aggregation morphology of chlorofazimine in the suspension is selected from crystal form I, crystal form II or crystal form III.

21. The application of a chlorfenapyridine suspension in the preparation of a lymphatic tracer, wherein the average particle size of the suspension is 10 nm to 500 nm.

22. The use according to claim 21, characterized in that, The active ingredient in the suspension is selected from chlorofazimine, chlorofazimine hydrochloride, chlorofazimine methanesulfonate, chlorofazimine oxalate, chlorofazimine sulfate, chlorofazimine sulfite, chlorofazimine phosphate, chlorofazimine acetate, chlorofazimine citrate, chlorofazimine isonicotinic acid, chlorofazimine nicotinic acid, chlorofazimine salicylate, chlorofazimine maleate, chlorofazimine hydrated methanesulfonate, chlorofazimine malonic acid, chlorofazimine dodecyl sulfate, chlorofazimine p-aminobenzoate, or chlorofazimine ethyl acetate solvate.

23. The use according to claim 21, characterized in that, Its application modes include at least one of fluorescence tracing, photoacoustic tracing, and dye tracing.

24. The use according to claim 21, characterized in that, The lymphatic tracer is either an NIR-II fluorescent lymphatic tracer or an NIR-I photoacoustic lymphatic tracer.

25. The use according to claim 21, characterized in that, The aggregation morphology of chlorofazimine in the suspension is selected from crystal form I, crystal form II, crystal form III or amorphous.

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