WZB117 nano preparation and preparation method thereof

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a WZB117 nano preparation and a preparation method thereof. In the treatment of uveal melanoma, an effective treatment scheme is lacked. According to the novel ophthalmic preparation and the preparation method thereof, hemoglobin and dopamine are used for carrying a glucose transporter 1 (Glut1) inhibitor WZB117 and copper ions at the same time, the nano preparation can form positive feedback circulation through active oxygen outbreak induced by copper death and anti-oxidation system collapse caused by WZB117 disulfide death, and tumor cell death is accelerated.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a WZB117 nano preparation and a preparation method thereof. Background Art

[0002] The posterior pole of the eye typically refers to the posterior retinal pole, the central area at the back of the eyeball. Specifically, it includes structures such as the optic nerve head (optic disc), macula, central retinal vasculature, and retinal pigment epithelium. The posterior pole of the eye is a common site for uveal melanoma (UM). Uveal melanoma primarily develops in the choroid, a part of the uvea located at the back of the eyeball.

[0003] Enucleation was once the mainstay of treatment for uveal melanoma, but with the development of other treatment options in recent years, surgical indications have evolved. Enucleation is now primarily reserved for patients with large tumors involving the optic nerve, blindness, or secondary glaucoma or retinal detachment. Local excision is suitable for smaller tumors, particularly those involving the iris and ciliary body, and can preserve partial vision. Radiotherapy is currently the mainstay of eye-sparing treatment for uveal melanoma. Brachytherapy, including radiotherapy with radionuclides such as iodine-125, palladium-103, and iridium-192, can deliver a maximum dose of 70 Gy over 5 days, achieving 98% tumor control and a 95% eye-sparing rate. Proton beam therapy, due to its Bragg peak properties, allows for more precise tumor irradiation and minimizes damage to surrounding tissue, making it particularly suitable for posterior pole tumors. Transscleral radiotherapy is also suitable for medium-sized tumors. Laser treatments include conventional laser photocoagulation, transpupillary thermotherapy (TTT), and photodynamic therapy. Conventional laser photocoagulation is suitable for choroidal malignant melanomas with a height ≤5D, a range <30°, and no surface retinal detachment. TTT is suitable for tumors ≤4mm thick and can be used alone or in combination with scleral surface application. Photodynamic therapy generates oxygen free radicals that kill tumor cells by injecting a photosensitive substance and then exposing it to a light source, but its efficacy requires further verification. Cryotherapy destroys tumor cells through low temperatures and is suitable for tumors in the equator and before the equator.

[0004] Recent studies have found that glucose transporter 1 (Glut1) inhibitors can inhibit the GLUT1-induced downregulation of glycolytic enzymes (such as hexokinase and phosphofructokinase), leading to NADPH deficiency and the inability to reduce cystine to cysteine. This leads to abnormal disulfide accumulation, which attacks cytoskeletal proteins such as actin, triggering cell membrane rupture and causing disulfide death. WZB117, a typical Glut1 inhibitor (CAS 1223397-11-2), suffers from poor water solubility and low tumor selectivity, resulting in poor therapeutic efficacy. Cuproptosis is a newly discovered novel controlled cell death mechanism. Its core mechanism is mitochondrial metabolic disturbances and proteotoxic stress triggered by intracellular copper ion (Cu⁺ / Cu²⁺) overload. Unlike traditional apoptosis, pyroptosis, or ferroptosis, cuproptosis targets lipoylated proteins and iron-sulfur cluster (Fe-S) proteins in the tricarboxylic acid (TCA) cycle, leading to metabolic dysfunction and cell death. Cancer cells that rely on mitochondrial respiration are more sensitive to copper-induced cell death, while those that prefer glycolysis may resist copper-induced cell death by inhibiting the TCA cycle. Disulfide-mediated cell death, which targets glucose metabolism (NADPH depletion), and copper-mediated cell death, which targets mitochondrial respiration (TCA inhibition), can simultaneously block complementary pathways of energy metabolism in tumor cells, leading to a "synthetic lethality" effect. The burst of reactive oxygen species (ROS) induced by copper-induced cell death and the collapse of the antioxidant system caused by disulfide-mediated cell death create a positive feedback loop, accelerating tumor cell death. As a poorly soluble drug component, WZB117 is difficult to effectively deliver with water-soluble copper ions. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the present invention provides a nanoformulation of WZB117 and its preparation method. This nanoformulation can simultaneously carry WZB117 and copper ions, solving the problem of co-delivery of poorly soluble drugs and water-soluble copper ions, and can be used to treat uveal melanoma.

[0006] In a first aspect, the present invention provides a WZB117 nanoformulation, characterized in that the nanoformulation contains nanoparticles formed by WZB117, hemoglobin, copper ions and polydopamine, with a particle size of less than 300 nm, and the WZB117 is a glucose transporter 1 inhibitor; The nanoformulation is prepared by the following method: WZB117 is dissolved in acetone as an organic phase, hemoglobin is dissolved in deionized water as an aqueous phase, the hemoglobin concentration is 10-50 mg / mL, the mass ratio of WZB117 to hemoglobin is 0.01-3:10-50, the volume ratio of acetone to deionized water is 1-10:1, the aqueous phase is added dropwise to the organic phase under stirring, the organic solvent is removed, dopamine hydrochloride and copper chloride are added, incubated and stirred, the mass ratio of dopamine hydrochloride to copper chloride is 1:0.1-3, a Tris solution with a pH of 8.5 is added and continued to stir to obtain nanoparticles, the volume ratio of the aqueous phase to the Tris solution is 1:10-20, and the stirring time is continued for 10-20 hours.

[0007] The nano preparation is used in the vitreous body and is administered by injection, and further, is administered by intravenous injection.

[0008] The mass ratio of the hemoglobin, dopamine hydrochloride and copper chloride is 10-50:5-30:1-30.

[0009] Preferably, the nanoparticles formed by WZB117, hemoglobin, copper ions, and polydopamine are incubated with the polypeptide Cys-Arg-Glu-Lys-Ala to form nanoparticles that can target fibronectin. The polypeptide Cys-Arg-Glu-Lys-Ala is a CREKA peptide that has a high affinity for fibronectin, which is highly expressed in tumor sites.

[0010] Further preferably, the nanoparticles formed by WZB117, hemoglobin, copper ions, and polydopamine are incubated simultaneously with the polypeptide Cys-Arg-Glu-Lys-Ala and povidone to form stable nanoparticles. Povidone acts as a stabilizer to improve the stability of the polypeptide-modified nanoformulation.

[0011] Compared with the prior art, the present invention has the following advantages: (1) Provides a WZB117 nanoformulation that solves the poor solubility problem of WZB117; (2) A method for preparing WZB117 nanoformulations is provided, which has uniform and stable particle size and high drug encapsulation efficiency.

[0012] (3) A nanoformulation that synergistically delivers copper apoptotic components and disulfide apoptotic drugs is provided for the effective treatment of uveal melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : Confocal imaging of cells taking up nanoparticles of the preparation of Example 1; Figure 2 : Particle size distribution diagram of the preparation of Example 2; Figure 3 : Transmission electron micrograph of the preparation of Example 4 (scale bar = 500 nm).

[0014] Figure 4 : The ability of the preparation of Example 1 to inhibit B16F10 cell migration in vitro. DETAILED DESCRIPTION

[0015] The present invention is further illustrated by the following examples. It should be understood that the examples are provided for illustration only and are not intended to limit the present invention. Therefore, simple modifications to the present invention based on the method of the present invention fall within the scope of the present invention.

[0016] Example 1 (1) 2.5 mg of WZB117 was dissolved in 5 mL of acetone as the organic phase, and 20 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase; (2) The aqueous phase was added dropwise to the organic phase under stirring for 1 hour, and the organic solvent was removed by rotary evaporation. 10 mg of dopamine hydrochloride and 10 mg of copper chloride were added and stirred; (3) Add 15 mL of Tris solution (pH 8.5) and continue stirring for 15 hours.

[0017] Example 2 (1) 0.1 mg of WZB117 was dissolved in 1 mL of acetone as the organic phase, and 30 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase; (2) The aqueous phase was added dropwise to the organic phase under stirring for 1 hour, and the organic solvent was removed by rotary evaporation. 10 mg of dopamine hydrochloride and 1 mg of copper chloride were added and stirred; (3) Add 10 mL of Tris solution (pH = 8.5) and continue stirring for 10 hours.

[0018] Example 3 (1) 3 mg of WZB117 was dissolved in 10 mL of acetone as the organic phase, and 50 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase; (2) The aqueous phase was added dropwise to the organic phase under stirring for 1 hour, and the organic solvent was removed by rotary evaporation. 10 mg of dopamine hydrochloride and 30 mg of copper chloride were added and stirred; (3) Add 20 mL of Tris solution (pH = 8.5) and continue stirring for 20 hours.

[0019] Example 4 (1) 1 mg of WZB117 was dissolved in 8 mL of acetone as the organic phase, and 10 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase; (2) The aqueous phase was added dropwise to the organic phase under stirring for 1.5 hours. The organic solvent was removed by rotary evaporation. 5 mg of dopamine hydrochloride and 3 mg of copper chloride were added and stirred. (3) Add 13 mL of Tris solution (pH = 8.5) and continue stirring for 14 hours.

[0020] Example 5 (1) 0.1 mg of WZB117 was dissolved in 3 mL of acetone as the organic phase, and 30 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase; (2) The aqueous phase was added dropwise to the organic phase under stirring for 2 hours, and the organic solvent was removed by rotary evaporation. 30 mg of dopamine hydrochloride and 10 mg of copper chloride were added and stirred; (3) Add 20 mL of Tris solution (pH = 8.5) and continue stirring for 15 hours.

[0021] Example 6 (1) 1 mg of WZB117 was dissolved in 8 mL of acetone as the organic phase, and 10 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase; (2) The aqueous phase was added dropwise to the organic phase under stirring for 1.5 hours. The organic solvent was removed by rotary evaporation. 5 mg of dopamine hydrochloride and 3 mg of copper chloride were added and stirred. (3) Add 13 mL of Tris solution (pH = 8.5) and continue stirring for 14 hours.

[0022] (4) Add 5 mg of the peptide Cys-Arg-Glu-Lys-Ala and incubate for 24 hours.

[0023] Example 7 (1) 1 mg of WZB117 was dissolved in 8 mL of acetone as the organic phase, and 10 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase; (2) The aqueous phase was added dropwise to the organic phase under stirring for 1.5 hours. The organic solvent was removed by rotary evaporation. 5 mg of dopamine hydrochloride and 3 mg of copper chloride were added and stirred. (3) Add 13 mL of Tris solution (pH = 8.5) and continue stirring for 14 hours.

[0024] (4) Add 5 mg of the peptide Cys-Arg-Glu-Lys-Ala and 20 mg of povidone and incubate for 24 hours.

[0025] Comparative Example 1 (1) 3 mg of paclitaxel was dissolved in 10 mL of acetone as the organic phase, and 50 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase; (2) The aqueous phase was added dropwise to the organic phase under stirring for 1 hour, and the organic solvent was removed by rotary evaporation. 10 mg of dopamine hydrochloride and 30 mg of copper chloride were added and stirred; (3) Add 20 mL of Tris solution (pH = 8.5) and continue stirring for 20 hours.

[0026] Comparative Example 2 (1) 3 mg of WZB117 was dissolved in 10 mL of acetone as the organic phase, and 50 mg of human serum albumin was dissolved in 1 mL of deionized water as the aqueous phase; (2) The aqueous phase was added dropwise to the organic phase under stirring for 1 hour, and the organic solvent was removed by rotary evaporation. 10 mg of dopamine hydrochloride and 30 mg of copper chloride were added and stirred; (3) Add 20 mL of Tris solution (pH = 8.5) and continue stirring for 20 hours.

[0027] Comparative Example 3 (1) 3 mg of WZB117 was dissolved in 10 mL of ethanol as the organic phase, and 50 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase; (2) The aqueous phase was added dropwise to the organic phase under stirring for 1 hour, and the organic solvent was removed by rotary evaporation. 10 mg of dopamine hydrochloride and 30 mg of copper chloride were added and stirred; (3) Add 20 mL of Tris solution (pH = 8.5) and continue stirring for 36 hours.

[0028] Comparative Example 4 (1) 0.5 mg of WZB117 was dissolved in 2 mL of acetone as the organic phase, and 20 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase; (2) The aqueous phase was added dropwise to the organic phase under stirring for 1 hour. The organic solvent was removed by rotary evaporation. 3.75 mg of dopamine hydrochloride and 3.75 mg of ferric chloride hexahydrate were added and stirred. (3) Add 5 mL of Tris solution (pH = 8.5) and continue stirring for 15 hours.

[0029] Comparative Example 5 (1) 3 mg of WZB117 was dissolved in 10 mL of acetone as the organic phase, and 10 mg of hemoglobin was dissolved in 5 mL of deionized water as the aqueous phase; (2) The aqueous phase was added dropwise to the organic phase under stirring for 1 hour, and the organic solvent was removed by rotary evaporation. 10 mg of dopamine hydrochloride and 30 mg of copper chloride were added and stirred; (3) Add 20 mL of Tris solution (pH = 8.5) and continue stirring for 48 hours.

[0030] Comparative Example 6 (1) 1 mg of WZB117 was dissolved in 10 mL of acetone as the organic phase, and 50 mg of hemoglobin was dissolved in 5 mL of deionized water as the aqueous phase; (2) The aqueous phase was added dropwise to the organic phase under stirring for 1 hour. The organic solvent was removed by rotary evaporation. 100 mg of dopamine hydrochloride and 1 mg of copper chloride were added and stirred. (3) Add 5 mL of Tris solution (pH = 8.5) and continue stirring for 20 hours.

[0031] Comparative Example 7 (1) 0.5 mg of Nile red was dissolved in 5 mL of acetone as the organic phase, and 20 mg of hemoglobin was dissolved in 1 mL of deionized water as the aqueous phase; (2) The aqueous phase was added dropwise to the organic phase under stirring for 1 hour, and the organic solvent was removed by rotary evaporation.

[0032] Verification Example 1. Place 2×10 5 B16F10 cells were seeded into confocal microplates and starved: DMEM / high glucose medium containing 0.2% FBS was replaced with serum-free DMEM / high glucose medium and cultured for 24 hours. Nile red was used to replace WZB117 to label the nanoparticles of Example 1. The Nile red-labeled nanoparticles of Example 1 and Comparative Example 7 were diluted with serum-free medium to an appropriate concentration of 0.015 mg / mL. The medium in the confocal microplates was removed, and the serum-free medium containing the nanoparticles was added, and the cells were incubated in the incubator for another 2 hours. After the incubation period, the medium containing the nanoparticles was aspirated, and the cells were washed three times with PBS to remove the uningested nanoparticles. 4% paraformaldehyde was then added to fix the cells for 15 minutes. After fixation, the cells were washed three times with PBS. An appropriate amount of DAPI stain was added to the cells and incubated at 37°C for 15 minutes to stain the cell nuclei. After staining, the cells were washed three times with PBS to remove excess DAPI stain. The cells were observed under a confocal microscope. Nile red-labeled nanoparticles emit red fluorescence under appropriate excitation light, while DAPI-stained nuclei produce blue fluorescence. Select an appropriate field of view to capture images of cells taking up nanoparticles. The results are as follows Figure 1 It was shown that the nanoformulation formed by polydopamine and hemoglobin had stronger red fluorescence than the hemoglobin nanoformulation, indicating a higher cellular uptake efficiency. 2. The solutions obtained in Examples 1-7 and Comparative Examples 1-6 were filtered through a 0.45 μm microporous filter membrane. The particle size and polydispersity index of the obtained liquid were monitored using a laser particle size analyzer.

[0033] Table 1. Particle size and polydispersity index (PDI) of various examples The results are as follows Figure 2 As shown in Table 1, the particle size of the nanoparticles prepared in the examples of the present invention conforms to the normal distribution, the particle size is less than 300 nm, the PDI is less than 0.3, and the particle size is uniform.

[0034] 3. The solution obtained in Example 4 was filtered using a 0.45 μm microporous filter membrane, and the resulting liquid was negatively stained with phosphotungstic acid, and the morphology was observed using a transmission electron microscope.

[0035] The results are as follows Figure 3 The nanoparticles prepared in the examples of the present invention are approximately spherical in shape and have relatively uniform particle sizes.

[0036] 4. The solutions obtained in Examples 1-7 and Comparative Examples 2-6 were filtered through a 0.45 μm microporous membrane. 200 μl of the resulting liquid was diluted 5-fold with methanol to break the emulsion. The WZB117 drug content was determined by high performance liquid chromatography. The chromatographic conditions were: the mobile phase was water: methanol (20:80 v / v ) solution at a flow rate of 0.5 mL / min, an injection volume of 10 μL, and a detection wavelength of 216 nm. Calculate the encapsulation efficiency of the nanoparticles.

[0037] Table 2. Encapsulation efficiency of Examples 1-7 and Comparative Examples 2-6 The results are shown in Table 2. The encapsulation efficiency of the preparation WZB117 prepared by the present invention is relatively high.

[0038] 5. The ability of the preparation of Example 1 to inhibit B16F10 cell migration in vitro was evaluated by cell scratch test. B16F10 cells were digested, centrifuged and resuspended, and the cells were plated at 2.4×10 5 Cells were seeded at a density of 100 cells / well in a 6-well cell culture plate. Observe the cell growth status and cell density. When the cells covered the bottom of the dish, streak with a 200 μL sterile pipette tip. After rinsing with PBS to remove floating cells, 2 mL of serum-free culture medium containing PBS, WZB117, and the preparation of Example 1 after passing through a 0.45 μm filter membrane were added and cultured in a 37 °C constant temperature incubator. Photos were taken at 0 h and 24 h, respectively. The scratch healing area was measured and analyzed using Image J software.

[0039] The results are as follows Figure 4As shown, the migration rate of B16F10 cells in Example 1 was 26.89%; the migration rate of B16F10 cells in free WZB117 was 67.45%; and the migration rate of B16F10 cells in PBS was 63.70%. The nanoformulation of the present invention effectively inhibited tumor cell migration.

[0040] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A WZB117 nanoformulation, characterized in that: The nanoformulation contains nanoparticles formed by WZB117, hemoglobin, copper ions and polydopamine, with a particle size of less than 300 nm, and the WZB117 is a glucose transporter 1 inhibitor; The nanoformulation is prepared by the following method: WZB117 is dissolved in acetone as an organic phase, hemoglobin is dissolved in deionized water as an aqueous phase, the hemoglobin concentration is 10-50 mg / mL, the mass ratio of WZB117 to hemoglobin is 0.01-3:10-50, the volume ratio of acetone to deionized water is 1-10:1, the aqueous phase is added dropwise to the organic phase under stirring, the organic solvent is removed, dopamine hydrochloride and copper chloride are added, incubated and stirred, the mass ratio of dopamine hydrochloride to copper chloride is 1:0.1-3, a Tris solution with a pH of 8.5 is added and continued to stir to obtain nanoparticles, the volume ratio of the aqueous phase to the Tris solution is 1:10-20, and the stirring time is continued for 10-20 hours.

2. The method for preparing the nanoformulation according to claim 1, wherein: The mass ratio of the hemoglobin, dopamine hydrochloride and copper chloride is 10-50:5-30:1-30.

3. The method for preparing the nanoformulation according to claim 1, wherein: The raw materials for preparing the nano preparation also include polypeptide Cys-Arg-Glu-Lys-Ala. The nano particles are co-incubated with the polypeptide Cys-Arg-Glu-Lys-Ala to obtain nano particles targeting fibronectin.

4. The method for preparing the nanoformulation according to claim 3, wherein: The raw materials for preparing the nano preparation also include polypeptide Cys-Arg-Glu-Lys-Ala and povidone. The nano particles, polypeptide Cys-Arg-Glu-Lys-Ala and povidone are incubated together to obtain stable nano particles.

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