A WZB117 nano-preparation and a preparation method thereof

Abstract

The application 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, there is a lack of effective treatment scheme. The application provides a novel ophthalmic preparation and a preparation method thereof, and utilizes hemoglobin and dopamine to simultaneously carry a glucose transporter 1 (Glut1) inhibitor WZB117 and copper ions. The nano preparation can form a positive feedback cycle through a copper death-induced active oxygen burst and an antioxidant system collapse caused by WZB117 double-sulfur death, so as to accelerate tumor cell death.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical preparations, and particularly relates to a WZB117 nano preparation and a preparation method thereof. BACKGROUND

[0002] The posterior pole of the eye generally refers to the posterior pole of the retina, i.e., the central region of the posterior part of the eyeball, and the specific range includes the optic nerve head (optic disc), macula, central retinal vascular system and retinal pigment epithelium and the like. The posterior pole of the eye is a common site of uveal melanoma (UM). Uveal melanoma mainly occurs in the choroid, which is a part of the uvea and is located at the back of the eyeball.

[0003] Enucleation of the eyeball was once the main means for treating uveal melanoma, but in recent years, with the development of other treatment methods, the surgical indications have changed. Now enucleation of the eyeball is mainly suitable for patients with large tumors, involvement of the optic nerve, blindness or secondary glaucoma, retinal detachment. Local resection is suitable for smaller tumors, especially tumors of the iris and ciliary body, and can retain part of the vision. Radiotherapy is currently the main method for retaining the eyeball to treat uveal melanoma. Brachytherapy includes the use of iodine-125, palladium-103, iridium-192 and other radionuclides for patch radiotherapy, which can reach a maximum dose of 70Gy within 5 days, 98% of the tumors are controlled, and the eyeball retention rate is as high as 95%. Proton beam therapy is particularly suitable for posterior pole tumors due to the Bragg peak characteristics, which can more accurately irradiate tumors and reduce damage to surrounding tissues. There is also transscleral patch radiotherapy, which is suitable for medium-sized tumors. Laser therapy includes ordinary laser photocoagulation, transpupillary thermotherapy (TTT) and photodynamic therapy. Ordinary laser photocoagulation is suitable for choroidal malignant melanoma with a height of ≤5D and a range of <30° without retinal detachment on the surface. TTT is suitable for tumors with a thickness of ≤4mm and can be used alone or in combination with scleral surface patch therapy. Photodynamic therapy kills tumor cells by injecting photosensitizers and exposing them to light sources to produce oxygen free radicals, but the efficacy still needs to be further verified. Cryotherapy is suitable for tumors in the equatorial region and before the equatorial region by destroying tumor cells at low temperature.

[0004] Recent studies have found that glucose transporter 1 (Glut1) inhibitors can inhibit the down-regulation of glycolytic enzymes (such as hexokinase and phosphofructokinase) caused by GLUT1, leading to insufficient NADPH, which cannot reduce cystine to cysteine, further causing abnormal accumulation of disulfides, attacking cytoskeletal proteins such as actin, causing cell membrane rupture, and causing disulfide death. WZB117 is a typical Glut1 inhibitor (CAS 1223397-11-2), which has the disadvantages of poor water solubility, low tumor selectivity, and poor therapeutic effect. Cuproptosis is a new type of controlled cell death discovered in recent years, and its core mechanism is the mitochondrial metabolic disorder and protein toxicity stress caused by copper ion (Cu⁺ / Cu²⁺) overload in cells. Unlike traditional apoptosis, pyroptosis or ferroptosis, cuproptosis targets sulfiredoxin proteins and iron-sulfur cluster (Fe-S) proteins in the tricarboxylic acid cycle (TCA), leading to metabolic dysfunction and cell death. Cancer cells that rely on mitochondrial respiration are more sensitive to cuproptosis, while cancer cells that prefer glycolysis may resist cuproptosis by inhibiting the TCA cycle. Disulfide death targets glycometabolism inhibition (NADPH depletion), and cuproptosis targets mitochondrial respiration (TCA inhibition), which can simultaneously block the complementary pathways of energy metabolism of tumor cells, resulting in a "synthetic lethal" effect. The positive feedback loop formed by the ROS explosion induced by cuproptosis and the collapse of the antioxidant system caused by disulfide death accelerates tumor cell death. WZB117, as a poorly soluble drug component, is difficult to form an effective synergistic delivery with water-soluble copper ions. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a nano preparation of WZB117 and a preparation method thereof. The nano preparation can simultaneously carry WZB117 and copper ions, solving the problem of synergistic delivery of poorly soluble drugs and water-soluble cuproptosis components, and can be used for treating uveal melanoma.

[0006] In the first aspect of the present application, a WZB117 nano preparation is provided, characterized in that the nano preparation contains nanoparticles formed by WZB117, hemoglobin, copper ions and polydopamine, the particle size is less than 300 nm, and the WZB117 is a glucose transporter 1 inhibitor;

[0007] The nano-preparation 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 concentration of hemoglobin 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 into the organic phase under stirring, the organic solvent is removed, dopamine hydrochloride and copper chloride are added and incubated under stirring, the mass ratio of dopamine hydrochloride to copper chloride is 1:0.1-3, Tris solution with pH=8.5 is added and continues to be stirred to obtain the nanoparticles, the volume ratio of the aqueous phase to Tris solution is 1:10-20, and the time for the continues stirring is 10-20 hours.

[0008] The above nano-preparation is used for intravitreal administration by injection, and further, is used for intravenous injection.

[0009] The mass ratio of the hemoglobin, dopamine hydrochloride and copper chloride is 10-50:5-30:1-30.

[0010] Preferably, the nanoparticles formed by WZB117, hemoglobin, copper ions and polydopamine are incubated with a polypeptide Cys-Arg-Glu-Lys-Ala to form nanoparticles that can target fibronectin. The polypeptide Cys-Arg-Glu-Lys-Ala is a CREKA peptide, which has high affinity for fibronectin highly expressed in tumor sites.

[0011] Further preferably, the nanoparticles formed by WZB117, hemoglobin, copper ions and polydopamine are incubated with a polypeptide Cys-Arg-Glu-Lys-Ala and povidone to form stable nanoparticles. Povidone is used as a stabilizer to improve the stability of the polypeptide-modified nano-preparation.

[0012] Compared with the prior art, the present application has the following advantages:

[0013] (1) A WZB117 nano-preparation is provided, which solves the problem of poor solubility of WZB117;

[0014] (2) A preparation method of the WZB117 nano-preparation is provided, which has uniform and stable particle size and high drug encapsulation efficiency.

[0015] (3) A nano-preparation for synergistically delivering copper death components and disulfide death drugs is provided, which can effectively treat uveal melanoma. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 : Figure 1 shows the image of the cell uptake of nanoparticles taken by confocal microscopy in Example 1;

[0017] Figure 2 Particle size distribution of the formulation of Example 2;

[0018] Figure 3 Transmission electron microscope image of the formulation of Example 4 (scale = 500 nm).

[0019] Figure 4 Example 1 formulation's ability to inhibit B16F10 cell migration in vitro. DETAILED DESCRIPTION

[0020] The application is further illustrated by the following examples. It should be understood that the examples of the application are given by way of illustration only and therefore should not be taken as limiting the application, so simple modifications to the method of the application are within the scope of the application.

[0021] Example 1

[0022] (1) WZB117 2.5 mg was dissolved in 5 mL of acetone as the organic phase, and hemoglobin 20 mg was dissolved in 1 mL of deionized water as the aqueous phase;

[0023] (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, and 10 mg of dopamine hydrochloride and 10 mg of copper chloride were added and stirred;

[0024] (3) 15 mL of Tris solution with pH = 8.5 was added and stirring was continued for 15 hours.

[0025] Example 2

[0026] (1) WZB117 0.1 mg was dissolved in 1 mL of acetone as the organic phase, and hemoglobin 30 mg was dissolved in 1 mL of deionized water as the aqueous phase;

[0027] (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, and 10 mg of dopamine hydrochloride and 1 mg of copper chloride were added and stirred;

[0028] (3) 10 mL of Tris solution with pH = 8.5 was added and stirring was continued for 10 hours.

[0029] Example 3

[0030] (1) WZB117 3 mg was dissolved in 10 mL of acetone as the organic phase, and hemoglobin 50 mg was dissolved in 1 mL of deionized water as the aqueous phase;

[0031] (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, and 10 mg of dopamine hydrochloride and 30 mg of copper chloride were added and stirred;

[0032] (3) Add 20 mL of Tris solution with pH=8.5 and continue stirring for 20 hours.

[0033] Example 4

[0034] (1) Dissolve 1 mg of WZB117 in 8 mL of acetone as the organic phase, and dissolve 10 mg of hemoglobin in 1 mL of deionized water as the aqueous phase;

[0035] (2) Add the aqueous phase drop by drop to the organic phase under stirring for 1.5 hours, remove the organic solvent by rotary evaporation, and then add 5 mg of dopamine hydrochloride and 3 mg of copper chloride and stir;

[0036] (3) Add 13 mL of Tris solution with pH=8.5 and continue stirring for 14 hours.

[0037] Example 5

[0038] (1) Dissolve 0.1 mg of WZB117 in 3 mL of acetone as the organic phase, and dissolve 30 mg of hemoglobin in 1 mL of deionized water as the aqueous phase;

[0039] (2) Add the aqueous phase drop by drop to the organic phase under stirring for 2 hours, remove the organic solvent by rotary evaporation, and then add 30 mg of dopamine hydrochloride and 10 mg of copper chloride and stir;

[0040] (3) Add 20 mL of Tris solution with pH=8.5 and continue stirring for 15 hours.

[0041] Example 6

[0042] (1) Dissolve 1 mg of WZB117 in 8 mL of acetone as the organic phase, and dissolve 10 mg of hemoglobin in 1 mL of deionized water as the aqueous phase;

[0043] (2) Add the aqueous phase drop by drop to the organic phase under stirring for 1.5 hours, remove the organic solvent by rotary evaporation, and then add 5 mg of dopamine hydrochloride and 3 mg of copper chloride and stir;

[0044] (3) Add 13 mL of Tris solution with pH=8.5 and continue stirring for 14 hours.

[0045] (4) Add 5 mg of polypeptide Cys-Arg-Glu-Lys-Ala to it and incubate for 24 hours.

[0046] Example 7

[0047] (1) Dissolve 1 mg of WZB117 in 8 mL of acetone as the organic phase, and dissolve 10 mg of hemoglobin in 1 mL of deionized water as the aqueous phase;

[0048] (2) The water phase was added dropwise into the organic phase under stirring for 1.5 hours, and the organic solvent was removed by rotary evaporation. 5 mg of dopamine hydrochloride and 3 mg of copper chloride were added and stirred;

[0049] (3) 13 mL of Tris solution with pH=8.5 was added and stirring was continued for 14 hours.

[0050] (4) 5 mg of polypeptide Cys-Arg-Glu-Lys-Ala and 20 mg of povidone were added and incubated for 24 hours.

[0051] Comparative Example 1

[0052] (1) Paclitaxel 3 mg was dissolved in 10 mL of acetone as the organic phase, and hemoglobin 50 mg was dissolved in 1 mL of deionized water as the water phase;

[0053] (2) The water phase was added dropwise into 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;

[0054] (3) 20 mL of Tris solution with pH=8.5 was added and stirring was continued for 20 hours.

[0055] Comparative Example 2

[0056] (1) WZB117 3 mg was dissolved in 10 mL of acetone as the organic phase, and human serum albumin 50 mg was dissolved in 1 mL of deionized water as the water phase;

[0057] (2) The water phase was added dropwise into 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;

[0058] (3) 20 mL of Tris solution with pH=8.5 was added and stirring was continued for 20 hours.

[0059] Comparative Example 3

[0060] (1) WZB117 3 mg was dissolved in 10 mL of ethanol as the organic phase, and hemoglobin 50 mg was dissolved in 1 mL of deionized water as the water phase;

[0061] (2) The water phase was added dropwise into 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;

[0062] (3) 20 mL of Tris solution with pH=8.5 was added and stirring was continued for 36 hours.

[0063] Comparative Example 4

[0064] (1) WZB117 0.5mg dissolved in 2mL acetone as organic phase, hemoglobin 20mg dissolved in 1mL deionized water as aqueous phase;

[0065] (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, and 3.75 mg of dopamine hydrochloride and 3.75 mg of ferric chloride hexahydrate were added and stirred;

[0066] (3) 5mL Tris solution with pH=8.5 was added and stirred for 15 hours.

[0067] Comparative Example 5

[0068] (1) WZB117 3mg dissolved in 10mL acetone as organic phase, hemoglobin 10mg dissolved in 5mL deionized water as aqueous phase;

[0069] (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, and 10mg of dopamine hydrochloride and 30mg of copper chloride were added and stirred;

[0070] (3) 20 mL Tris solution with pH=8.5 was added and stirred for 48 hours.

[0071] Comparative Example 6

[0072] (1) WZB117 1mg dissolved in 10mL acetone as organic phase, hemoglobin 50mg dissolved in 5mL deionized water as aqueous phase;

[0073] (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, and 100mg of dopamine hydrochloride and 1mg of copper chloride were added and stirred;

[0074] (3) 5 mL Tris solution with pH=8.5 was added and stirred for 20 hours.

[0075] Comparative Example 7

[0076] (1) Nile red 0.5mg dissolved in 5mL acetone as organic phase, hemoglobin 20mg dissolved in 1mL deionized water as aqueous phase;

[0077] (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.

[0078] Verification Example

[0079] 1. 2x105 B16F10 cells were seeded into confocal dishes for starvation treatment: DMEM / high glucose medium containing 0.2% FBS was replaced with serum-free DMEM / high glucose medium, and incubated 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 a suitable concentration of 0.015 mg / mL. The medium in the confocal dish was removed, and the serum-free medium containing the nanoparticles was added, and incubated in the incubator for 2 hours. After incubation, the medium containing the nanoparticles was aspirated, and the cells were washed with PBS for 3 times to remove the nanoparticles that were not taken up. Then, 4% paraformaldehyde was added to fix the cells for 15 minutes, and after fixation, the cells were washed with PBS for 3 times. An appropriate amount of DAPI staining solution was added to the cells, and incubated at 37°C for 15 minutes to stain the cell nuclei. After staining, the cells were washed with PBS for 3 times to remove excess DAPI staining solution. The cells were observed under a confocal microscope. The Nile red-labeled nanoparticles emitted red fluorescence under a suitable excitation light, and the cell nuclei emitted blue fluorescence after DAPI staining. An appropriate field of view was selected, and the images of the cells taking up the nanoparticles were taken.

[0080] The results are shown in Table 1. Figure 1 As shown, the nanoscale preparation formed from polydopamine and hemoglobin has stronger red fluorescence than the hemoglobin nanoscale preparation, indicating higher cell uptake efficiency.

[0081] 2. The solutions obtained in Examples 1-7 and Comparative Examples 1-6 were filtered using a 0.45 micron microporous filter. The obtained liquid was monitored for particle size and polydispersity index using a laser particle size analyzer.

[0082] Table 1. Particle size and polydispersity index (PDI) of each example

[0083]

[0084] The results are shown in Table 1. Figure 2 As shown in Table 1, the particle size of the nanoscale preparation prepared in the examples of the present application conforms to a normal distribution, the particle size is less than 300 nm, the PDI is less than 0.3, and the particle size is uniform.

[0085] 3. The solution obtained in Example 4 was filtered using a 0.45 micron microporous filter, and the obtained liquid was negatively stained with phosphotungstic acid, and the morphology was observed using a transmission electron microscope.

[0086] The results are shown in Table 1. Figure 3 As shown in Table 1, the nanoscale preparation prepared in the examples of the present application is approximately spherical, and the particle size is uniform.

[0087] 4. The solutions obtained in Examples 1-7 and Comparative Examples 2-6 were filtered through 0.45 micron microfiltration membranes, and 200 microliters of the obtained liquid was diluted 5 times with methanol to break the emulsion, and the WZB117 drug content was detected by high performance liquid chromatography, with the following chromatographic conditions: the mobile phase was a solution of water:methanol (20:80), the flow rate was 0.5 mL / min, the sample volume was 10 μL, and the detection wavelength was 216 nm. The encapsulation rate of the nanoparticles was calculated. v / v

[0088] Table 2. Encapsulation rates of Examples 1-7 and Comparative Examples 2-6

[0089]

[0090] The results are shown in Table 2, and the encapsulation rate of the preparation WZB117 prepared according to the present application is higher.

[0091] 5. The ability of the preparation of Example 1 to inhibit the migration of B16F10 cells in vitro was evaluated by a cell scratch test. After B16F10 cells were digested, centrifuged and resuspended, they were seeded in a 6-well cell culture plate at a cell density of 2.4 x 10 5 cells per well. The cell growth state and cell density were observed, and when the cells had grown to cover the bottom of the plate, a 200 μL sterile gun tip was used to make a scratch. After the floating cells were removed by rinsing with PBS, 2 mL of serum-free medium containing PBS, WZB117 and the preparation of Example 1 filtered through a 0.45 um filter were added, respectively, and placed in a 37 °C incubator for culture. Photographs were taken at 0 h and 24 h, respectively. The scratch healing area was measured using Image J software and analyzed.

[0092] The results are shown in Table 2, and the encapsulation rate of the preparation WZB117 prepared according to the present application is higher. Figure 4 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 nano-preparation of the present application effectively inhibited the migration of tumor cells.

[0093] The above only describes preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.​

Claims

1. A WZB117 nano-formulation, characterized in that, The nano-formulation contains nanoparticles formed by WZB117, hemoglobin, copper ions and polydopamine, with a particle size of less than 300 nm. WZB117 is a glucose transporter 1 inhibitor. The nanoparticles are prepared as follows: WZB117 is dissolved in acetone as the organic phase, and hemoglobin is dissolved in deionized water as the aqueous phase. The hemoglobin concentration is 10-50 mg / mL, the mass ratio of WZB117 to hemoglobin is 0.1-3:10-50, and the volume ratio of acetone to deionized water is 1-10:

1. The aqueous phase is added dropwise to the organic phase under stirring to remove the organic solvent. Dopamine hydrochloride and copper chloride are added and stirred during incubation. The mass ratio of dopamine hydrochloride to copper chloride is 1:0.1-3. Tris solution with pH=8.5 is added and stirring is continued to obtain nanoparticles. The volume ratio of the aqueous phase to the Tris solution is 1:10-20, and the stirring time is 10-20 hours.

2. The method for preparing nano-formulations as described in claim 1, characterized in that, The raw materials for preparing the nano-formulation also include the polypeptide Cys-Arg-Glu-Lys-Ala. The nanoparticles are co-incubated with the polypeptide Cys-Arg-Glu-Lys-Ala to obtain nanoparticles targeting fibronectin.

3. The method for preparing nano-formulations as described in claim 2, characterized in that, The raw materials for preparing the nano-formulation also include the polypeptide Cys-Arg-Glu-Lys-Ala and povidone. The nanoparticles, the polypeptide Cys-Arg-Glu-Lys-Ala and povidone are incubated together to obtain stable nanoparticles.

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