Sound-sensitive agent as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a sound-sensitive agent and a preparation method and application thereof, and the sound-sensitive agent has a structure as shown in a formula (I), methacrylate-temozolomide (MA-TMZ) has good sound sensitivity and blood brain barrier penetrability, and ultrasonic and MA-TMZ can induce generation of singlet oxygen so as to enhance the toxic effect of the medicine on brain glioma and melanoma cells. The new therapy for developing the anti-tumor effect by utilizing sonodynamic development of MA-TMZ and active products through ultrasonic excitation has immeasurable clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a sonosensitizer and a preparation method and application thereof. Background Art

[0002] Malignant tumors continue to threaten human health and well-being. Glioblastomas (especially glioblastomas (GBMs)) and melanomas are particularly challenging due to their highly invasive nature and resistance to chemotherapy and radiotherapy. Temozolomide (TMZ), an oral imidazole tetrazine prodrug, has rapidly become a first-line chemotherapeutic agent for glioblastomas and malignant melanoma since its launch in 1999. As a new generation of small molecule alkylating agent prodrug, TMZ has a molecular weight of only 194.15 Da, good lipid solubility, approximately 30%-40% blood-brain barrier penetration, and oral bioavailability approaching 100%. At physiological pH, the drug spontaneously hydrolyzes to its active product, monomethyltriazene 5-(3-methyltriazene-1-yl)-imidazole-4-carboxamide (MTIC). MTIC further reacts with water to release 5-aminoimidazole-4-carboxamide (AICA) and a methyldiazonium cation, which transfers a methyl group to the N7 position of DNA bases guanine (N7-MeG), N3 adenine (N3-MeA), and O6-guanine (O6-MeG), forming mismatches. If O6-methylguanine-DNA methyltransferase (MGMT) activity is low or depleted, continued repair failure triggers DNA breakage, cell cycle arrest, and apoptosis. Although TMZ has been on the market for over 20 years, it remains the preferred chemotherapy drug due to its oral convenience and potent antitumor activity. However, in actual clinical treatment, only half of patients are sensitive to the drug, and some develop resistance with continued treatment, resulting in poor efficacy. Therefore, the development of new, safe, and reliable therapies to enhance the anti-tumor efficacy of TMZ is an urgent issue.

[0003] Sonodynamic therapy (SDT) is a new, non-invasive tumor treatment that utilizes the synergistic effect of ultrasound and sonosensitizers to generate large amounts of highly cytotoxic reactive oxygen species (ROS), which in turn kill tumor cells. Low-frequency ultrasound has the advantage of noninvasively penetrating tissue at the centimeter level, reaching deep into tumors. Furthermore, the focused ultrasound beam can precisely target the irradiated area, achieving localized treatment while maximally sparing surrounding normal tissue. Sonosensitizers are crucial for the effectiveness of sonodynamic therapy, but currently, there is a lack of sonosensitizers suitable for gliomas in clinical practice. Therefore, the development of highly effective, safe, blood-brain barrier-penetrating, and targeted sonosensitizers remains an urgent challenge. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a sonosensitizer and a preparation method and application thereof.

[0005] To achieve the above object, the technical solution adopted by the present invention is: A sonosensitizer having the structure of the following formula (I); (I).

[0006] The present invention also includes a method for preparing the sonosensitizer, which is prepared using the following formula (II): (II).

[0007] The method specifically comprises the following steps: mixing TMZ-COOH with hydroxyethyl methacrylate, adding 4-dimethylaminopyridine DMAP and 1-ethylcarbonyl diimmonium hydrochloride EDCI as catalysts, and obtaining the product MA-TMZ.

[0008] The present invention also includes an application of the sonosensitizer. Using ultrasound in conjunction with temozolomide to treat tumors can induce singlet oxygen 1 The production of O2 further enhances the tumor therapeutic effect.

[0009] Specifically, the excited ultrasonic frequency is 20kHz-1MHz; the ultrasonic intensity is 0.5W / cm 2 -2W / cm 2 ; Ultrasonic duty cycle is 10%-50%, and ultrasonic time is 30-60s.

[0010] Preferably, the excited ultrasonic frequency is 1 MHz; the ultrasonic intensity is 1 W / cm 2 ; Ultrasonic duty cycle is 20%; Ultrasonic time is 60s.

[0011] The application is to prepare for sonodynamic therapy of tumors, wherein the tumor is glioma and / or melanoma.

[0012] Compared with the prior art, the present invention has the following beneficial effects: Methacrylate-temozolomide (MA-TMZ) exhibits excellent sonosensitivity and blood-brain barrier penetration. Ultrasound synergistically activates MA-TMZ, enhancing its toxicity against glioma and melanoma cells by inducing singlet oxygen production. This invention utilizes ultrasound to stimulate the sonodynamics of MA-TMZ and its active product to develop novel anti-tumor therapies, which will have inestimable clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the MA-TMZ H NMR spectrum; Figure 2 is the blood-brain barrier penetration rate of MA-TMZ; Figure 3Middle A: Detection graph of singlet oxygen generation at different time points after ultrasound activation of MA-TMZ; Figure 3 Middle B is the detection diagram of singlet oxygen generation by ultrasound-activated MA-TMZ with different concentrations; Figure 4 Ultrasonic stability diagram of MA-TMZ for MS detection; Figure 5 This is the ROS detection diagram in MA-TMZ cells; Figure 6 This is the IC50 graph of ultrasound synergistic effect of MA-TMZ; Figure 7 A is the effect of ultrasound synergistically with MA-TMZ on tumor cell apoptosis; B is the effect of ultrasound synergistically with MA-TMZ on tumor cell cycle arrest; Figure 8 Figure A shows the inhibitory effect of ultrasound synergistically with MA-TMZ on the growth of intracranial tumors in mice; Figure B shows the effect of ultrasound synergistically with MA-TMZ on the survival of mice. DETAILED DESCRIPTION

[0014] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below with reference to the accompanying drawings and the best embodiments.

[0015] Example 1: Synthesis of temozolomide derivative MA-TMZ.

[0016] 1. Synthesis of TMZ-COOH: Dissolve temozolomide (9.7 g) in concentrated sulfuric acid (50 ml) to form a yellow solution; cool the mixture to 0°C under nitrogen, and slowly add an aqueous solution of sodium nitrite (6.9 g, 50 ml) dropwise to catalyze the reaction; return the mixture to room temperature, stir in the dark for 24 h, and then cool to 0°C. Quench with ice (100 g) and continue stirring to produce a white solid precipitate. Vacuum filter, wash with cold water, and vacuum dry to obtain carboxylated temozolomide (TMZ-COOH).

[0017] 2. Synthesis of MA-TMZ: TMZ-COOH (9 g) was dissolved in dichloromethane (DCM, 300 mL) to form a turbid solution. Hydroxyethyl methacrylate (5.76 g) was added and mixed thoroughly. DMAP (0.56 g) and EDCI (10.61 g) were then added to catalyze the reaction. Under nitrogen, the mixture was stirred at room temperature for 3 h. The reaction mixture was then diluted with DCM (500 mL) and washed with 0.1 M aqueous HCl (5 x 800 mL). The organic layer was dried over MgSO₄, filtered, and concentrated under reduced pressure to yield a residue. The residue was purified by C₁₈ (0.1% FA) to yield a white solid (8.07 g), temozolomide methacrylate.

[0018] The structure of TMA-MA was identified by hydrogen nuclear magnetic resonance spectroscopy: 1H NMR (400 MHz, DMSO-d6) δ 8.85 (s, 1H), 6.07-6.02 (m, 1H), 5.71-5.66 (m, 1H), 4.66-4.58 (m, 2H), 4.48- 4.40 (m, 2H), 3.88 (s, 3H), 1.90-1.84 (m, 3H) (see Figure 1 ).

[0019] Example 2: Detection of the blood-brain barrier penetration rate of MA-TMZ.

[0020] Using C57 black mice as a model, they were randomly divided into TMZ group and MA-TMZ group, with 3 mice in each group. 250mmol / kg of temozolomide and methacrylate temozolomide were administered orally, and blood and brain tissue samples were collected from the mice 1.5 hours later. The supernatant was taken after centrifugation at 3000 r·min*5 min, and methanol was added at a ratio of 1:3 and vortexed for 1 min to remove protein. Then, the supernatant was centrifuged at 10000r / min*10min, and an appropriate amount of supernatant was collected for detection. The blood-brain permeability of the drug was calculated by mass spectrometry. The blood-brain barrier penetration rate of the drug in the MA-TMZ group was about 57.25%, which was better than the 40.94% of TMZ (see Figure 2 ).

[0021] Example 3: MA-TMZ singlet oxygen generation detection diagram and time dependence.

[0022] Use PBS to prepare TMZ and MA-TMZ solutions with a concentration of 200μM. After vortexing and mixing, stand in the dark at room temperature for 1 hour. Add singlet oxygen detection reagent SOSG to the above reagents at a ratio of 1:1000. After vortexing and mixing, place on the ultrasound probe with ultrasound parameters (1MHz, 1W / cm2, 20%). Take out 200μl of liquid at 0, 2, 4, 6, 8, and 10 minutes of ultrasound, add PBS to dilute it at a ratio of 1:10, and detect the ultraviolet absorption of the solution at excitation wavelength Ex=504 nm and emission wavelength Em=525 nm in a spectrophotometer to obtain the absorbance value. The results showed that the sonodynamic force of MA-TMZ began to be stronger than TMZ at 2 minutes of ultrasound, and gradually increased with the extension of ultrasound time, indicating that the drug has stronger sonosensitivity (see Figure 3 Middle A).

[0023] MA-TMZ solutions at concentrations of 20, 50, 100, 200, 400, and 800 μM were prepared using PBS. Singlet oxygen detection reagent SOSG was added at a ratio of 1:1000 and then stimulated with ultrasound (parameters: 1 MHz, 1 W / cm2, 20%, 1 min). The absorbance of each group was measured using a spectrophotometer. The results showed that the sonodynamic intensity of MA-TMZ was positively correlated with drug concentration (see Figure 3 Middle B).

[0024] Example 4: Using MS to detect the ultrasonic stability of MA-TMZ.

[0025] A 200 μM MA-TMZ solution was prepared using PBS and divided into two groups. One group was treated with ultrasound (ultrasound parameters: 1 MHz, 1 W / cm2, 20%, 5 min), while the other group was not treated. Mass spectrometry was then used to detect that ultrasound did not affect the structure of MA-TMZ (see Figure 4 ).

[0026] Example 5: Detection of ROS in MA-TMZ cells.

[0027] Using glioma cells LN229 as a model, LN229 glioma cells in the logarithmic growth phase were obtained, digested with trypsin, and seeded into 6-well plates at a density of 5 x 105. They were divided into 6 groups: control group, TMZ group, MA-TMZ group, simple ultrasound group, TMZ ultrasound group, and MA-TMZ ultrasound group. After the cells adhered to the wall and the serum-free culture medium was replaced, 200 μM TMZ and MA-TMZ were added respectively. After 1 hour, ultrasound stimulation was given (ultrasound parameters: 1 MHz, 1 W / cm2, 20%, 2 min), and DCFH-DA probes were loaded at 1:1000. After incubation for 1 hour, ROS were detected by flow cytometry. The results showed that both TMZ and MA-TMZ activated by ultrasound could produce reactive oxygen species in cells, but the fluorescence intensity of MA-TMZ was stronger, indicating that MA-TMZ has a stronger ability to generate reactive oxygen species in cells (see Figure 5 ).

[0028] Example 6: IC50 of ultrasound synergistic effect of MA-TMZ.

[0029] Using LN229 glioma cells as a model, logarithmically growing LN229 cells were trypsinized and seeded at a density of 3,000 cells / well in 96-well plates. After cell attachment, various concentrations of TMZ and MA-TMZ (0.31, 0.92, 2.74, 8.23, 24.69, 74.07, 222.22, 666.67, and 2000 μM) were added. One hour later, ultrasound was applied (ultrasound parameters: 1 MHz, 1 W / cm², 20%, 1 min). After 72 hours, CCK-8 solution was added at a ratio of 1000:1. After incubation in a cell culture incubator for 2 hours, the OD450 value was measured using a microplate reader. The results showed that the IC50 of MA-TMZ was 681.6 μM, and the IC50 of ultrasound-assisted MA-TMZ was 192.5 μM, both significantly exceeding the 1675 μM of TMZ alone. Compared with TMZ, MA-TMZ has a stronger tumor cell killing effect, and the sonodynamic effect of MA-TMZ can be further enhanced by combining it with ultrasound to activate its sonodynamic effect (see Figure 6 ).

[0030] Example 7: Effects of ultrasound synergistically with MA-TMZ on tumor cell apoptosis and cell cycle arrest.

[0031] Using glioma cells LN229 as a model, the cells were divided into 6 groups, including control group, TMZ group, MA-TMZ group, simple ultrasound group, TMZ ultrasound group, and MA-TMZ ultrasound group, and three replicate wells were set. LN229 glioma cells in the logarithmic growth phase were obtained, digested with trypsin, and seeded into 6-well plates at a density of 5 x 105. After the cells adhered to the wall, they were given different treatments. After 24 hours, the cell apoptosis rate and cell cycle arrest were detected by flow cytometry. The results of flow apoptosis showed that the apoptosis rate of the TMZ group was 8.68%, the apoptosis rate of MA-TMZ cells was 10.97%, the apoptosis rate of the TMZ ultrasound group was 22.62%, and the apoptosis rate of the MA-TMZ ultrasound group was 30.25% (see Figure 7 This indicates that MA-TMZ has a better killing effect on tumor cells than TMZ, and combined with ultrasound treatment can further enhance the killing effect. Flow cytometry results also showed that the tumor cell cycle arrest in the MA-TMZ treatment group increased significantly, and combined with ultrasound treatment can further increase the cell cycle arrest rate (see Figure 7 Middle B).

[0032] Example 8: Effects of ultrasound and MA-TMZ on intracranial tumors in mice.

[0033] Bal-c nude mice were used as a model and divided into 4 groups, namely control group, MA-TMZ group, simple ultrasound group and MA-TMZ ultrasound group, with 6 mice in each group. Lucifer-bearing LN229 cells were implanted into the skull of 5-week-old Bal-c nude mice at a density of 305 cells / 5 μL (position: 2.0 mm right of bregma, 1.0 mm posterior, 2.0 mm depth). On the 7th day after tumor implantation, mice were gavaged with MA-TMZ (250 mmol / kg). One hour after gavage, the mice in the MA-TMZ ultrasound group were treated with ultrasound (ultrasound parameters: 1.0 MHz, 2 W / cm2, 20%, 2 min). Ultrasound was administered 5 days a week, on days 1, 3 and 5 respectively. The therapeutic effect of ultrasound synergistically with MA-TMZ was significant, and the growth of intracranial tumors was significantly inhibited. In vivo experimental results showed that the use of MA-TMZ to treat glioma-bearing mice could significantly inhibit tumor growth, and the ultrasound combined with MA-TMZ treatment group had the best effect (see Figure 8 Further statistics on the survival of mice in each group showed that MA-TMZ treatment could effectively prolong the survival of mice, and the tumor suppression effect of the combined ultrasound treatment group was more significant (see Figure 8 Middle B).

[0034] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A sonosensitizer, characterized in that Having the structure of the following formula (I); （I）。 2. A method for preparing the sonosensitizer according to claim 1, characterized in that: The following formula (II) is used for preparation: (II)。 3. The method for preparing the sonosensitizer according to claim 2, wherein: The method specifically comprises the following steps: mixing TMZ-COOH with hydroxyethyl methacrylate, adding 4-dimethylaminopyridine DMAP and 1-ethylcarbonyl diimmonium hydrochloride EDCI as catalysts, and obtaining the product MA-TMZ.

4. Use of the sonosensitizer according to claim 1.

5. The use according to claim 4, characterized in that Using ultrasound in combination with temozolomide to treat tumors can induce singlet oxygen 1 The production of O2 further enhances the tumor therapeutic effect.

6. The use according to claim 5, characterized in that The excited ultrasonic frequency is 20kHz-1MHz; the ultrasonic intensity is 0.5W / cm 2 -2W / cm 2 ; Ultrasonic duty cycle is 10%-50%, and ultrasonic time is 30-60s.

7. The use according to claim 5, characterized in that The excited ultrasonic frequency is 1 MHz; the ultrasonic intensity is 1 W / cm 2 ; Ultrasonic duty cycle is 20%; Ultrasonic time is 60s.

8. The use according to claim 4, characterized in that Preparation for sonodynamic therapy of tumors.

9. The use according to claim 8, characterized in that The tumor is glioma and / or melanoma.

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