Monomeric compound dta and use thereof in the preparation of a drug for inhibiting temozolomide-sensitive glioma

Abstract

The application discloses a monomer compound DTA and application thereof in preparation of a temozolomide-sensitive glioma inhibiting drug. Through a glioma primary cell model, it is proved that the monomer compound Demethylenedelcoine A (DTA) can produce a better anti-chemotherapy resistant glioma effect than a clinical first-line chemotherapy drug temozolomide and carmustine. Therefore, in clinical treatment of temozolomide chemotherapy resistance and radiotherapy failure recurrent glioma, the monomer compound DTA can have a potential treatment advantage, and therefore has a potential drug development value.

Description

Technical Field

[0001] This invention relates to the field of temozolomide-sensitive glioma treatment technology, and more specifically to the monomeric compound DTA and its use in the preparation of drugs that inhibit temozolomide-sensitive gliomas. Background Technology

[0002] Gliomas are the most common primary intracranial tumors in adults, accounting for approximately 30%-50% of all brain tumors, and have the highest recurrence rate among intracranial tumors. These tumors often exhibit invasive growth, making complete surgical resection difficult, and currently there is no definitively effective treatment. The current clinical approach primarily involves surgery combined with radiotherapy and chemotherapy, but the prognosis for patients is extremely poor, and this approach cannot effectively prolong patient survival.

[0003] Chemotherapy is one of the main treatments for gliomas. Currently, the alkylating agent temozolomide is mainly used in clinical practice for chemotherapy in glioma patients.

[0004] However, glioma cells easily develop resistance (tolerance) to chemotherapy drugs, severely limiting the effectiveness of chemotherapy and reducing the efficacy of treatment. Therefore, reversing chemotherapy resistance in glioma cells is of great significance for the treatment of gliomas. Numerous studies have demonstrated that many chemotherapy drugs exert their anti-tumor effects by inducing tumor cell apoptosis. However, the heterogeneity and molecular variations of glioma cells cause them to evade and inhibit apoptosis, significantly reducing the cell-killing ability of anti-tumor drugs. Therefore, the screening, research, and development of lead compounds that can reverse the inhibition of apoptosis in glioma cells, i.e., induce tumor cell apoptosis, is an important approach to improving current clinical drug treatments for gliomas.

[0005] Therefore, how to provide a compound that induces tumor cell apoptosis and apply it to reverse the inhibition of glioma cell apoptosis is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a monomeric compound DTA and applies it to the inhibition of temozolomide-sensitive gliomas.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A monomeric compound, DTA, has the structural formula shown in Formula I:

[0009]

[0010] As having the same inventive concept as the above-described technical solution, this invention also claims protection for a method for preparing the monomeric compound DTA, comprising the following process:

[0011] (1) Select the whole plant of Delphinium, dry it at a constant temperature, pulverize it, and pass it through a 20-40 mesh sieve to obtain the raw material;

[0012] (2) Add methanol to the raw material and extract it by cold soaking 5 times, each time for 24 hours. After the extract is concentrated under reduced pressure, it is suspended in distilled water and extracted with n-butanol. After concentration under reduced pressure, n-butanol extract is obtained.

[0013] (3) The n-butanol extract and silica gel were mixed in a ratio of 1:1-2. After the solvent was evaporated, 30g of silica gel was used to dry-load the column. The column was first eluted with chloroform:methanol = 10:1, then eluted with Sepedex LH-20 chloroform:methanol = 2:1, and then eluted with reversed-phase silica gel water:methanol = 3-1:1-4. The water:methanol = 1:1 eluent was collected and concentrated under reduced pressure to obtain compound DTA.

[0014] As having the same inventive concept as the above-described technical solution, the present invention also claims protection for the use of the monomeric compound DTA as shown in claim 1 in the preparation of a drug for inhibiting temozolomide-sensitive gliomas.

[0015] Preferably, the monomeric compound DTA inhibits the proliferation of chemotherapeutic and chemotherapeutic-resistant primary glioma cells.

[0016] Preferably, the monomeric compound DTA inhibits the growth rate of primary organoids of temozolomide-resistant gliomas.

[0017] Preferably, the monomeric compound DTA promotes apoptosis in primary temozolomide-resistant glioma cells.

[0018] Preferably, the monomeric compound DTA inhibits the proliferation of temozolomide-resistant gliomas in vivo.

[0019] Preferably, the concentration of the monomeric compound DTA is 10-20 μM.

[0020] As can be seen from the above technical solution, compared with the prior art, the monomeric compound Demethyle nedelcoine A (DTA) isolated from the whole herb of Delphinium honanense (Ranunculaceae family) has shown through activity screening experiments based on primary organoid tissue and cell models of temozolomide-sensitive and temozolomide-resistant glioblastoma that, compared with temozolomide-sensitive glioma organoid tissue and cells, DTA can produce the same growth inhibition and pro-apoptotic effects on primary organoid tissue and cells of temozolomide-resistant glioma. In addition, in vivo experiments further demonstrated that the DMSO solution of DTA has a significant inhibitory effect on subcutaneous tumorigenesis of temozolomide-resistant glioma cell lines.

[0021] Using primary glioma cell models, the monomeric compound Demethylenedelcoine A (DTA) of this product demonstrated better efficacy against chemotherapy-resistant gliomas than first-line clinical chemotherapy drugs temozolomide and carmustine. Therefore, it may have potential therapeutic advantages in the clinical treatment of temozolomide-resistant and radiotherapy-failed recurrent gliomas, thus possessing potential drug development value. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 The attached figure shows the inhibitory effects of TMZ and DTA provided by this invention on primary glioma cells that are sensitive and resistant to chemotherapy.

[0024] Figure 2 The attached figure shows the inhibitory effect of DTA on primary organoids of TMZ-resistant gliomas.

[0025] Figure 3 The attached figure shows the pro-apoptotic effect of DTA on primary TMZ-resistant glioma cells.

[0026] Figure 4 The attached figure shows the in vivo inhibitory effect of DTA on TMZ chemotherapy-resistant gliomas. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] The chemotherapy-sensitive U251 cells, chemotherapy-resistant T98G cells, and reagents used in this embodiment are all commercially available.

[0029] Example 1 verifies the effects of TMZ and DTA on the activity of chemotherapeutic and chemotherapeutic glioma cells. The specific steps are as follows:

[0030] (1) U251 cells and T98G cells were placed in a culture medium containing 10% fetal bovine serum and 100 U / mL penicillin / streptomycin, respectively, at 37°C and 5% CO2.2 The conditions for cultivation.

[0031] (2) U251 cells and T98G cells were respectively subjected to 5×10 3 Cells were seeded at a density of 100 cells / well in 96-well plates and incubated for 24 hours under standard culture conditions.

[0032] (3) Add different concentrations of TMZ and DTA to each well of the culture plate and incubate for 48 hours in an incubator. Then add 10 μL / well of CCK-8 reagent to the 96-well plate and incubate at 37°C for 1 hour.

[0033] (4) Use a multi-functional microplate reader to detect the optical density (OD) value at 450 nm.

[0034] The results are as follows Figure 1 As shown, the inhibitory effect on glioma cell proliferation increases with increasing concentrations of TMZ and DTA. However, the half-inhibitory concentrations (WICs) of temozolomide on chemosensitive and chemoresistant glioma cells were 30.46 μM and 213.2 μM, respectively; while the WICs of DTA on chemosensitive and chemoresistant glioma cells were 11.12 μM and 13.72 μM, respectively. It is evident that, compared to TMZ, the compound DTA in this invention has a significant inhibitory effect on both chemosensitive and chemoresistant glioma cells.

[0035] Example 2 verifies the effect of DTA on primary organoids of TMZ-resistant gliomas. The specific method is as follows:

[0036] Select 1mm 3 Patient-derived glioma tissue of various sizes was minced and suspended in a mixed medium of DMEM / F12 and Neurobasal (volume ratio = 1:1) containing Gluta Max (1X), NEAA (1X), N2 (1X), B27 (1X), 2-mercaptoethanol (1X), and 2.5 μg / ml insulin. The medium was then incubated at 37°C in a 5% CO2 incubator. 2 After two months of culture and growth under specific conditions, glioma organoids were obtained. These organoids were then treated with DTA (14 μM) for days 1, 9, and 18, with physiological saline serving as a control. The growth of organoids in the two groups was compared. Specific results are as follows: Figure 2 As shown.

[0037] Depend on Figure 2 It can be seen that in the control group, the growth rate of glioma organoids increased significantly with the extension of treatment time; while after treatment with the compound DTA of this invention, the glioma organoids did not increase with the extension of time, which can effectively inhibit organ growth.

[0038] Example 3 verifies the effect of DTA on apoptosis of TMZ-resistant glioma cells. The specific method is as follows:

[0039] (1) Take T98G chemoresistant glioma cells in the logarithmic growth phase and use 1.5×10 5 1 / ml was inoculated into a 6-well culture plate;

[0040] (2) After the cells were fully attached to the plate 24 hours after plating, different concentrations of DTA (5 μM, 15 μM, 20 μM) were added. At the same time, a solvent control group was set up, and the cells were cultured for another 48 hours.

[0041] (3) Harvest cells after 48 hours, wash twice with pre-cooled PBS, and prepare 1×10⁻⁶ cells using 1×Binding Buffer. 6 Take 100 μL of cell suspension per mL and place it in a flow cytometry tube. Add 5 μL of 7-AAD and 5 μL of PE for staining according to the kit instructions. Gently vortex the cells and incubate at room temperature in the dark for 15 min. Then add 200 μL of 1×Binding Buffer to the tube and perform flow cytometry analysis within 1 h.

[0042] Depend on Figure 3 The results showed that DTA treatment (5 μM, 15 μM, 20 μM) induced a concentration-dependent increase in the percentage of apoptotic glioma cells, with even 5 μM DTA increasing the percentage of apoptotic glioma cells resistant to TMZ chemotherapy. This indicates that DTA can induce apoptosis in TMZ chemotherapy-resistant glioma cells.

[0043] Example 4 verifies the in vivo inhibitory effect of DTA on TMZ-resistant gliomas.

[0044] Glioma cells T98G were cultured in 10% 1640 medium, and the cells were collected and suspended. Cell viability was counted by trypan blue staining and was >95%. The suspended cells were stored at 4°C for later inoculation. Twenty-four healthy Wistar rats (half male and half female) were randomly divided into three groups (n=8 per group): a control group (2 ml saline), a 10 μM DTA group, and a 20 μM DTA group. 100 μl of DTA containing 2 × 10⁻⁶ cells was subcutaneously injected into the right thigh. 6 A suspension of T98G glioma cells was collected and then housed in a sterile, 25°C environment. Seven days after inoculation, rats were injected intraperitoneally daily for seven consecutive days. Tumor growth was observed in each group of rats. Fourteen days after inoculation, the rats were sacrificed, and the tumors were removed and their weights were measured.

[0045] Depend on Figure 4It was found that, compared with the control group, the tumor weight in the 10 μM DTA group and the 20 μM DTA group was significantly reduced, and the difference was statistically significant. This demonstrates that the compound DTA in this invention can significantly inhibit tumor growth, thereby reducing tumor weight.

[0046] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0047] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The use of a monomeric compound DTA in the preparation of drugs to inhibit temozolomide-sensitive or resistant gliomas, characterized in that, The structural formula of the compound is shown below: 。 2. The application according to claim 1, characterized in that, The preparation method of the compound includes the following process: using Delphinium hainanense, a plant of the Ranunculaceae family, as raw material, the monomeric compound DTA is extracted; (1) Select the whole plant of Delphinium, dry it at a constant temperature, pulverize it, and pass it through a 20-40 mesh sieve to obtain the raw material; (2) Add methanol to the raw material and extract it by cold soaking 5 times, each time for 24 h. After the extract is concentrated under reduced pressure, it is suspended in distilled water and extracted with n-butanol. After concentration under reduced pressure, n-butanol extract is obtained. (3) Mix the n-butanol extract with silica gel at a ratio of 1:1-2, evaporate the solvent, and then dry-load 30g of silica gel onto a column. First, elute with chloroform:methanol at a ratio of 10:1, then elute with Sepedex LH-20 chloroform:methanol at a ratio of 2:1, and then elute with reversed-phase silica gel at a ratio of water:methanol at a ratio of 3-1:1-4. Collect the water:methanol eluent at a ratio of 1:1 and concentrate under reduced pressure to obtain compound DTA.

3. The application according to any one of claims 1-2, characterized in that, The concentration of the monomeric compound DTA is 10-20 μM.

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