CN113476609A

CN113476609A - Composition of temozolomide and hypoxanthine-guanine nucleoside phosphate transferase micromolecule inhibitor and application thereof

Info

Publication number: CN113476609A
Application number: CN202110896450.3A
Authority: CN
Inventors: 钱旭; 尤永平; 施祝梅; 尹建星; 葛新; 汪秀星
Original assignee: Nanjing Medical University
Current assignee: Nanjing Medical University
Priority date: 2021-08-05
Filing date: 2021-08-05
Publication date: 2021-10-08

Abstract

The invention discloses a TMZ and HPRT1 small-molecule inhibitor composition and application thereof, wherein the molar ratio of the TMZ to the HPRT1 small-molecule inhibitor is 1-20: 1, the drug sensitivity of TMZ can be obviously improved by the combined administration of the two drugs. Experiments prove that compared with single TMZ administration, the combined use of the HPRT1 inhibitor can obviously enhance the inhibitory action of TMZ on glioma cell activity, is expected to become a new chemotherapy scheme for chemotherapy-resistant glioma, and has wide application prospect.

Description

Composition of temozolomide and hypoxanthine-guanine nucleoside phosphate transferase micromolecule inhibitor and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a temozolomide and hypoxanthine-guanine nucleoside phosphate transferase small-molecule inhibitor composition and application thereof.

Background

In nearly twenty-three years, with the development and application of advanced instruments and equipment, the growth and fusion of related subjects and the continuous progress of new diagnosis and treatment concepts, people gradually deepen the research on neurosurgical diseases. Brain glioma is the most common malignant tumor of the central nervous system, accounts for about 80%, because of the characteristics of high invasiveness, easy recurrence and the like, the mortality of patients is high, and particularly for high-grade glioma patients, half of the life cycle is less than 14 months. Currently, the standard treatment regimen for glioma primarily involves surgical resection combined with post-operative chemoradiotherapy, where post-operative chemotherapy is particularly critical to improving patient prognosis. Temozolomide (TMZ) is a new generation of oral alkylating agent, which can cause DNA alkylation of tumor cells to promote apoptosis due to its advantages such as easy passage through the blood brain barrier, etc., and has been used as a first-line drug for chemotherapy after glioma operation. However, in clinical treatment, TMZ chemotherapy resistance is an important reason for poor later-stage curative effect, researchers do not have new research breakthrough aiming at the problem for many years, and the search for new research breakthrough is particularly urgent for clinical treatment of glioma.

HPRT1 (hypoxanthine-guanine nucleoside phosphate transferase) is a key enzyme in guanine or hypoxanthine ribosylation, and is involved mainly in the salvage synthesis pathway of intracellular purine nucleotides in the cytoplasm. The production of guanylic acid or inosine monophosphate is catalyzed by xanthine or hypoxanthine and phosphoribosyl pyrophosphate (PRPP) by HPRT 1. Inactivation or reduction of the HPRT1 enzyme activity may lead to various degrees of metabolic disease, such as Lesch-Nyhan syndrome. Defects in HPRT1 can lead to metabolic reprogramming of tumor cells, regulating tumor progression.

Antimetabolites of purine nucleotides are mainly analogs of purine, amino acids or folic acid, etc. They interfere or block the anabolism of purine nucleotides, primarily in a competitive manner, or "spurious" or the like, thereby preventing nucleotide biosynthesis. As the purine analogues, 6-MP (6-mercaptopurine), 6-TG (6-thioguanine) and the like can be mentioned. The structures of 6-MP and 6-TG are similar to those of hypoxanthine and guanine, and can directly influence HPRT1 through competitive inhibition, so that phosphoribose in PRPP molecules cannot be transferred to guanine and hypoxanthine, and purine synthesis is prevented.

Failure of DNA methylation and mismatch repair is currently believed to be the major mechanism of TMZ cytotoxicity. Under physiological conditions, TMZ does not require enzymatic catalysis, and when a water molecule acts on the 4 th carbon atom with positive charge, the closed ring is opened immediately, carbon dioxide is released, and an active product 5- (3-methyltriazalen-1-) imidazole-4-amide (MTIC) is generated. MTIC is converted to cationic methyl diazonium salt, which transfers the methyl group to DNA and degrades to the final product 5-aminoimidazole-4-carboxamide- (AICA), which is excreted by the kidney. The site of DNA methylation is the 7 th nitrogen atom of guanine and the 3 rd oxygen atom of adenine in sequence. Although the proportion of the methyl guanine adduct occupying the oxygen atom at the 6 th position is the least, the methyl guanine adduct plays a key role in resisting tumors. During DNA replication, although the mismatch repair system recognizes mismatched base pairs, it cannot find a matched base for methylguanine, which is the 6 th oxygen atom, and thus the daughter strand DNA is nicked. The gap gradually accumulates with cell division, eventually preventing replication initiation, thus arresting the cell cycle at G2/M and the cell undergoes apoptosis.

The chemotherapy resistance mechanism of glioma cells mainly focuses on MGMT abnormal expression, glioma stem cells and the like. Previous studies show that the expression level of MGMT in recurrent glioma tissues and TMZ drug-resistant glioma cells is remarkably increased. The high-expression MGMT is used as methyltransferase and can transfer methyl on O6 site to itself, thereby weakening the killing of TMZ on tumor cells. Research shows that the interference measures aiming at the MGMT, such as the scheme of knocking down the MGMT, the use of an MGMT inhibitor and the like, can obviously enhance the chemotherapy effect of the TMZ. Glioma stem cells are a small fraction of tumor cells in tumor tissue that have the ability to self-renew and proliferate indefinitely, and are considered the "seed source" for glioma recurrence because of their higher resistance to chemoradiotherapy as compared to glioma cells. The targeting GSC key gene can inhibit glioma stem cell proliferation and promote cell apoptosis, thereby enhancing chemotherapy sensitivity and improving glioma patient prognosis.

Although adjusting the treatment regimen to the results of the above study may partially increase TMZ sensitivity, the improvement was not significant. Therefore, the TMZ resistance mechanism needs to be further explored on the basis of the above, so as to increase the sensitivity of glioma chemotherapy.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

The invention aims to find a medicament which can be combined with a chemotherapeutic medicament TMZ to be applied to glioma, improve the curative effect of the TMZ on treating the glioma, reduce the occurrence of chemotherapy tolerance and improve the survival time of a tumor patient.

In one aspect, the invention provides a composition of temozolomide and a small molecule inhibitor of hypoxanthine-guanine nucleoside phosphate transferase: the molar ratio of the temozolomide to the hypoxanthine-guanine nucleoside phosphate transferase micromolecule inhibitor is 0.5-100: 1, the small molecule is an organic compound with a molecular weight of less than 900 daltons.

As a preferred scheme of the composition of the temozolomide and hypoxanthine-guanine nucleoside phosphate transferase small molecule inhibitor, the invention comprises the following steps: the molar ratio of the temozolomide to the hypoxanthine-guanine nucleoside phosphate transferase micromolecule inhibitor is 1-80: 1.

as a preferred scheme of the composition of the temozolomide and hypoxanthine-guanine nucleoside phosphate transferase small molecule inhibitor, the invention comprises the following steps: the molar ratio of the temozolomide to the hypoxanthine-guanine nucleoside phosphate transferase micromolecule inhibitor is 1-40: 1.

as a preferred scheme of the composition of the temozolomide and hypoxanthine-guanine nucleoside phosphate transferase small molecule inhibitor, the invention comprises the following steps: the hypoxanthine-guanine nucleoside phosphate transferase small molecule inhibitor comprises 6-MP and/or 6-TG.

As a preferred scheme of the composition of the temozolomide and hypoxanthine-guanine nucleoside phosphate transferase small molecule inhibitor, the invention comprises the following steps: the concentration of the temozolomide is 100-400 mu M; the concentration of the 6-MP is 5-20 mu M, and the concentration of the 6-TG is 5-20 mu M.

As a preferred scheme of the composition of the temozolomide and hypoxanthine-guanine nucleoside phosphate transferase small molecule inhibitor, the invention comprises the following steps: the administration concentration of the temozolomide is 20-50mg/kg, the administration concentration of the 6-MP is 1-20mg/kg, and the administration concentration of the 6-TG is 1-20 mg/kg.

As another aspect of the present invention, the present invention provides an application of the composition of temozolomide and a small molecule inhibitor of hypoxanthine-guanine nucleoside phosphate transferase: the composition is used for preparing medicines for treating and inhibiting the growth of tumor cells. The tumor, including glioma. Wherein, the hypoxanthine-guanine nucleoside phosphate transferase small molecule inhibitor can inhibit the activation of temozolomide on an AMPK signal path and increase the chemotherapy sensitivity of glioma cells.

The invention has the beneficial effects that: the invention provides a TMZ and HPRT1 small-molecule inhibitor composition, and the combination of the two synergistically enhances the treatment effect of TMZ. Experiments prove that in glioma cells, TMZ and 6-MP can be used together to inhibit cell proliferation by inhibiting AMPK signal pathway activation and DNA damage repair, and the synergistic inhibition effect of the two drugs is still remarkable in a nude mouse in-situ glioma model. The small molecule inhibitor composition and the pharmaceutical preparation can be applied to preparing antitumor drugs and have wide application prospects.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:

FIG. 1 is a graph showing the effect of various concentrations of TMZ on the AMPK signaling pathway in various glioma cell lines; in glioma cells and glioma stem cells, TMZ at a concentration of 200 μ M can significantly activate the AMPK signaling pathway in glioma cells.

FIG. 2 is a graph of the effect of knockdown of HPRT1 on the glioma cell line AMPK signaling pathway; after TMZ treatment at a concentration of 200 μ M for 24 hours on the control or HPRT1 knockdown group of glioma cells, AMPK signaling pathway was significantly activated in the control group cells, while activation of AMPK signaling pathway was inhibited in the HPRT1 knockdown group cells.

FIG. 3 is a mass spectrometric detection of the reaction of AICA with PRPP mediated by HPRT1 to produce AICAR. After 100ng of active wild type or mutant HPRT1 protein, 10mM PRPP and 10mM AICA were reacted at 37 ℃ for 2 hours, the presence or absence of AICAR production was determined by mass spectrometry.

FIG. 4 is a mass spectrometric detection of the reaction of HPRT1 mediated AICA and PRPP to produce AICAR in glioma cells. The wild-type or mutant HPRT1 plasmid was knocked down and complemented back in glioma cells and glioma stem cells. After 24 hours of treatment of the cells with isotope-labeled TMZ, cell samples were collected and the cells were examined for changes in the levels of isotope-labeled AICA and AICAR by mass spectrometry.

Fig. 5 is a graph demonstrating the effect of HPRT1 knockdown on glioma cell sensitivity to TMZ in a cell viability assay. Glioma cells and glioma stem cells were treated with TMZ at a concentration of 200 μ M for 1, 3, and 5 days, respectively. Compared with the control group of cells, TMZ has stronger inhibition effect on the growth of the HPRT1 knockdown group of cells.

FIG. 6 is a graph of the effect of TMZ on tumor growth in a mouse intracranial in situ tumorigenic model of U87 cells; TMZ treatment was administered continuously in mouse models of U87 cells in the control or HPRT1 knockdown group. Compared with the control group of mice, the tumor growth of the HPRT1 knock-down group is obviously inhibited, and the survival time of the mice is obviously prolonged.

FIG. 7 is a graph of the effect of TMZ on tumor growth in a mouse intracranial in situ tumorigenic model of glioma stem cell MES 28; TMZ treatment was administered continuously in mouse models of control or HPRT1 knockdown group MES 28 cells. Compared with the control group of mice, the tumor growth of the HPRT1 knock-down group is obviously inhibited, and the survival time of the mice is obviously prolonged.

FIG. 8 shows mass spectrometry detection of the reaction of TMZ-derived AICA and PRPP inhibited by HPRT1 inhibitor in glioma cells to produce AICAR. After treatment of glioma cells for 24 hours with 200 μ M concentrations of isotope-labeled TMZ in combination with different concentrations of HPRT1 inhibitor, cell samples were collected and the changes in the levels of isotope-labeled AICA and AICAR were detected by mass spectrometry.

Fig. 9 is a graph of cell viability assays demonstrating the effect of HPRT1 inhibitors on glioma cell TMZ sensitivity. Glioma cells and glioma stem cells were treated with TMZ at a concentration of 200 μ M in combination with HPRT1 inhibitor at a concentration of 10 μ M for 1, 3, and 5 days, respectively. TMZ in combination with an inhibitor of HPRT1 was more potent in inhibiting cell growth than TMZ alone.

FIG. 10 is a graph of the combined effect of TMZ and HPRT1 inhibitors at different concentrations in glioma cells; the TMZ and HPRT1 inhibitors were treated at different concentrations in combination for 24 hours and the Compuyn software was used to calculate the synergy index of the two drugs, where the horizontal line indicates CI 1 and CI <1 indicates synergy; the results show a synergistic inhibitory effect in the U87 cell line.

FIG. 11 is a graph of the effect of TMZ alone and TMZ in combination with an inhibitor of HPRT1 (6-MP) on tumor growth in a mouse intracranial in situ tumorigenic model of U87 cells; compared with the mice in the group using TMZ alone, the tumor growth of the TMZ combined with the HPRT1 inhibitor is remarkably inhibited, and the survival time of the mice is remarkably prolonged.

FIG. 12 is a graph of the effect of TMZ alone and TMZ in combination with an inhibitor of HPRT1 (6-TG) on tumor growth in a mouse intracranial in situ tumorigenic model of U87 cells; compared with the mice in the group using TMZ alone, the tumor growth of the TMZ combined with the HPRT1 inhibitor is remarkably inhibited, and the survival time of the mice is remarkably prolonged.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

Example 1:

western blot is adopted to detect the change of signal paths of U87, U251, T98G, LN18, MES 28 and GSC 3028 glioma cell lines under the single-drug treatment of TMZ with different concentrations, and the specific method is as follows:

1. the U87, U251, T98G, LN18, MES 28, and GSC 3028 cell lines were 6X 10 in advance one day⁵The cells were plated in 6cm dishes, 7 dishes for each cell.

2. Single dose TMZ concentrations were prepared at 0. mu.M, 200. mu.M, 400. mu.M.

3. After the cells are well paved, 5mL of the medicine in the step 2 is added into each dish 24h, and DMSO with the concentration of 2 per mill is added into a control group for treatment for 24 h.

4. After 24h of drug treatment, the cells were washed twice with PBS, drained, 200. mu.L of cell lysate was added to each dish, the lysed cells were collected into 1.5mL EP tubes with a scraper, lysed for half an hour at 4 ℃, centrifuged at 13000rpm for 15min, and the supernatants were stored at-80 ℃.

5. The protein content of each sample was measured by BCA method.

6. Preparing 10% polyacrylamide gel for protein separation, loading 20 mu g of protein samples into each hole, performing 100V constant-pressure electrophoresis until bromophenol blue disappears, performing 250mA wet rotation for 2h, sealing 3% BSA for 3 hours, incubating at 4 ℃ for 6h, washing the membrane with 1 xTBST for 3 times, 10min for each time, incubating at room temperature for 1h with a secondary antibody, washing the membrane with 1 xTBST for 5 times, 6min for each time, and developing and exposing with ECL developing solution.

As shown in FIG. 1, as the treatment concentration of TMZ increased, the expression of p-ACC1(Ser79) and p-AMPK alpha (Thr172) in U87, U251, T98G, LN18, MES 28, GSC 3028 increased significantly, indicating that TMZ significantly activated the AMPK signaling pathway in glioma cells.

Example 2

Western blot is adopted to detect the influence of HPRT1 knock-down in U87 cells and MES 28 glioma cells on a signal channel under single TMZ drug treatment, and the specific implementation scheme is as follows:

1. the control group and the HPRT1 knock-down group U87 and MES 28 cells were 6X 10 cells one day in advance⁵The cells were plated in 6cm dishes, 7 dishes for each cell.

2. A single dose of TMZ was prepared at a concentration of 200. mu.M.

5. The protein content of each sample was measured by BCA method.

As shown in FIG. 2, TMZ treatment of U87 and MES 28 resulted in a significant increase in p-ACC1(Ser79) and p-AMPK α (Thr172) expression in cells, while HPRT1 knockdown inhibited activation of the AMPK signaling pathway.

Example 3

Mass spectrometry was used to detect the generation of AICAR by HPRT1 mediated AICA and PRPP reactions, and the specific embodiment was as follows:

1. the active wild-type and mutant HPRT1 enzymes were purified in bacteria.

2. The purified protein concentration was measured by BCA method, and 100ng of the wild type and mutant HPRT1 enzyme was aspirated and reacted with 10mM PRPP and 10mM AICA at 37 ℃ for 2 hours.

3. An 18C filter column is used for filtering out impurities, and a 10KDa filter column is used for filtering out redundant protein. The rest sample is used for liquid chromatography and mass spectrum detection.

As shown in fig. 3, HPRT1 can catalyze the conversion of AICA to AICAR.

Example 4

Mass spectrometry was used to detect the generation of AICAR by HPRT1 mediated AICA and PRPP reactions in glioma cells, and the specific embodiment is as follows:

1. synthesis of 15N-labeled TMZ.

2. The control group, HPRT1 knock-down group, HPRT1 wild-type and mutant glioma cells were arranged at 1X 10⁴Each well was seeded in 6-well plates.

3. A single-drug isotopically-labeled TMZ concentration of 200 μ M was formulated.

4. After 24 hours of TMZ treatment of the cells, cell samples were collected, and 18C filter column was used to remove impurities and 10kDa filter column was used to remove excess protein. The rest sample is used for liquid chromatography and mass spectrum detection.

As shown in fig. 4, HPRT1 knockdown or mutation can significantly inhibit the production of TMZ-derived AICAR in cells.

Example 5

CCK-8 is adopted to detect the influence of HPRT1 knock-down on the TMZ sensitivity of glioma cells, and the specific implementation scheme is as follows:

1. u87, MES 28 were plated in duplicate wells at 1000 cells per well in 96-well plates per cell per concentration and controls were set up the day before.

2. A single dose of TMZ was prepared at 200. mu.M. Cells in the control and HPRT1 knockdown groups were treated for 1, 3, and 5 days, respectively.

3. And (3) after TMZ treatment is finished, removing the old culture medium, adding 10 mu L of CCK-8 detection reagent into each hole, incubating for 1h in a cell culture box with 5% carbon dioxide at 37 ℃, detecting absorbance under the condition of excitation light wavelength of 450nm by using a chemiluminescence apparatus, and recording data of each hole.

As shown in FIG. 5, TMZ continued treatment of the cells for 5 days inhibited the growth of control tumor cells. And the growth inhibition effect of TMZ on the HPRT1 knockdown cells is stronger when TMZ is continuously used for treating the cells for 5 days, which indicates that the knockdown of HPRT1 can obviously increase the killing effect of TMZ on tumor cells.

Example 6

A mouse intracranial in-situ tumor formation model is adopted to verify that the chemotherapy sensitivity of the glioma in-situ model can be increased by knocking down HPRT1, and the specific embodiment is as follows:

1. the cells of the control group and the HPRT1 knockdown group are transfected with luciferase virus, so that the subsequent tumor size detection is facilitated.

2. Will be 1 × 10⁶U87 cells were injected into the brain parenchyma of mice in situ, TMZ was injected intraperitoneally at a dose of 20mg/kg starting on day 4 after injection, and in vivo imaging was performed on

days

4, 14, 25, and 36 to count the size of intracranial tumors in mice.

3. And (5) counting the death time of the mouse and drawing a survival curve.

As shown in fig. 6, compared with the control mice, TMZ has a stronger effect of inhibiting the growth of intracranial tumors in the HPRT 1-knocked-down mice, and the HPRT 1-knocked-down mice have a better prognosis and a longer survival time, suggesting that knocking-down of HPRT1 can significantly increase the tumor killing effect of TMZ.

Example 7

2. Will be 1 × 10⁵ MES 28 cells were injected in situ into the brain parenchyma of mice, TMZ was injected intraperitoneally at a dose of 20mg/kg starting on day 30 after injection, and in vivo imaging detection was performed on

days

30, 40, 50, and 60 to count the sizes of intracranial tumors in the mice.

3. And (5) counting the death time of the mouse and drawing a survival curve.

As shown in fig. 7, compared with the control mice, TMZ has a stronger effect of inhibiting the growth of intracranial tumors in the HPRT 1-knocked-down mice, and the HPRT 1-knocked-down mice have a better prognosis and a longer survival time, suggesting that knocking-down of HPRT1 can significantly increase the tumor killing effect of TMZ.

Example 8

HPRT1 and TMZ were administered in combination and mass spectrometry was used to detect changes in AICA and AICAR levels in glioma cells. The specific embodiment is as follows:

1. preparing a single medicine TMZ 200 mu M; 5 μ M of HPRT1 inhibitor + TMZ 200 μ M and 10 μ M of HPRT1 inhibitor + TMZ 200 μ M.

2. Culturing the cells in the culture solution prepared in the step 1 for 24 hours, and collecting cell samples.

3. Filtering out impurities by using an 18C filter column, and filtering out redundant protein by using a 10KDa filter column. The rest sample is used for liquid chromatography and mass spectrum detection.

As shown in fig. 8, HPRT1 inhibitors can significantly inhibit TMZ-derived AICAR production in cells.

Example 9

CCK-8 is adopted to detect the influence of the HPRT1 inhibitor on the TMZ sensitivity of glioma cells, and the specific embodiment is as follows:

1. u87 cells were plated at 1000 cells per well in 96-well plates at 3 replicates per cell concentration and controls were set on the previous day.

2. Preparing a single medicine TMZ 200 mu M; 10 μ M of HPRT1 inhibitor (6-MP or 6-TG) and 10 μ M of HPRT1 inhibitor plus 200 μ M of TMZ. U87 cells were treated for 1, 3, and 5 days, respectively.

As shown in FIG. 9, TMZ continued treatment of the cells for 5 days inhibited the growth of control tumor cells. And the growth inhibition effect of TMZ on cells is stronger when the TMZ is combined with the HPRT1 inhibitor to continuously treat the cells for 5 days, which indicates that the HPRT1 inhibitor can obviously increase the killing effect of the TMZ on tumor cells.

Example 10

Alamar blue is adopted to detect the cell proliferation of TMZ and HPRT1 inhibitor under the conditions of single medicine and combination of two medicines, and CI values are calculated, and the specific embodiment is as follows:

1. u87 cells were plated in 96-well plates at 1000 cells per well, 3 replicates per cell concentration, and controls were set on the day before.

2. Preparing single medicine TMZ 0 μ M, 10 μ M, 50 μ M, 100 μ M, 150 μ M, 200 μ M and 250 μ M; single drug HPRT1 inhibitor (6-MP or 6-TG)0 μ M, 1 μ M, 2.5 μ M, 5 μ M, 7.5 μ M, 10 μ M and 15 μ M; and any combination of the concentrations of both drugs.

3. And (2) absorbing the old culture medium after the cells adhere to the wall in the step 1, adding a mixed solution of 100 mu L of complete culture medium and 10 mu L of Alamar Blue into each hole, incubating for 4 hours in a cell culture box with 37 ℃ and 5% carbon dioxide, detecting absorbance by using a chemiluminescence instrument under the conditions of excitation light wavelength 534nm and emission light wavelength 584nm, and recording data of each hole as Day0 data.

4. Cells which were tested for Day0 data were aspirated away from the Alamar Blue containing mixture, added to the drug formulated in step 2 on each well, and monitored for cell growth on the third Day by Alamar Blue as described in step 2 and recorded as Day3 data.

5. The relative proliferation rates of the obtained concentrations are plotted in a bar chart, and the Compuyn software is used for calculating the synergy index of the two drugs and then analyzing and plotting.

As shown in fig. 10, the combined concentration of TMZ and HPRT1 inhibitors decreased significantly with increasing concentration relative to the proliferation rate of the same single drug. Calculating the synergy index of the two drugs by using Compuyn software, wherein the horizontal line in the figure indicates that CI is 1, and when CI is more than 1, the two drugs have antagonism; when CI is 1, the two medicines have the addition function; when CI <1, the two medicines have synergistic effect. The results show that the TMZ and HPRT1 inhibitors have strong synergistic inhibition effect in glioma cell lines.

Example 11

The mouse intracranial in situ tumor model is adopted to verify that the combination of the HPRT1 inhibitor (6-MP) and TMZ can increase the chemotherapy sensitivity of the glioma in situ model, and the specific embodiment is as follows:

1. the luciferase virus is transfected in U87 cells, so that the subsequent tumor size detection is facilitated.

2. Will be 1 × 10⁶U87 cells were injected into the brain parenchyma of mice in situ, beginning intraperitoneal injection of TMZ (20mg/kg) or TMZ (20mg/kg) in combination with HPRT1 inhibitor (20mg/kg) on day 4 after injection, and in vivo imaging detection was performed on

days

4, 14, 25 and 36 to count the sizes of intracranial tumors in mice.

3. And (5) counting the death time of the mouse and drawing a survival curve.

As shown in fig. 11, TMZ inhibited intracranial tumor growth in mice compared to control mice. The combined use of the HPRT1 inhibitor can further inhibit the tumor growth and prolong the survival time of mice, which suggests that the HPRT1 inhibitor can significantly increase the killing effect of TMZ on tumors.

Example 12

A mouse intracranial in-situ tumor formation model is adopted to verify that the combination of the HPRT1 inhibitor (6-TG) and TMZ can increase the chemotherapy sensitivity of the glioma in-situ model, and the specific embodiment is as follows:

days

4, 14, 25 and 36 to count the sizes of intracranial tumors in mice.

3. And (5) counting the death time of the mouse and drawing a survival curve.

As shown in figure 12, TMZ inhibited intracranial tumor growth in mice compared to control mice. The combined use of the HPRT1 inhibitor can further inhibit the tumor growth and prolong the survival time of mice, which suggests that the HPRT1 inhibitor can significantly increase the killing effect of TMZ on tumors.

TMZ is taken as a second-generation oral alkylating agent, although the TMZ has better killing effect on tumor cells, because of factors such as MGMT, glioma stem cells and the like, the tumor is easy to develop chemotherapy resistance on the TMZ, and the curative effect of the TMZ is severely limited. Therefore, chemotherapy resistance is a problem that needs to be solved at present. We found that the TMZ metabolic byproduct AICA is structurally very similar to the nucleotide de novo synthetic pathway intermediate AICAR, which has one more 5' -ribose phosphate than AICA. AICAR is an AMPK agonist. AMPK is the cell energy metabolism center, maintains the intracellular energy metabolism balance mainly by regulating and controlling the synthesis and transformation of glucose, fat and protein, and plays an important role in the cell energy metabolism process. The research shows that AMPK has important regulation and control effects on a plurality of life activities of tumor cells, such as autophagy, apoptosis, energy metabolism and the like. AMPK and tumor chemotherapy resistance have been studied in a variety of tumors. Studies have shown that activation of AMPK can significantly contribute to glioma tolerance to TMZ. Thus, inhibition of AICA conversion to AICAR can significantly inhibit AMPK activation, thereby improving glioma cell chemotherapy tolerance.

In the present invention, we found that knocking down HPRT1 significantly enhanced the sensitivity of glioma cells and glioma stem cells to TMZ. Furthermore, in this project, we found through experiments such as mass spectrometry that AICA and PRPP can catalyze the generation of AICAR via HPRT1, similarly to the reaction of xanthine or hypoxanthine to generate guanylic acid or hypoxanthine. Knock-down of HPRT1 inhibited the conversion of AICA to AICAR in cells. Therefore, we were asked whether the sensitivity of glioma cells to TMZ could be enhanced using HPRT1 inhibitors. The combined use of TMZ and HPRT1 inhibitors in various glioma cells and intracranial in situ tumorigenic nude mice significantly inhibited tumor progression. In the research of a synergistic inhibition mechanism, the HPRT1 inhibitor can inhibit the conversion of TMZ metabolite AICA to AICAR, thereby inhibiting the activation of AMPK signal pathway and further increasing the chemotherapy sensitivity of tumor cells.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims

1. A composition of temozolomide and hypoxanthine-guanine nucleoside phosphate transferase small molecule inhibitors is characterized in that: the molar ratio of the temozolomide to the hypoxanthine-guanine nucleoside phosphate transferase micromolecule inhibitor is 0.5-100: 1, the small molecule is an organic compound with a molecular weight of less than 900 daltons.

2. A temozolomide and hypoxanthine-guanine nucleoside phosphate transferase small molecule inhibitor composition according to claim 1, wherein: the molar ratio of the temozolomide to the hypoxanthine-guanine nucleoside phosphate transferase micromolecule inhibitor is 1-80: 1.

3. a temozolomide and hypoxanthine-guanine nucleoside phosphate transferase small molecule inhibitor composition according to claim 1, wherein: the molar ratio of the temozolomide to the hypoxanthine-guanine nucleoside phosphate transferase micromolecule inhibitor is 1-40: 1.

4. a temozolomide and hypoxanthine-guanine nucleoside phosphate transferase small molecule inhibitor composition according to any one of claims 1 to 3, wherein: the hypoxanthine-guanine nucleoside phosphate transferase small molecule inhibitor comprises 6-MP and/or 6-TG.

5. A temozolomide and hypoxanthine-guanine nucleoside phosphate transferase small molecule inhibitor composition according to claim 4, wherein: the concentration of the temozolomide is 100-400 mu M; the concentration of the 6-MP is 5-20 mu M, and the concentration of the 6-TG is 5-20 mu M.

6. A temozolomide and hypoxanthine-guanine nucleoside phosphate transferase small molecule inhibitor composition according to claim 5, wherein: the administration concentration of the temozolomide is 20-50mg/kg, the administration concentration of the 6-MP is 1-20mg/kg, and the administration concentration of the 6-TG is 1-20 mg/kg.

7. The use of temozolomide in combination with a small molecule inhibitor of hypoxanthine-guanine nucleoside phosphate transferase according to claim 1, wherein: the composition is used for preparing medicines for treating and inhibiting the growth of tumor cells.

8. The use of temozolomide in combination with a small molecule inhibitor of hypoxanthine-guanine nucleoside phosphate transferase according to claim 7, wherein: the tumor, including glioma.

9. The use of temozolomide in combination with a small molecule inhibitor of hypoxanthine-guanine nucleoside phosphate transferase according to claim 8, wherein: the hypoxanthine-guanine nucleoside phosphate transferase small molecule inhibitor can inhibit activation of temozolomide on AMPK signal path, and increase chemotherapy sensitivity of glioma cells.