CN111346096B - Pharmaceutical composition for preventing or treating glioma - Google Patents

Abstract

The invention provides a pharmaceutical composition for treating glioma, which comprises a therapeutically effective amount of Tamatinib or a prodrug or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of temozolomide or a prodrug or a pharmaceutically acceptable salt thereof. The invention also provides a kit for treating glioma, comprising a first pharmaceutical composition comprising a therapeutically effective amount of Tamatinib or a prodrug or a pharmaceutically acceptable salt thereof and a second pharmaceutical composition comprising a therapeutically effective amount of temozolomide or a prodrug or a pharmaceutically acceptable salt thereof. The invention further provides application of the Tamatinib or the prodrug or the pharmaceutically acceptable salt thereof as a sensitizer of chemotherapeutic drugs for treating glioma.

Description

Pharmaceutical composition for preventing or treating glioma

Technical Field

The invention relates to chemotherapy of glioma, in particular to a pharmaceutical composition for preventing or treating glioma. The invention also relates to the use of said pharmaceutical composition in the treatment of gliomas with gliomas stem cells, and in the prevention or treatment of the recurrence of gliomas.

Background

According to the molecular pathological characteristics and biological behaviors of tumors, the malignant degree of gliomas can be classified into WHO I-IV grades from low to High, wherein the gliomas of WHO grade III and WHO grade IV are collectively called High-grade gliomas (HGGs), including anaplastic astrocytomas, anaplastic oligodendrogliomas and most malignant Glioblastomas (GBMs). HGG is the most common invasive intracranial primary tumor, and is clinically combined with surgery and chemoradiotherapy for comprehensive treatment, but the treatment effect is still not ideal, and the recurrence is very easy to occur. The different differentiation degrees and self-replication abilities of tumor cells form a gradient hierarchical structure of cells in tumor tissues of high-grade Glioma, and the gradient hierarchical structure shows obvious cell heterogeneity, wherein a small part of tumor cell subsets with stem cell characteristics mediate the occurrence, development and recurrence of tumor tissues and the resistance to the existing therapeutic measures through the inherent molecular biological mechanism of the small part of cells and the interaction with the tumor microenvironment, and the small part of cells are called Glioma Stem Cells (GSCs).

GSCs mediate heterogeneity and drug resistance in tumor tissues and are one of the sources of glioma recurrence. The research shows that the GSCs have low proliferation level in the tumor development process, are basically in the G0/G1 stage and are insensitive to radiotherapy and chemotherapy. In addition, GSCs can further increase resistance to treatment through self-drug resistance mechanisms, such as high expression of adenosine triphosphate-binding cassette (ABC) transporters. During treatment, when non-GSCs are partially killed, GSCs can rapidly enter the proliferative cycle, maintain their numbers through asymmetric self-division, and mediate short-term recurrence and progression of tumor tissue. Therefore, GSCs are considered to be critical for HGG treatment. Finding a means for effectively killing GSCs is expected to improve the clinical prognosis of HGG patients.

Spleen Tyrosine Kinase (SYK) is an unresponsive protein Tyrosine Kinase that is commonly expressed in hematopoietic cells. Syk plays a different role in different classes of tumors. On one hand, clinical data show that the decrease of syk expression in breast cancer, lung cancer and pancreatic cancer is related to the occurrence of tumor and poor survival prognosis, meanwhile, immunohistochemical experiments in gastric cancer show that the expression of syk in nucleus is related to the stage, invasion capacity and lymph node metastasis of tumor, and in addition, functional studies show that the abnormal expression of syk is negatively related to the proliferation and metastasis capacity of tumor cells, so that the syk is considered as a candidate cancer suppressor gene. On the other hand, syk plays a role in promoting cancer in cancer tissues such as head and neck squamous cell carcinoma, nasopharyngeal carcinoma, small cell lung cancer and the like, the high expression level of Syk in tumor tissues is higher than that in normal tissues, and the prognosis is poor, and after the Syk is silenced, the proliferation and migration capacity of tumor cells are inhibited. The research results show that Syk plays an important role in controlling the occurrence and development of part of tumors to a certain extent. Recent studies have shown that Syk may play a role of oncogenes in glioma, and is a new target for glioma therapy.

Tamatinib, also known as R406, is an ATP competitive Syk inhibitor whose disodium phosphate salt prodrug, fostamatinib (or R788, trade name tavalise), has been approved by the FDA in the united states for clinical treatment of chronic Immune Thrombocytopenia (ITP). Fostamatinib can block the endocytosis of macrophage mediated by inhibiting Syk activity, so as to slow down the speed of platelet degradation and achieve the therapeutic effect. In addition, syk is involved in the signaling pathway of B cell receptors and may be involved in regulating autoantibody production. Fostamatinib was able to significantly increase platelet levels in some patients with ITP.

Targeted drugs are the trend in the future for treating tumors. At present, breakthrough results are obtained for targeted therapy of lung cancer, leukemia, breast cancer and colorectal cancer, but targeted drugs for HGG have a few fingers and limited effects.

Disclosure of Invention

The technical problem to be solved by the invention is to reduce the viability of GSCs, inhibit the proliferation and/or kill GSCs, thereby treating glioma and preventing the recurrence thereof. The inventor finds that the Tamatinib or the prodrug Fostamatiib thereof can induce glioma stem cells to undergo apoptosis, and the glioma stem cells can be effectively killed whether positive or negative to Syk protein expression, so that the survival activity of the glioma stem cells is reduced with high titer, and the cell proliferation is inhibited.

Therefore, one aspect of the present invention provides the use of Tamatinib or a prodrug or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of glioma. Another aspect of the present invention provides a use of Tamatinib or a prodrug or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing glioma recurrence.

Another aspect of the present invention provides the use of Tamatinib or a prodrug or a pharmaceutically acceptable salt thereof in the preparation of a sensitizer for a chemotherapeutic drug for the treatment of glioma. In some embodiments of the invention, the chemotherapeutic agent for treating glioma is temozolomide. In some embodiments of the invention, the glioma stem cells have reduced or eliminated resistance to temozolomide following administration of Tamatinib, or a prodrug or pharmaceutically acceptable salt thereof.

Another aspect of the invention provides a method of treating glioma or preventing or treating glioma recurrence comprising administering to a subject having glioma a therapeutically effective amount of Tamatinib, or a prodrug or pharmaceutically acceptable salt thereof. In some embodiments, the subject is a mammal, preferably a human. In some embodiments, the glioma has glioma stem cells. In some embodiments, the subject has undergone surgery, radiation therapy, or chemotherapy.

Another aspect of the present invention provides a method of killing or inhibiting glioma stem cells, said method comprising contacting said glioma stem cells with an effective amount of Tamatinib, or a prodrug or pharmaceutically acceptable salt thereof. In some embodiments, the glioma stem cell is comprised in a glioma. In some embodiments, the glioma is in a subject. In some embodiments, the subject is a mammal, preferably a human. In some embodiments, the subject has undergone surgery, radiation therapy, or chemotherapy.

Another aspect of the invention provides Tamatinib or a prodrug or pharmaceutically acceptable salt thereof for use in the treatment of glioma. One aspect of the present invention provides Tamatinib, or a prodrug or pharmaceutically acceptable salt thereof, for use in the prevention or treatment of glioma recurrence.

For each of the above aspects, in some embodiments, the glioma has glioma stem cells. For each of the above aspects, in some embodiments, the glioma stem cells comprise glioma stem cells negative for Syk protein expression. For each of the above aspects, in some embodiments, the glioma stem cells comprise Syk protein-negative glioma stem cells and Syk protein-positive glioma stem cells. For each of the above aspects, in some embodiments, the glioma is selected from the group consisting of anaplastic astrocytoma, anaplastic oligodendroglioma, primary glioblastoma and secondary glioblastoma. For each of the above aspects, in some embodiments, the glioma is a WHO grade III or WHO grade IV glioma.

Another aspect of the present invention provides a pharmaceutical composition for treating glioma, comprising a therapeutically effective amount of Tamatinib or a prodrug or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of temozolomide or a prodrug or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

Another aspect of the present invention provides a kit for treating glioma comprising a first pharmaceutical composition comprising a therapeutically effective amount of Tamatinib, or a prodrug or pharmaceutically acceptable salt thereof, and a second pharmaceutical composition comprising a therapeutically effective amount of temozolomide, or a prodrug or pharmaceutically acceptable salt thereof.

Another aspect of the present invention provides a use of Tamatinib or a prodrug or a pharmaceutically acceptable salt thereof in combination with temozolomide or a prodrug or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating glioma.

Another aspect of the present invention provides a use of Tamatinib or a prodrug or a pharmaceutically acceptable salt thereof in combination with temozolomide or a prodrug or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating glioma recurrence.

Another aspect of the present invention provides a method of treating glioma or preventing or treating glioma relapse, comprising administering to a subject having glioma a pharmaceutical composition of the present invention comprising a therapeutically effective amount of Tamatinib, or a prodrug or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of temozolomide, or a prodrug or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

Another aspect of the invention provides a method of treating glioma or preventing or treating glioma relapse, said method comprising administering to a subject suffering from glioma a kit of the invention comprising a first pharmaceutical composition comprising a therapeutically effective amount of Tamatinib, or a prodrug or pharmaceutically acceptable salt thereof, and a second pharmaceutical composition comprising a therapeutically effective amount of temozolomide, or a prodrug or pharmaceutically acceptable salt thereof.

Another aspect of the present invention provides a method for treating glioma or preventing or treating glioma relapse, comprising administering to a subject having glioma a first pharmaceutical composition comprising a therapeutically effective amount of Tamatinib, or a prodrug or pharmaceutically acceptable salt thereof, and a second pharmaceutical composition comprising a therapeutically effective amount of temozolomide, or a prodrug or pharmaceutically acceptable salt thereof. In some embodiments, the method further comprises radiation therapy. In some embodiments, the first pharmaceutical composition is administered before, simultaneously with, or after the administration of the second pharmaceutical composition. In some embodiments, the first pharmaceutical composition is not administered simultaneously with the second pharmaceutical composition, the administration of the first pharmaceutical composition and the administration of the second pharmaceutical composition are separated by 0.1 to 72 hours.

In some embodiments, the subject is a mammal, preferably a human. In some embodiments, the glioma has glioma stem cells. In some embodiments, the subject has undergone surgery, radiation therapy, or chemotherapy.

Another aspect of the invention provides a method of killing or inhibiting a population of glioma cells, said method comprising contacting an effective amount of Tamatinib, or a prodrug or pharmaceutically acceptable salt thereof, with said population of cells and prior to, simultaneously with, or subsequent to, contacting temozolomide, or a prodrug or pharmaceutically acceptable salt thereof, with said population of cells. In some embodiments, the glioma has glioma stem cells. In some embodiments, the glioma stem cell is comprised in a population of glioma cells. In some embodiments, the population of glioma cells is in a subject. In some embodiments, the subject is a mammal, preferably a human. In some embodiments, the subject has undergone surgery, radiation therapy, or chemotherapy.

Another aspect of the present invention provides a pharmaceutical composition for treating glioma, comprising a therapeutically effective amount of Tamatinib or a prodrug or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of temozolomide or a prodrug or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In some embodiments, the glioma has glioma stem cells.

For each of the above aspects, in some embodiments, the glioma stem cell comprises a Syk protein-negative glioma stem cell. For each of the above aspects, in some embodiments, the glioma stem cells comprise Syk protein-negative glioma stem cells and Syk protein-positive glioma stem cells. For each of the above aspects, in some embodiments, the glioma is selected from the group consisting of anaplastic astrocytoma, anaplastic oligodendroglioma, anaplastic astrocytoma, anaplastic ductoma, primary glioblastoma, and secondary glioblastoma. For each of the above aspects, in some embodiments, the glioma is a WHO grade III or WHO grade IV glioma.

Based on the discovery that the Tamatinib and the prodrug thereof can effectively kill glioma stem cells, the Tamatinib is applied to treatment of glioma and prevention and treatment of glioma recurrence, and experimental data prove that the Tamatinib has specificity on killing and inhibition of glioma stem cells, has no obvious toxic or side effect on normal nerve cells, and shows a strong killing effect no matter whether the glioma stem cells express Syk protein or not. In addition, when Tamatinib is combined with temozolomide, a first-line chemotherapy drug of glioma, experiments prove that the Tamatinib and temozolomide have strong synergistic effect, and the tumor volume is continuously and remarkably reduced compared with the single use of Tamatinib or the single use of temozolomide.

Drawings

FIG. 1.R406 is effective in inhibiting cell viability of GSC-1 and GSC-2. Effect of r406 on GSCs morphological changes. Cells were treated with R406 at each concentration for 48 hours, and then phase contrast microscopy images (40 ×) were captured. B. Cell viability of GSC-1 and GSC-2 was measured 48 hours after treatment with R406 at each concentration. C. The number of neurospheres of GSC-1 and GSC-2 was counted 48 hours after R406 treatment for each concentration. C17.2 mouse neural progenitor cells were insensitive to R406. R406 at concentrations up to 10. Mu.M is not toxic to C17.2 cells. Scale bar, 100mM. Mean ± SD (n = 3/group) is shown. ns, no significance; * P is less than 0.05; * P < 0.01, p < 0.001, compared to control.

Figure 2. Effect of r406 on morphological changes of GSCs and their nuclei. A. GSCs were treated with 1uM rh406 for 48 hours before fluorescence microscopy images were captured. Scale bar, 50um. B. Cleaved caspase3 and PARP expression levels were analyzed by Westem blot 48 hours after GSC were treated with each concentration (0 uM,0.01uM,0.1uM, 1uM) of R406. Beta-actin served as loading control. The percentage of annexin V/PI positive cells was determined by flow cytometry. GSCs were treated with 1 μ M R for different durations (12 hours, 24 hours, 48 hours) (C) and positive cells were quantified (D). Mean ± SD (n = 3/group) is shown. ns, no significance; * P is less than 0.05; * P < 0.01; * P < 0.001; compared to the control group.

FIG. 3 expression levels of SYK protein in each cell line. Western blot analysis of syk expression. The cell line SW620 was used as a positive control and the cell line HepG-2 was used as a negative control. Beta-actin was used as loading control. R406 cannot effectively inhibit cell viability of U87 and U251. Cell viability was measured for U87 (B) and U251 (C) 48 hours after treatment with R406 at each concentration. Mean ± SD (n = 3/group) is shown. ns, no significance; * P is less than 0.05; * P < 0.01, p < 0.001, compared to the control group.

FIG. 4. Biological effects of SYK interference induced by GSC-1. A. The extent of RNA interference was examined by Western blotting. GSC were transfected with negative control siRNA (siNC) or siSYK001,002,003, where siSYK-002 acted. B. Cell viability of GSC-1 after treatment with siSYK-002 was measured. Mean ± SD (n = 3/group) is shown. ns, no significance; * P is less than 0.05; * P < 0.01, p < 0.001, compared to si-NC group. C. The mean fluorescence intensity of each group was determined by flow cytometry. GSC were treated by siSYK-002. D. GSC was treated with GSSYK-002 and then OCR was monitored in real time by using a Seahorse Bioscience intercellular flux analyzer. The dashed line indicates incubation of cells with the indicated compounds.

Figure 5 biological effects induced by pi3k inhibitor ZSTK474 in GSCs. Westem blot analysis of P-P13K, PI3K, P-Akt and Akt expression, each cell line was treated with R406 for 48 hours. Beta-actin was used as loading control. B. Cell viability of GSCs was measured 48 hours after treatment with ZSTK474 at each concentration. Mean ± SD (n = 3/group) is shown. ns, no significance; * P is less than 0.05; * P < 0.01, p < 0.001, compared to control. C. The mean fluorescence intensity of each group was determined by flow cytometry. GSCs were treated with 0.5 uzmzstk474 for 12 hours, 24 hours, and 48 hours. D. GSCs were treated with 0.5 uzmzstkk474 for 12 and 24 hours, then monitored for OCR in real time by using a Seahorse Bioscience extracellular flux analyzer. The dashed line indicates the incubation of the cells with the indicated compounds.

Figure 6.R406 synergistically enhances TMZ cytotoxicity on GSC in vitro and in vivo. (A) Cell viability of GSCs after treatment with R406 alone, TMZ alone, or a combination of both reagents was measured using a Muse cell analyzer. Quantitative analysis indicated that the combination of R406 and TMZ was superior to monotherapy with a single drug. (B) The combination of R406 and TMZ significantly delayed subcutaneous tumor growth in vivo compared to monotherapy. (C) IHC of tumor sections demonstrated that addition of R406 to TMZ reduced the number of proliferating cells, which were positive for Ki-67 (white arrows). (D) Proteins were extracted from xenograft specimens and distributed for western blotting. Enhanced activation of caspase3 was found in mice treated with a combination of R406 and TMZ. (E) R406+ TMZ significantly prolonged survival of mice in the in situ model compared to monotherapy with either R406 or TMZ alone. NS, no significance; * P is less than 0.05; * P < 0.001, statistically different from the control group. #, p < 0.01, # #, p < 0.001, statistically different from the group treated with the combination of R406 and TMZ.

Detailed Description

Definition of

As used herein, the term "composition" refers to a formulation suitable for administration to a desired animal subject for therapeutic or prophylactic purposes, which contains at least one pharmaceutically active ingredient, e.g., a compound. Optionally, the composition further comprises

As used herein, the terms "therapeutically effective amount" and "effective amount" mean that the substance or amount of substance is effective to prevent, alleviate or ameliorate one or more symptoms of a disease or disorder, and/or to prolong survival of the subject being treated.

As used herein, "treating" includes administering a compound, pharmaceutically acceptable salt, or composition thereof of the present application to alleviate symptoms or complications of a disease or disorder, or to eliminate a disease or disorder. The term "alleviating" as used herein is used to describe the process of reducing the severity of signs or symptoms of a disorder. Symptoms can be reduced without elimination. In one embodiment, administration of the pharmaceutical composition of the present application results in the elimination of the signs or symptoms.

A "prodrug" is a compound or a pharmaceutically acceptable salt thereof that when metabolized or converted by solvolysis under physiological conditions to yield the desired active compound. Prodrugs include, but are not limited to, esters, amides, carbamates, carbonates, ureides, solvates or hydrates of the active compound. In general, prodrugs are inactive, or less active than the active compound, but are capable of providing one or more beneficial therapeutic, administration, and/or metabolic properties. For example, some prodrugs are esters of the active compound; during metabolism, the ester group is cleaved to yield the active drug. Also, some prodrugs are activated enzymatically to yield the active compound, or are compounds which upon further chemical reaction yield the active compound. A prodrug may be changed from a prodrug form to an active form in one step, or may have one or more intermediate forms that may or may not be active themselves.

Conceptually, prodrugs can be divided into two non-exclusive categories: a bioprecursor prodrug and a carrier prodrug. In general, a bioprecursor prodrug is a compound that is inactive or less active than the corresponding active pharmaceutical compound, contains one or more protecting groups and is converted to the active form by metabolic or solvolytic action. The active pharmaceutical form and any metabolites released should have acceptably low toxicity. Carrier prodrugs are pharmaceutical compounds containing a transport moiety that can improve uptake and/or local delivery to the site(s) of action. For example, a carrier prodrug may be used to improve one or more of the following properties: increased lipophilicity, increased duration of pharmacological effect, increased site specificity, reduced toxicity and adverse reactions, and/or improvements in pharmaceutical formulations (e.g., stability, water solubility, inhibition of unwanted organoleptic or physicochemical properties). For example, lipophilicity can be increased by esterifying hydroxyl groups with lipophilic carboxylic acids, or esterifying carboxylic acid groups with alcohols, such as aliphatic alcohols.

By "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" is meant that the compound can be formulated as or in the form of a pharmaceutically acceptable salt. Contemplated pharmaceutically acceptable salt forms include, but are not limited to, mono-, di-, tri-, tetra-, and the like. Pharmaceutically acceptable salts are non-toxic in the amounts and concentrations at which they are administered. The preparation of such salts may facilitate pharmacological applications by altering the physical properties of the compounds without preventing them from exerting their physiological effects. Useful changes in physical properties include lowering the melting point for transmucosal administration, and increasing solubility for administration of higher concentrations of drug.

Pharmaceutically acceptable salts include acid addition salts such as those containing sulfate, chloride, hydrochloride, fumarate, maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, mesylate, esylate, benzenesulfonate, p-toluenesulfonate, cyclamate, and quinic acid salts. Pharmaceutically acceptable salts can be obtained from acids such as hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclamic acid, fumaric acid, and quinic acid. Pharmaceutically acceptable salts also include base addition salts when acidic functional groups such as carboxylic acids or phenols are present, such as those containing benzathine, chloroprocaine, choline, diethanolamine, ethanolamine, t-butylamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamine, and zinc.

Pharmaceutically acceptable salts can be prepared by standard techniques. For example, the free base form of the compound is dissolved in a suitable solvent, such as an aqueous or aqueous-alcoholic solution containing the appropriate acid, and the solution is evaporated for isolation. In another example, salts are prepared by reacting the free base and the acid in an organic solvent. Unless stated to the contrary, the scope of the present invention is intended to include prodrugs and pharmaceutically acceptable salts of these compounds.

As used herein, the terms "subject," "patient," and similar terms refer to both humans and non-human vertebrates, e.g., mammals, e.g., non-human primates; sports and commercial animals, such as horses, cattle, pigs, sheep, rodents; and pets such as dogs and cats.

Tamatinib

Tamatinib, also called R406, has a chemical structural formula shown in formula I, a molecular weight of 470.45, a CAS number of 841290-80-0, and a molecular formula C ₂₂ H ₂₃ FN ₆ O ₅ Chemical name 6- [ [ 5-fluoro-2- [ (3,4,5-trimethoxyphenyl) amino]-4-pyrimidinyl]Amino group]-2,2-dimethyl-2H-pyrido [3,2-b]-1,4-oxazin-3 (4H) -one.

Tamatinib is a Syk Inhibitor (IC) ₅₀ :41 nM) and its prodrug, fostamatinib (or R788, trade name tavalise), has been approved by the FDA in the united states for the clinical treatment of chronic Immune Thrombocytopenia (ITP). The structural formula of Fostamatinib is shown in formula II, and CAS number 901119-35-5. Oral administration of the disodium salt of Fostamatinib metabolizes to Tamatinib in vivo.

In addition, tamatinib can also form salts with other acids, such as the Tamatinib besylate salt with benzenesulfonic acid, formula III, CAS number 841290-81-1, available from MedKoo bioscience (MedKoo Cat #: 406779).

Temozolomide

Temozolomide, the name Temozolomide, abbreviated as TMZ, has a chemical structural formula shown in formula IV, a molecular weight of 194.15 and a molecular formula C ₆ H ₆ N ₆ O ₂ CAS number 85622-93-1, chemical name 3,4-dihydro-3-methyl-4-oxoimidazole [5,1-d]And 1,2,3,5-tetrazine-8-carboxamide. Temozolomide is a first-line medicine for treating brain glioma, is generally used for treating newly diagnosed glioblastoma multiforme clinically, is an oral novel alkylating agent type anti-tumor medicine, has broad-spectrum anti-tumor activity, can pass through a blood brain barrier, has bioavailability close to 100%, can treat newly diagnosed and recurrent glioblastoma multiforme and anaplastic astrocytoma, prolongs the life cycle of a patient, and has good safety and tolerance.

Use and therapy

One aspect of the present invention relates to the use of Tamatinib or a prodrug or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of glioma. In some embodiments, the prodrug is Fostamatinib or a sodium salt thereof. In this aspect, preferably, the glioma is a glioma having glioma stem cells. Glioma stem cells are present in the glioma bulk as a sub-population. Gliomas with glioma stem cells include, but are not limited to, anaplastic astrocytomas, anaplastic oligodendrogliomas, primary glioblastoma, and secondary glioblastoma.

In some embodiments, the glioma is a WHO grade III or WHO grade IV glioma. WHO grade III and WHO grade IV gliomas are collectively referred to as High-grade gliomas (HGGs), including anaplastic astrocytomas, anaplastic oligodendrogliomas, and the most malignant Glioblastoma (GBM), among others. High-grade gliomas often have no complete envelope, grow infiltratively, are not clearly demarcated with normal brain tissues, are difficult to completely cut off by operations, have the characteristics of necrosis, hemorrhage, cystic change, anaplasia and the like in histopathology, increase the division images of microscopic cytopathogenic nuclei, increase the nuclear-to-cytoplasmic ratio and the like. GBM consists of mitotically active, highly anaplastic glial cells, and tumors have high cell density with marked microvascular proliferation and necrosis, and characteristic "pseudo-palisade" necrotic areas.

The specific biological characteristics of glioma stem cells comprise suspension balling growth; can differentiate to form tumors and maintain the number of self populations through asymmetric self-replication; capable of differentiating into multiple cell lineages, i.e., possessing multipotentiality; tumors can be formed by xenografting in immunodeficient animal models, and the tumor tissue formed will conform to the phenotype and heterogeneity of the primary tumor. Glioma stem cells often express specific biological markers, such as nestin, sox-2, CD133, and A2B5, and are regulated by specific signal transduction pathways.

In an embodiment of the invention, the glioma having glioma stem cells comprises non-dry glioma cells and glioma stem cells. In some embodiments, the glioma stem cells comprise Syk protein-negative glioma stem cells and Syk protein-positive glioma stem cells. Thus, in some embodiments, the use of the invention involves gliomas having non-dry glioma cells, syk protein-negative glioma stem cells and Syk protein-positive glioma stem cells. In some embodiments, the use of the invention relates to gliomas having non-dry glioma cells and Syk protein positive glioma stem cells. In some embodiments, the use of the invention relates to gliomas having non-dry glioma cells and Syk protein negative glioma stem cells.

Another aspect of the present invention relates to the use of Tamatinib or a prodrug or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the prevention or treatment of glioma relapse. In some embodiments, the prodrug is Fostamatinib or a sodium salt thereof. In this aspect, the glioma recurrence is at least partially due to the presence of glioma stem cells, or the glioma recurrence is primarily due to glioma stem cells. The recurrent glioma has glioma stem cells, and the glioma stem cells are present in the glioma body in the form of a sub-population of cells.

In an embodiment of the invention, the recurrent glioma comprises non-dry glioma cells and glioma stem cells. In some embodiments, the glioma stem cells comprise Syk protein-negative glioma stem cells and Syk protein-positive glioma stem cells. Thus, in some embodiments, a relapsed glioma has non-dry glioma cells, syk protein-negative glioma stem cells, and Syk protein-positive glioma stem cells. In some embodiments, the relapsed glioma has non-dry glioma cells and Syk protein positive glioma stem cells. In some embodiments, the relapsed glioma has non-dry glioma cells and Syk protein-negative glioma stem cells.

In some embodiments, the relapsed glioma includes, but is not limited to, anaplastic astrocytoma, anaplastic oligodendroglioma, anaplastic astrocytoma, anaplastic ductoma, primary glioblastoma, and secondary glioblastoma. In some embodiments, the relapsed glioma is a WHO grade III or WHO grade IV glioma.

Another aspect of the invention provides a method of treating a glioma, comprising administering to a subject suffering from said glioma a therapeutically effective amount of Tamatinib, or a prodrug or pharmaceutically acceptable salt thereof. In some embodiments, the prodrug is Fostamatinib or a sodium salt thereof. In some embodiments, the glioma has glioma stem cells. Gliomas with glioma stem cells are as defined above. In some embodiments, the subject is a mammal, preferably a human. In some embodiments, the subject has undergone or is undergoing surgery, radiation therapy, or chemotherapy.

Another aspect of the present invention provides a method for preventing or treating glioma relapse, comprising administering to a subject suffering from said glioma a therapeutically effective amount of Tamatinib, or a prodrug or pharmaceutically acceptable salt thereof. In some embodiments, the prodrug is Fostamatinib or a sodium salt thereof. Recurrent gliomas are as defined above. In some embodiments, the subject is a mammal, preferably a human. In some embodiments, the subject has undergone surgery, radiation therapy, or chemotherapy.

In some embodiments, the methods can be used in combination with other glioma therapies. For example, the method can be used in combination with glioma surgery, radiation therapy, or chemotherapy. Thus, in some embodiments, the method of treating a glioma of the present invention further comprises administering to a subject having said glioma a therapeutically effective amount of a second chemotherapeutic agent. In other embodiments, the methods of preventing or treating glioma recurrence of the present invention further comprise administering to a subject having a relapsed glioma a therapeutically effective amount of a second chemotherapeutic agent.

In some embodiments, the second chemotherapeutic agent is temozolomide or a prodrug or pharmaceutically acceptable salt thereof. In these embodiments, the second chemotherapeutic agent is administered prior to, concurrently with, or subsequent to the administration of Tamatinib, or a prodrug or pharmaceutically acceptable salt thereof.

When the second chemotherapeutic agent is not administered simultaneously with Tamatinib, or a prodrug or pharmaceutically acceptable salt thereof, the administration is separated by about 0.1 hour to about 72 hours, e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,2,3, 4,5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 72 hours.

When a second chemotherapeutic agent is administered concurrently with Tamatinib or a prodrug or pharmaceutically acceptable salt thereof, in some embodiments, tamatinib or a prodrug or pharmaceutically acceptable salt thereof and the second chemotherapeutic agent are provided separately in separate pharmaceutical compositions. In some embodiments, the separate pharmaceutical compositions are provided in the same kit. When a second chemotherapeutic agent is administered concurrently with Tamatinib or a prodrug or pharmaceutically acceptable salt thereof, in other embodiments, tamatinib or a prodrug or pharmaceutically acceptable salt thereof is provided with the second chemotherapeutic agent in the form of a pharmaceutical composition.

In some embodiments, the prevention of glioma recurrence according to the present invention refers to administering a therapeutically effective amount of Tamatinib or a prodrug or a pharmaceutically acceptable salt thereof alone after administering a glioma therapy to a patient with glioma, thereby killing glioma stem cells that may remain, so as to achieve the purpose of preventing glioma recurrence.

Another aspect of the present invention provides a method of killing or inhibiting glioma stem cells, said method comprising contacting said glioma stem cells with an effective amount of Tamatinib, or a prodrug or pharmaceutically acceptable salt thereof. In some embodiments, the glioma stem cell is comprised in a glioma. In some embodiments, the glioma is in a subject. In some embodiments, the subject is a mammal, preferably a human. In some embodiments, the subject has undergone surgery, radiation therapy, or chemotherapy. In some embodiments, the glioma stem cells are present in the glioma tumor body as a subcellular population. In some embodiments, the glioma stem cell is a Syk protein-negative glioma stem cell. In some embodiments, the glioma stem cell is a Syk protein positive glioma stem cell. In some embodiments, the glioma stem cell is a mixture of a Syk protein-negative glioma stem cell and a Syk protein-positive glioma stem cell.

Pharmaceutical composition and kit

One aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of Tamatinib or a prodrug or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of temozolomide or a prodrug or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Thus, the pharmaceutical composition of the present invention comprises both active principles, which are contained in the same pharmaceutical composition in a suitable form, and the subjects are administered the two active principles simultaneously or sequentially when administering the pharmaceutical composition. For example, tamatinib, temozolomide and a carrier are present in a pharmaceutical composition in a mixture in a predetermined ratio. Alternatively, tamatinib and carrier are combined in a predetermined ratio to form one part of the pharmaceutical composition, temozolomide and carrier are combined in a predetermined ratio to form another part of the pharmaceutical composition, and the two parts are combined to form the pharmaceutical composition, for example, in a core-shell structure. Other methods available in pharmacy or pharmaceutical engineering may also be used to combine the two active ingredients together without affecting the performance of the active ingredients after administration.

Another aspect of the invention provides a kit comprising a first pharmaceutical composition comprising a therapeutically effective amount of Tamatinib, or a prodrug or pharmaceutically acceptable salt thereof, and a second pharmaceutical composition comprising a therapeutically effective amount of temozolomide, or a prodrug or pharmaceutically acceptable salt thereof, that are separate. Thus, in some embodiments of the kit, the first pharmaceutical composition can be present in a separate dosage form and the second pharmaceutical composition can be present in another separate dosage form, both dosage forms being the same or different. In some embodiments, the first and second pharmaceutical compositions in the kit are each contained in separate containers.

A therapeutically or prophylactically effective amount of the active ingredient to be administered can be determined by standard procedures, and factors considered can include, for example, compound IC ₅₀ Biological half-life, age, size, and weight of the subject, and a condition associated with the subject. The importance of these and other factors is well known to those of ordinary skill in the art. In general, the dose will be between about 0.01mg/kg and 50mg/kg, preferably between 0.1mg/kg and 20mg/kg of the subject to be treated.

The carrier or excipient may be used in the manufacture of a pharmaceutical composition. The carrier or excipient may be selected to facilitate administration of the compound. Examples of carriers include calcium carbonate, calcium phosphate, various sugars (e.g. lactose, glucose or sucrose), or starch types, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents. Examples of physiologically compatible solvents include sterile water for injection (WFI), saline solutions and glucose.

Suitable dosage forms depend, in part, on the route of administration, e.g., oral, transdermal, transmucosal, inhalation, or by injection (parenteral). Such dosage forms should allow the active ingredient to reach the target cells. The medicaments or pharmaceutical compositions of the invention may be administered by different routes, including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, transmucosal, rectal, transdermal or inhalation. In some embodiments, oral administration is preferred. For oral administration, for example, the compounds may be formulated in conventional oral dosage forms such as capsules, tablets, as well as liquid preparations such as syrups, elixirs, and concentrated drops.

Examples

R406 can effectively inhibit cell viability of glioma stem cells and cell proliferation

Experimental Material

Glioma stem cell lines GSC-1 and GSC-2 (isolated culture of tumor tissue specimens of neurosurgery in the center of tumor prevention and treatment of Zhongshan university). R406 (free base) 50mg of freeze-dried powder is prepared into a stock solution with the concentration of 40mM by DMSO, diluted into a 10mM mother solution by DMSO as required, filtered and stored at the temperature of minus 20 ℃. Preparing a culture medium: 500ml DMEM/F12+2% by weight B27+20ng/ml bFGF +20ng/ml EGF +50U/ml penicillin + 50. Mu.g/ml streptomycin. The C17.2 mouse neural stem cell line was generously provided by professor Pi Rongbiao, pharmacology of zhongshan medical college, zhongshan university. C17.2 cells were routinely cultured in DMEM supplemented with 10% FBS (Gibco), 5% horse serum (Gibco) and 2mM L-glutamine.

Experimental methods

Culturing glioma stem cells: the glioma stem cell lines were cultured in a well-prepared DMEM/F12 medium, incubated in a constant-temperature closed incubator with 5% of CO2, 37 ℃ and 95% of relative humidity, and the growth was observed using an inverted microscope. And (4) carrying out passage when the cell density reaches about 80%, carrying out passage once in about 2-4 days, and taking the cells in the logarithmic growth phase for formal experiments.

Passage of glioma stem cells: when the cell density reaches about 80%, centrifuging at 800-1200 rpm to collect cells, removing the supernatant culture medium, adding a proper amount of Stempro, gently blowing to beat cell masses, incubating in an incubator for 3-5 minutes to fully digest the cell masses into single cell suspension, centrifuging at 800-1200 rpm to collect cells again, discarding the supernatant digest, rinsing with a proper amount of culture medium, and passaging according to the requirement of 1: 4-1: 5.

Determination of the R406 titer: taking cells in logarithmic phase, digesting, counting, inoculating to a 96-well plate according to 4000 cells/well, adding drugs into the 96-well plate according to dosage gradients of 0uM,0.01uM,0.1uM,1uM, 1.25uM, 2.5uM, 5uM, 10uM and 20uM, setting 3-5 auxiliary wells for each dosage, and incubating for 48 hours in an incubator. Adding the CCK-8 kit into a 96-well plate according to the proportion of 1: 10 in the specification, incubating for 2-4 hours in an incubator, and measuring the absorbance at the wavelength of 450nm by using an enzyme-labeling instrument. IC50 values were calculated for each of the two glioma stem cells using SPSS software based on the CCK-8 assay.

The cell morphology was observed by inverted phase contrast microscopy: after the cells were treated as described above, the morphological changes of the cells were observed by inverted phase contrast microscope at different magnifications and photographed. The images were taken by an inverted phase contrast microscope (40 Xcamera), 3-5 visual fields were randomly taken for each concentration gradient, the size of the colony spheres was observed, and the colony spheres consisting of 10 cells were used as a standard, and counted manually by ipwin32 software and recorded.

The statistical method comprises the following steps: quantitative data corresponding to normal distribution are expressed as mean ± sd, and differences of P < 0.05 are considered statistically significant using SPSS 13.0 statistical software using the t test or one-way ANOVA analysis of variance combined with Dunnett's multiple tests.

Results of the experiment

The effect of R406 is verified in two glioma stem cells GSC-1 and GSC-2, the concentration gradient is refined, the morphological change of cells is observed under a mirror after administration, and the phenomena of cell number reduction, clone sphere volume reduction, under-mirror cell fragment increase and the like appear in the two glioma stem cells (figure 1A). The CCK-8 assay results suggest that R406 is effective in inhibiting cell survival in both cell lines in a concentration-dependent manner, with IC 50's of 0.751uM and 0.891uM in GSC-1 and GSC-2, respectively (FIG. 1B). According to the clone counting results after the treatment of the two strains of cells, it is suggested that R406 also has the effects of reducing the cell viability and inhibiting the proliferation (FIG. 1C).

Furthermore, to determine the effect of R406 on normal neural stem cells, we incubated the inhibitor with C17.2 cells, which were immortalized mouse neural progenitor cells. Viability analysis showed that R406 was not toxic to C17.2 cells even at a concentration of 10 μ M (fig. 1D).

Dose-and time-dependent induction of apoptosis of GSCs by R406

Experimental method

R406 treatment of cells: taking cells in logarithmic phase for digestion, counting, and inoculating the cells into culture bottles according to 2-5 multiplied by 105 cells/bottle. The reverse time course of R406 was 1uM final concentration and the incubations were carried out in the incubator for 48, 24 and 12 hours, respectively. R406 was added in a gradient of 0.01uM,0.1uM,1uM and incubated in an incubator for 48 hours.

Hoechst33342 nucleic acid dye staining: after the cells are treated by R406, hoechst33342 is added according to the concentration of 1X, the cells are incubated for 20 to 30 minutes in the dark, the culture medium containing the dye is removed by suction, and the cells are washed for 2 to 3 times by using the culture medium or PBS and observed under a fluorescence microscope. After Hoechst33342 and double-stranded DNA are combined, the maximum excitation wavelength is 350nm, and the maximum emission wavelength is 461nm. And selecting a proper visual field for photographing record.

Western blot (Western blot): after R406 treatment of cells, cells were harvested by centrifugation, 4 ℃ pre-cooled PBS (0.01M pH 7.2-7.3) was added, the cells were washed by gentle shaking for 1min, and then the wash solution was discarded, and the above procedure was performed on ice and repeated three times. Adding a certain volume of M-PER mammalian cell protein extraction reagent according to cell density, vortex and shake, centrifuging at 4 deg.C for 10min at 12,000 Xg, removing cell precipitate, collecting supernatant to a new precooled EP tube, and further performing next protein quantification or storing at-80 deg.C. Protein quantification was performed by BCA method. SDS-PAGE gel electrophoresis: the gel concentration is chosen according to the molecular weight of the protein of interest, generally 12%, and the gel thickness is chosen according to the molecular weight and the protein concentration, generally 1.5mm. Adjusting the sample to the same concentration by using a protein lysate, adding 5Xloadingbufer, heating in boiling water at 100 ℃ for 5min to fully denature the protein, taking out the sample from the boiling water bath, rapidly placing the sample in an ice bath, uniformly mixing the sample by vortex, and centrifuging the mixture for a short time. 15-hole plate glue, and each hole is loaded with 20-30ul of sample; 10-well plate glue, each well loading 30-40ul. Running concentrated glue at 80-100V. Running separation gel at 100-180V. Carefully lift the glass plate, cut off the concentrated gel, remove the gel and soak in the transfer buffer. The PVDF membrane was activated in methanol for 5min and then soaked in transfer buffer. The transfer device is installed in the following order: (negative) sponge-transfer filter paper-gel-PVDF membrane-transfer filter paper-sponge (positive), carefully remove air bubbles, 100V constant pressure transfer 180mins in transfer buffer. After the transfer, the PVDF membrane was carefully removed, soaked in TBST for 5min, then blocked with 5% skim milk and blocked at room temperature for 1h. After blocking was complete, the membrane was washed 3 times 5min each with TBST on a decolourisation shaker. Primary antibody was then added and incubated overnight or after 12h at 4 ℃ in a shaker. Washing with TBST for 3 times, 5min each time, adding secondary antibody, incubating at room temperature for 1h with shaking table, washing with TBST for 3 times, 5min each time. And mixing 1mL of the solution B with 1mL of the solution LA to prepare the ECL chemiluminescent solution. Taking out the PVDF membrane, sucking the membrane washing solution on paper, and then incubating with ECL chemiluminescence solution at room temperature. Imaging was performed in a Bio-Rad ChemiDoc XRS + chemiluminescence imaging system.

Determination of apoptosis by Annexin V/PI double staining method: r406, centrifuging to collect cells, adding a proper amount of Stempro digestive juice to incubate for 5-10 minutes, centrifuging to remove the digestive juice, washing with PBS for 2-3 times, and centrifuging to remove the PBS. According to the kit specification, in the experiment, after washing cells, 100ul of Binding Buffer is used for flushing and suspending the cells, the cells are shaken and mixed evenly, 5ul of annexin V reagent and 5ul of PI reagent are added, the mixture is lightly blown and mixed evenly, incubation is carried out for 15mins in a dark place, 400ul of Binding Buffer is added for stopping staining, and the cells are put into a machine for detection. After the staining was terminated, FITC channel and PE channel were selected for detection according to the kit instructions and recorded.

The statistical method comprises the following steps: quantitative data corresponding to normal distribution are expressed as mean ± sd, and differences of P < 0.05 are considered statistically significant using SPSS 13.0 statistical software using the t test or one-way ANOVA analysis of variance combined with Dunnett's multiple tests.

Results of the experiment

To investigate the mechanism of R406 biological effect, we treated GSCs with time gradient and concentration gradient, respectively, and first stained with Hoechst33342 to observe the change of cells, especially nuclei, and observed under a fluorescence microscope, and the proportion of cells showing the phenomena of nuclear shrinkage and nuclear fragmentation increased significantly after the treatment (fig. 2A). After the above phenomena are observed, we guess that the effect of R406 induces the occurrence of GSCs apoptosis, so we examine the expression level of apoptosis-related proteins clear caspase3 and PARP by the Western Blot method, and after the cells are treated by different concentrations of R406, the expression level of clear caspase3 and PARP is up-regulated to different degrees, and the effect is most obvious at 1uM concentration (FIG. 2B). Further we explored the time course effect of R406 at a dose of 1uM concentration, and we found that the proportion of early apoptotic cells represented by either Annexin V single-staining positive or Annexin V/PI double-staining positive increased significantly from 12 hours after R406 treatment of the cells (fig. 2C and 2D).

Inhibition of SYK and PI3K-Akt signaling pathways involved in R406-induced metabolic remodeling and apoptosis of glioma stem cells

Experimental Material

Glioma stem cell lines GSC-1 and GSC-2 (isolated culture of tumor tissue specimens of central neurosurgery of tumor prevention and treatment of Zhongshan university). Malignant glioma cell lines U87 and U251 were purchased from ATCC (American Type Culture Collections, USA).

R406 (free, base) Selleck, USA. ZSTK474, available from Selleck, USA. R406 (free basa) is prepared as before; the PI3K inhibitor ZSTK474 is prepared into a mother solution with the concentration of 10mM by DMSO, stored at the temperature of-20 ℃, and diluted into a working solution as required.

Specific sirnas targeting Syk (# siB 09122384015) and PI3K (# siB 171206112648) were purchased from RiboBio, china. The siRNAs were transfected using Lipofectamine RNAiMAX (Life Technologies) and OPTI-MEM (Life Technologies) according to the manufacturer's instructions.

Preparing a culture medium of U87 and U251 cell strains: DMEM medium, 50mL fetal bovine serum, 50U/mL penicillin, 50. Mu.g/mL streptomycin per 450mL liquid medium before use.

Experimental methods

Cell culture: the GSCs culture method is the same as the previous method. Culturing malignant glioma cell strains U87 and U251: the cell lines were cultured in a constant-temperature closed incubator containing 5% CO2, 37 ℃ and 95% relative humidity for subculture, and cultured in DMEM medium containing 10% FBS, 50U/ml penicillin and 50. Mu.g/ml streptomycin, and the growth was observed using an inverted microscope. And (4) carrying out passage when the cell density reaches about 80%, carrying out passage once in about 2-4 days, and taking the cells in the logarithmic growth phase for formal experiments.

Treatment of cells with drugs: r406 processing of U87 and U251: cells in the logarithmic growth phase were seeded in 96-well plates at approximately 2000-4000 cells/well, and R406 was allowed to act on the cells in a concentration gradient of 0uM, 1.25uM, 2.5uM, 5uM, 10uM, 20uM for 48 hours. ZSTK474 treatment of GSCs: cells in logarithmic growth phase are inoculated into a 96-well plate, about 2000-4000 cells/well, and the ZSTK474 is acted on the cells for 48 hours according to the concentration gradient of 0uM, 1.25uM, 2.5uM, 5uM, 10uM and 20 uM. According to the dose-effect relationship, the concentration of 0.5uM is selected for subsequent experiments.

Knock-down of SYK expression with siRNA: the method is operated according to the Lipofectamine RNAiMAX specification, and comprises the following specific steps: (1) taking a 35mm cell culture dish as an example, cells were seeded onto the dish (without double antibody, medium containing 10% FBS was used) and allowed to adhere overnight with a cell density of about 50% at transfection; (2) dilution Lipofectamine RNAiMAX: the transfection reagent is shaken gently and mixed evenly, diluted by 200 mu L of optimized culture medium (Opti-MEM) according to the amount of 2 mu L per dish, and gently and evenly mixed; (3) dilution of siRNA: the siRNA was diluted with 200. Mu.L of Opti-MEM at a final concentration of 50nM at 3.6. Mu.L per dish; (4) mixing the diluted Lipofectamine RNAiMAX and siRNA, and standing for 5min at room temperature; (5) 400. Mu.L of the above mixture was dropped on a cell culture dish, shaken well and cultured normally.

The statistical method comprises the following steps: quantitative data corresponding to normal distribution are expressed as mean ± standard deviation, using SPSS 13.0 statistical software, using t test or one-way ANOVA analysis of variance coupled with Dunnett's multiple test, P < 0.05 is considered statistically significant for the differences. The quantitative data of the abnormal distribution adopts a rank sum test.

Results of the experiment

SYK expression varies between different cell lines: in order to further clarify the properties and expression levels of SYK genes in High-grade gliomas (HGGs) and GSCs, the expression levels of SYK proteins in two Glioma stem cell strains GSC-1 and GSC-2 and two High-grade Glioma cell strains U87 and U251 are verified by a Western Blot method, and the results show that the SYK proteins are positively expressed only in the GSC-1 and the rest three strains are negatively expressed (FIG. 3A).

Killing of GSCs by R406 is specific: we verified that R406 has a high killing effect on either GSC-1 or GSC-2, and based on the verification of expression level of SYK protein, the biological function of R406 seems to depend on expression of SYK, in other words, R406 may have multi-target effect, so to clarify the specificity, we treated the U87 and U251 cell strains with R406 according to dose gradient and measured their cell viability, and CCK-8 results show that R406 can not effectively inhibit the survival of U87 and U251 cells, and the IC50 is more than 1mM, indicating that R406 has relative specificity to GSCs (FIGS. 3B and 3C).

R406 has a multi-target effect, and besides SYK targets, PI3K/Akt signal channels mediate the play of biological functions: r406 acts as an inhibitor of SYK, and to further investigate the signal transduction mechanisms involved in the biological function of SYK, we first verified the function of SYK in GSC-1. We knocked down SYK protein expression levels in GSC-1 by means of RNA interference and preliminarily verified the interference efficiency by Western Blot. Through verification, the 002 # fragment in the siba kit in leber can effectively knock down expression of SYK (fig. 4A). Furthermore, we used CCK-8, cellRox staining flow cytometry and Seahorse cytometabolome to detect the activity of GSC-1 cells, changes in intracellular ROS content and OCR changes after SYK knockdown, respectively. The results show that interfering with SYK expression has the same biological effect as SYK inhibitor R406, namely decreased cell viability, increased intracellular ROS levels and increased oxidative phosphorylation levels (fig. 4B, 4C, 4D). It is suggested that in GSC-1, R406 may act by inhibiting SYK.

We further verify the change of the expression level of key molecules of a PI3K/Akt signal pathway after R406 treats each cell strain, and the Western Blot result proves that the expression level of phosphorylated PI3K and Akt proteins of an administration group is reduced averagely and the expression level of total PI3K proteins is up-regulated in two glioma stem cell strains. The expression level of either phosphorylated PI3K protein, akt protein or total PI3K, akt protein was not significantly changed in two non-dry high-grade glioma cell lines U87 and U251 (FIG. 5A). Further, in order to determine the influence of a PI3K/Akt pathway on glioma stem cells, GSCs are treated by a PI3K inhibitor ZSTK474 according to concentration gradient, the titer of the GSCs is determined, and the concentration of 0.5uM is selected for carrying out subsequent experiments. After 0.5um zsttk474 acted on GSCs, the same biological effects as SYK inhibitor R406, i.e. decreased cell viability, increased intracellular ROS levels and increased oxidative phosphorylation levels, were produced (fig. 5B, 5C, 5D). Suggesting that the PI3K/Akt signaling pathway mediates the biological effects of R406 in GSCs.

R406 synergistically enhances TMZ cytotoxicity on GSCs in vitro and in vivo

Experimental Material

Experimental animals: female BALB/c-nu/nu nude mice, 4-6 weeks old, purchased from the animal testing center, university of Nanjing.

In vitro cell experiments: r406 is prepared as before; TMZ was prepared as a 100mM stock solution in DMSO, protected from light and stored at-20 ℃. Dilution was performed as required for subsequent experiments. Animal experiments: r406: adding 2.5ml DMSO into 50mg standard to prepare 20mg/ml; diluting with PEG400 to 5mg/ml, and storing at-20 deg.C; TMZ:100mg size, 3.33ml DMSO is added to prepare about 30mg/ml; diluting with hydroxypropyl cyclodextrin to 10mg/ml, and storing at-20 deg.C under dark condition.

Experimental methods

Subcutaneous transplantation tumor of nude mice: 4-6 weeks old female BALB/c-nu/nu nude mice are selected. Mice were housed in a pathogen-free animal facility. Taking glioma stem cells in logarithmic growth phase, digesting, counting, centrifuging at low speed, collecting cells, suspending, adjusting cell density to appropriate value, injecting about 4.3 × 10 to left costal part of each nude mouse ⁵ cell,100 ul/cell (cell concentration 4.3X 10) ⁶ cell/mL, DMEM/F12 pure medium dilution and resuspension). Tumors formed after 72 hours and animals were randomized into four groups, control, R406 alone, TMZ alone and combination. Measuring the volume of the xenograft tumor and the body weight of the animal every day, and administering according to the body weight in a specific administration mode: intraperitoneal injection, R40620mg/kg and TMZ 50mg/kg. TMZ group and combination group were discontinued after 5 consecutive days of Temozolomide administration after neoplasia, R406 group and combination group were continued after neoplasia administration R406, i.e. combination group was treated with R406 and TMZ together five days after neoplasia followed by continued treatment with R406 alone.

In situ animal models: 4-week-old female BALB/c-nu/nu mice were anesthetized and 1X 10 mice were anesthetized ^s GSC-1 cells were resuspended in 2% methylcellulose and injected intracranially into the surface of the skull at 1mm from the lateral and 2mm from bregma using a microinjector and 27G needle, 4mm from the posterior. ImplantAfter 3 days, 28 mice were randomly divided into 4 groups, and the vehicle, TMZ (50 mg/kg daily for 5 consecutive days), R406 (20 mg/kg daily), and R406+ TMZ were intraperitoneally injected, respectively. Survival of the mice was recorded and Kaplan-Meier survival curves were generated.

Calculation of statistical methods and Combination Index (CI): quantitative data corresponding to normal distribution are expressed as mean ± standard deviation, using SPSS 13.0 statistical software, using ttest test or one-way ANOVA analysis of variance coupled with Dunnett's multiple test, P < 0.05 is considered statistically significant for the differences. The quantitative data of the abnormal distribution adopts a rank sum test. The Combination Index (CI) is calculated by Calcusyn software with reference to Chou-Talalay formula, and when the CI value is less than 1, it is suggested that there is a synergistic effect.

Results of the experiment

Temozolomide (TMZ) is a DNA alkylating agent and is the standard chemotherapeutic agent for GBM. However, significant resistance is often observed in clinical practice. Therefore, we further investigated whether R406 can enhance the efficacy of TMZ on GSC. To this end, we assessed cell viability after treatment of GSCs with R406, TMZ or a combination of R406 and TMZ, respectively. R406 or TMZ monotherapy moderately reduced the viability of GSCs, while combination treatment resulted in a significant increase in GSC cell death (fig. 6A). The calculated Combination Index (CI) was 0.262 for GSC-1 and 0.280 for GSC-2. CI of less than 1 for both GSCs indicates that R406 and TMZ have a synergistic effect.

Our in vitro results strongly suggest that addition of R406 in the treatment of GBM in vivo may be beneficial for TMZ. We first assessed this possibility by injecting GSC subcutaneously into immunocompromised mice. After tumor establishment, mice were given either vector alone, R406 or TMZ, or a combination of R406 and TMZ, respectively. As shown in fig. 6B and 6C, GSC was effective in initiating glioma in animal models. Treatment with TMZ alone was effective at an early stage, but failed to delay tumor growth at a later stage. At 15 days post-implantation, tumors proliferated at an accelerated rate, with tumor size exceeding that of the control group. Rebound growth of gliomas in vivo is similar to the recurrence observed in GBM patients. Monotherapy with R406 was more effective in delaying tumor growth than TMZ alone. Notably, a stable and significant reduction in tumor size was found in mice receiving the combination of R406 and TMZ compared to the control group and monotherapy with either R406 or TMZ alone. Analysis of tumor sections from treated animals demonstrated a decrease in cell proliferation (Ki-67) for R406+ TMZ therapy (fig. 6C). Significant activation of caspase3 in xenograft tissues indicated enhanced apoptosis in the combination treatment group (fig. 6D).

Since the brain microenvironment strongly affects glioma progression and the cerebral blood barrier (BBB) significantly affects the entry of therapeutic agents into the CNS, we established an in situ animal model to determine the efficacy of combination therapy on GSC. Similarly, we found that R406+ TMZ was more effective in prolonging the survival of tumor-bearing mice than either R406 (p < 0.05) or TMZ (p < 0.01) alone (FIG. 6E). Together, these data suggest that R406 attenuates resistance of GSCs to TMZ in vitro and, in synergy with TMZ, inhibits GSC-induced tumors by promoting apoptosis in vivo.

The invention discovers that high-grade glioma cell lines such as U87, U251, U118, T98G and LN18 do not express SYK, expression of SYK in GSC-2 is negative, but glioma stem cell lines can be inhibited by R406, and non-dry high-grade glioma cell lines are insensitive to R406, which indicates that R406 has relative specificity to GSCs. In order to explore the mechanism of the phenomenon, the expression level of SYK is firstly knocked down, the effect of R406 can be achieved through verification, the SYK is suggested to be one of the targets of R406, in addition, the signal transduction pathway PI3K/Akt for regulating and controlling various biological phenotypes of cells is only obviously changed in glioma stem cell strains, the cell strains such as U87 and U251 are not changed, the PI3K/Akt signal pathway is suggested to mediate the exertion of the biological effect of R406, and the P13K inhibitor is subsequently utilized to block the pathway, so that the experimental result consistent with the R406 is obtained, and the point is also proved. Taken together, we suggest that R406 function in GSCs is not completely dependent on the expression level of SYK, which has potential multi-target effects and relative specificity of GSCs. More importantly, temozolomide (TMZ) is a primary chemotherapy drug for HGG at present, and the synergistic effect of R406 and TMZ, as confirmed by in vitro experiments and xenograft tumor animal experiments in the subject, shows that R406 has great potential clinical application value.

Claims (5)

1. Use of Tamatinib or a pharmaceutically acceptable salt thereof and temozolomide for the preparation of a pharmaceutical composition for the treatment of glioma, said pharmaceutical composition comprising a therapeutically effective amount of Tamatinib or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of temozolomide, wherein said glioma is caused by glioma stem cells.
2. 2. The use of claim 1, wherein the glioma stem cells comprise Syk protein-negative glioma stem cells.
3. 3. The use of claim 1, wherein the glioma stem cells comprise Syk protein-negative glioma stem cells and Syk protein-positive glioma stem cells.
4. 4. Use according to any one of claims 1 to 3, wherein the glioma is selected from the group consisting of anaplastic astrocytoma, anaplastic oligodendroglioma, primary glioblastoma and secondary glioblastoma.
5. 5. Use according to any one of claims 1 to 3, wherein the glioma is a WHO grade III or WHO grade IV glioma.

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