WO2023113538A1

WO2023113538A1 - Griseofulvin combination therapy for treating brain tumor

Info

Publication number: WO2023113538A1
Application number: PCT/KR2022/020588
Authority: WO
Inventors: 장창영; 백선하; 박순범; 홍지희
Original assignee: 숙명여자대학교산학협력단
Priority date: 2021-12-17
Filing date: 2022-12-16
Publication date: 2023-06-22

Abstract

The present invention relates to a pharmaceutical composition comprising griseofulvin and temozolomide for treating or preventing brain tumors. When temozolomide was used alone, survival period prolongation was insignificant and temozolomide-resistant patients received little effect, but using griseofulvin in combination therewith exhibits synergistic effects such that better effects are exhibited, and thus can be used in the treatment of brain tumors.

Description

Glyceoflavin Combination Therapy for Brain Tumor Treatment

The present invention provides glyceofulvin combination therapy for the treatment of brain tumors.

Glioblastoma Multiforme is a type of brain tumor arising from glial cells and is a malignant tumor with the highest fatality rate occurring in the brain. Usually, radiotherapy and temozolomide, an oral treatment, are used as standard treatments for brain tumors, and the treatment effect is insignificant due to drug resistance. Temozolomide is an anticancer drug that attaches a methyl group to DNA. Damaged DNA such as N7-meG or O6-meG cannot be repaired, causing double strand breaks and eventually causing apoptosis, thereby killing cancer cells. Based on this mechanism, it is reported that resistance to temozolomide is acquired by overexpression of O(6)-methylguanine-DNA methyltransferase (MGMT), which removes methyl groups in cancer cells. As a strategy for overcoming resistance, development of a treatment using an inhibitor of demethylation of the MGMT promoter, which is expected to be the cause of MGMT overexpression, is being developed.

Glioblastoma, which accounts for about 12-15% of all brain tumors, has an average survival time of only 14 months even after surgery, radiation, and chemotherapy, so the development of new treatments is urgent.

An object of the present invention is to provide a pharmaceutical composition for treating or preventing brain tumors containing griseofulvin and temozolomide.

Another object of the present invention is to provide a biomarker composition for predicting brain tumor prognosis comprising PCNT (pericentrin) protein or a gene encoding the protein as an active ingredient.

Another object of the present invention is to provide a biomarker composition for predicting brain tumor prognosis containing, as an active ingredient, an agent capable of detecting PCNT (pericentrin) protein expression or activity level, or the expression level of a gene encoding the protein. is in

Another object of the present invention is to predict the prognosis of brain tumors, including the step of comparing the expression or activity level of PCNT (pericentrin) protein or the expression level of a gene encoding the protein from a sample isolated from a sample and a normal control group. It is about providing a way to present information.

Another object of the present invention is (1) measuring the expression level of PCNT (pericentrin) protein or the expression level of a gene encoding the protein from a sample isolated from a brain tumor patient; (2) comparing the expression level of PCNT (pericentrin) protein or the expression level of the gene encoding the protein in step (1) with a control sample; And (3) a method for providing information for temozolomide and glyceofulvin combination therapy for brain tumor treatment, including determining the dose of glyceofulvin to be used in combination with temozolomide treatment is to provide

In order to achieve the above object, the present invention provides a pharmaceutical composition for treating or preventing brain tumors containing griseofulvin and temozolomide.

In addition, the present invention provides a biomarker composition for predicting brain tumor prognosis comprising a PCNT (pericentrin) protein or a gene encoding the protein as an active ingredient.

In addition, the present invention provides a biomarker composition for predicting brain tumor prognosis containing, as an active ingredient, an agent capable of detecting the expression or activity level of PCNT (pericentrin) protein or the expression level of a gene encoding the protein.

In addition, the present invention provides information for predicting brain tumor prognosis, including the step of comparing the expression or activity level of PCNT (pericentrin) protein or the expression level of a gene encoding the protein from a sample isolated from a sample and a normal control group. provide a way to provide

In addition, the present invention comprises the steps of (1) measuring the expression level of PCNT (pericentrin) protein or the expression level of a gene encoding the protein from a sample isolated from a brain tumor patient; (2) comparing the expression level of PCNT (pericentrin) protein or the expression level of the gene encoding the protein in step (1) with a control sample; And (3) a method for providing information for temozolomide and glyceofulvin combination therapy for brain tumor treatment, including determining the dose of glyceofulvin to be used in combination with temozolomide treatment to provide.

The present invention relates to a pharmaceutical composition for treating or preventing brain tumors containing griseofulvin and temozolomide. Although the effect was low for patients with temozolomide resistance, synergistic effects can be achieved by using glyceofulvin in combination.

1 is a result confirming that multipolar spindles are formed when apoptosis is induced by treating human glioblastoma cell lines U87 cell line and U373 cell line with 10 μM temozolomide for 24 hours.

Figure 2 shows the results confirmed by staining with Plk1 antibody and centrin antibody after treating human glioblastoma cell lines U87 cell line and U373 cell line with 10 μM temozolomide for 24 hours.

Figure 3 shows the results of confirming the cells by staining with PCNT (pericentrin) antibody and beta-tubulin antibody after treating the U87 and U373 cell lines with the drug for 24 hours.

Figure 4 shows the results of counting multipolar spindles and unaligned chromosomes by observing cells after antibody staining after treating 7 glioblastoma cell lines with drugs for 24 hours.

5 is a result of confirming the amount of PCNT (pericentrin) present in extracts of glioblastoma cell lines by Western blotting.

6 shows the results of colony formation assay after treatment of U87 and U373 cell lines with drugs.

7 shows the results of colony formation assay after treatment of glioblastoma cell lines with drugs.

Hereinafter, the present invention will be described in more detail.

The present invention provides a pharmaceutical composition for treating or preventing brain tumors containing griseofulvin and temozolomide.

The concentration of griseofulvin may be 0.0353 mg/kg to 3.528 mg/kg, and the concentration of temozolomide may be 0.971 mg/kg to 1.94 mg/kg, but is not limited thereto.

The brain tumor may be any one selected from the group consisting of glioma, low-grade glioma, glioma multiforme, glioblastoma, glioblastoma multiforme, astrocytoma, anaplastic astrocytoma, oligodendrocyte tumor, and ependymocyte tumor, It is not limited to this.

The pharmaceutical composition may be administered to a patient overexpressing MGMT (O(6)-methylguanine-DNA methyltransferase).

In the pharmaceutical composition, temozolomide weakens centrosomes to induce multipolar spindles to induce apoptosis, and griseofulvin can induce centrosome declustering. .

In another embodiment of the present invention, the pharmaceutical composition is a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, lubricant, lubricant, flavoring agent, antioxidant, buffer, bacteriostatic agent, One or more additives selected from the group consisting of diluents, dispersants, surfactants, binders and lubricants may be further included. Specifically, carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil may be used, and solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules. These solid preparations may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for oral use, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. As a base material of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used. According to one embodiment of the present invention, the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or It can be administered to a subject in a conventional manner via the intradermal route. The dosage of the active ingredient according to the present invention may vary depending on the condition and weight of the subject, the type and severity of the disease, the drug type, the route and duration of administration, and may be appropriately selected by a person skilled in the art, and the daily dosage is 0.01 mg. /kg to 200 mg/kg, preferably 0.1 mg/kg to 200 mg/kg, and more preferably 0.1 mg/kg to 100 mg/kg. Administration may be administered once a day or divided into several times, and the scope of the present invention is not limited thereby.

The "biomarker" of the present invention is a substance that can be diagnosed by distinguishing tissues or cells of a subject with a brain tumor from tissues or cells of a normal control group, and is a pattern of increase or decrease in tissues or cells of a subject with a disease compared to a normal control group. It includes organic biomolecules such as proteins or nucleic acids, lipids, glycolipids, glycoproteins, etc.

As used herein, the term "prediction of prognosis" refers to the act of predicting the course and outcome of a disease in advance. More specifically, prognosis prediction is interpreted as meaning that the course of a disease after treatment may vary depending on the patient's physiological or environmental state, and that it refers to any act of predicting the course of a disease after treatment by comprehensively considering the patient's condition. It can be.

For the purpose of the present invention, the prediction of prognosis may be interpreted as an act of predicting the disease-free survival rate or survival rate of a cancer patient by predicting in advance the course of a disease and whether or not it is completely cured after treatment of a cancer patient. For example, predicting "good prognosis" means that the disease-free survival rate or survival rate of cancer patients after treatment is high, so that cancer patients are likely to be cured, and predicting "poor prognosis" means that cancer patients are likely to be treated. Since the disease-free survival rate or survival rate of cancer patients after treatment is at a low level, it means that there is a high possibility of cancer recurrence or death due to cancer from cancer patients.

The agent may be selected from the group consisting of primers, probes, antibodies, peptides and aptamers.

As used herein, the term "primer" is a short gene sequence that is the starting point of DNA synthesis, and refers to an oligonucleotide synthesized for the purpose of diagnosis, DNA sequencing, and the like. The primers may be synthesized and used in a length of 15 to 30 base pairs, but may vary depending on the purpose of use, and may be modified by methylation, capping, or the like by a known method.

As used herein, the term “probe” refers to a nucleic acid capable of specifically binding to mRNA of several to several hundred bases in length prepared through enzymatic chemical separation and purification or synthesis. The presence or absence of mRNA can be confirmed by labeling a radioactive isotope or an enzyme, and it can be designed and modified using a known method.

As used herein, the term “antibody” is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to the PCNT of the present invention, and the antibody can be prepared according to a conventional method in the art. The type of antibody includes polyclonal antibodies or monoclonal antibodies, and all immunoglobulin antibodies are included. The antibody is meant in its complete form with two full-length light chains and two full-length heavy chains. In addition, the antibody also includes special antibodies such as humanized antibodies.

The term "peptide" used in the present invention has the advantage of high binding ability to a target substance, and does not undergo denaturation even during heat/chemical treatment. In addition, because of its small molecular size, it can be attached to other proteins and used as a fusion protein. Specifically, since it can be used by attaching it to a polymer protein chain, it can be used as a diagnostic kit and a drug delivery material.

As used herein, the term “aptamer” is a special kind of single-stranded nucleic acid (DNA, RNA or modified nucleic acid) that has a stable tertiary structure and can bind to a target molecule with high affinity and specificity. It means a kind of composed polynucleotide. As described above, aptamers can specifically bind to antigenic substances in the same way as antibodies, but are more stable than proteins, have a simple structure, and are composed of polynucleotides that are easy to synthesize, so they can be used instead of antibodies. can

When the PCNT (pericentrin) protein or gene expression level in step (1) is overexpressed compared to the control group, the dose of griseofulvin to be used in combination can be determined to be 0.0176 mg/kg to 0.352 mg/kg.

When the level of PCNT (pericentrin) protein or gene expression in step (1) is lower than that of the control group, the dose of griseofulvin to be used in combination can be determined to be 0.0176 mg/kg to 0.352 mg/kg.

In the present invention, “overexpression” refers to a case in which the PCNT (pericentrin) protein or gene expression level is increased by 50 to 200% compared to the control group, and “low expression” refers to a case in which the PCNT (pericentrin) protein or gene expression level is increased by 20 to 200% compared to the control group. This means a 50% reduction.

Hereinafter, examples and the like will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. Examples of the present invention and the like are provided to more completely explain the present invention to those skilled in the art.

<실시 예 1> <Example 1> 테모졸로마이드temozolomide 처리에 따른 according to processing 다극성multipolarity 방추사가 형성 여부 및 세포사멸을 일으키는 기전 확인 Confirmation of spindle formation and mechanism of apoptosis

(1) Immunofluorescence microscopy

Cells treated with solvent (DMSO) and temozolomide for 24 hours were fixed in 100% ethanol at -20 °C for 20 minutes. After 20 minutes, PBS-BT (PBS, 3% BSA, 0.1% Triton X-100) was added and left at room temperature for 30 minutes. After 30 minutes, the cells were stained with PCNT (pericentrin) and beta-tubulin antibodies for 30 minutes. After 30 minutes, the cells were washed three times with PBS-BT, and stained with Alexa Fluor 594 or 488-linked secondary antibodies for 30 minutes. After 30 minutes, it was washed three times with PBS-BT, and DNA was stained with DAPI and attached to a slide glass using Mowiol (Polyvinyl alcohol). Photographs were taken with a Zeiss Axiovert 200 M microscope and imaged with AutoVisualizer v9.1.

(2) Results

When apoptosis was induced by treating human glioblastoma cell lines U87 cell line and U373 cell line with 10 μM temozolomide for 24 hours, it was confirmed that multipolar spindles were formed, according to FIG. 1 . This phenomenon was suppressed by ATR inhibitor (NU6027) and CHK1 inhibitor (UCN-01), which are upper kinases of the gene damage response, and ATM inhibitor (KU55933) did not prevent multipolar spindle formation by temozolomide. couldn't Therefore, it can be seen that multipolar spindle formation by temozolomide is induced by ATR and CHK1.

<실시 예 2> 테모졸로마이드 처리에 따른 미성숙 중심립 분리 현상 확인<Example 2> Confirmation of immature centriole separation according to temozolomide treatment

(1) Immunofluorescence microscopy

Cells treated with solvent (DMSO) and temozolomide for 24 hours were fixed in 100% ethanol at -20 °C for 20 minutes. After 20 minutes, PBS-BT (PBS, 3% BSA, 0.1% Triton X-100) was added and left at room temperature for 30 minutes. After 30 minutes, the cells were stained with Plk1 antibody and centrin antibody. After 30 minutes, the cells were washed three times with PBS-BT, and stained with Alexa Fluor 594 or 488-linked secondary antibodies for 30 minutes. After 30 minutes, it was washed three times with PBS-BT, and DNA was stained with DAPI and attached to a slide glass using Mowiol (Polyvinyl alcohol). Photographs were taken with a Zeiss Axiovert 200 M microscope and imaged with AutoVisualizer v9.1.

(2) Results

Human glioblastoma cell lines, U87 cell line and U373 cell line, were treated with 10 μM temozolomide for 24 hours, stained with Plk1 antibody and centrin antibody, DNA was stained with DAPI, and Merge was observed by three staining results, according to FIG. 2, temozolomide Premature centriole disengagement from centrosomes was confirmed by Zolomide.

<실시 예 3> <Example 3> 글리세오플빈glyceofulvin 및 and 테모졸로마이드temozolomide 병용처리에 의한 by combined treatment 다극성multipolarity 방추사 형성 여부 확인 Check for spindle formation

(1) Immunofluorescence microscopy

(2) Counting

The ratio was calculated by counting the number of multipolar cells out of 100 metaphase cells, and the same experiment was repeated three times to count a total of 300 cells.

(3) Results

After treating the U87 and U373 cell lines with the drug for 24 hours, the cells were observed by staining with PCNT (pericentrin) antibody and beta-tubulin antibody. As a result, according to FIGS. 3 and 4, when the U87 cell line and the U373 cell line were treated with 10 μM temozolomide for 24 hours, multipolar spindle fibers were formed at a low rate, and glyceofulvin was treated with 0.1 μM in U87 and 10 μM in U373. Multipolar spindles do not form. However, when temozolomide and glyceofulvin were combined, the formation rate of multipolar spindles and pseudo-bipolar spindles increased rapidly (50%), indicating that there was a synergistic effect. It was confirmed that unaligned chromosomes were also generated in the U373 cell line by glyceofulvin.

<실시 예 4> 교모세포종 세포주들의 추출물에 존재하는 <Example 4> Present in extracts of glioblastoma cell lines PCNT(pericentrin)의PCNT (pericentrin) 양 확인 Check amount

(1) Western blot

Glioblastoma cell lines were treated with LAP200 lysis buffer (50 mM Hepes, pH 7.4, 200 mM KCl, 0.3% NP-40, 10% glycerol, 1 mM EGTA, 1 mM MgCl ₂ , 0.5 mM DTT, 0.5 μM microcystin, 10 μg/ml each of leupeptin, pepstatin). and chymostatin). Proteins were separated from each cell lysate using an SDS-PAGE gel, and the proteins in the gel were transferred to a membrane and then detected with PCNT, MAD2, MGMT, and Actin antibodies to compare the protein expression levels.

(2) Results

As a result, according to FIGS. 4 and 5, in the case of U87, U251, LN428, T98G, and Snb19 cell lines with high PCNT protein expression, multipolar spindles and pseudo-bipolar spindles were induced by the combined treatment with 0.1 μM glyceofulvin, but PCNT It was confirmed that multipolar spindles and pseudobipolar spindles were induced in U373, F98, and HT22 cell lines with low protein expression levels when 10 μM of glyceofulvin was co-treated. In addition, as a result of comparative analysis of Mad2 and MGMT, it was confirmed that there was no correlation with the phenotype.

It was found that the PCNT (pericentrin) protein expression level was proportional to the resistance of glioblastosis cells to glyceofulvin. The lower the PCNT protein expression level, the higher the concentration of glyceofulvin required for multipolar spindle induction and apoptosis. It was confirmed that can be used as a prognostic factor for brain tumor patients.

<실시 예 5> <Example 5> 글리세오플빈glyceofulvin 및 and 테모졸로마이드temozolomide 병용처리에 의한 by combined treatment 집락colony (colony) 형성 확인Confirm colony formation

(1) Colony Forming cell assay

2 ml of methylcellulose-based media was dispensed into 15 ml conical tubes for each group. Glyeofulvin and temozolomide were added to each group, mixed well, and then waited for 20 minutes. At this time, air bubbles in the media caused by high viscosity were removed. After 20 minutes, 5.0 x 10² cells of each cell line were added to each group (control group, temozolomide group, glyceofulvin group, and combination treatment group), mixed, and then waited another 20 minutes to remove air bubbles. Using a syringe, exactly 1.5 ml was slowly added to the 6-well plate without bubbles. Put sterilized water (H ₂ O) between the wells. Colonies were observed after culturing in a 37°C, 5% CO ₂ environment for 14 to 16 days.

(2) Results

As a result, according to FIGS. 6 and 7, it was confirmed that the number of colonies in the group treated with temozolomide and glyceofulvin was significantly reduced compared to the control group, the temozolomide group, and the glyceofulvin group.

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

A pharmaceutical composition for treating or preventing brain tumors comprising griseofulvin and temozolomide.
The method of claim 1,

The pharmaceutical composition, characterized in that the concentration of griseofulvin is 0.0353 mg / kg to 3.528 mg / kg, and the concentration of temozolomide is 0.971 mg / kg to 1.94 mg / kg.
The method of claim 1,

The brain tumor is any one selected from the group consisting of glioma, low-grade glioma, glioma multiforme, glioblastoma, glioblastoma multiforme, astrocytoma, anaplastic astrocytoma, oligodendrocyte tumor and ependymocyte tumor, characterized in that A pharmaceutical composition to be.
The method of claim 1,

The pharmaceutical composition is a pharmaceutical composition, characterized in that for administration to a patient overexpressed MGMT (O (6) -methylguanine-DNA methyltransferase).
The method of claim 1,

The pharmaceutical composition is characterized in that temozolomide weakens centrosomes to induce multipolar spindles to induce apoptosis, and griseofulvin induces centrosome declustering. A pharmaceutical composition to be.
A biomarker composition for predicting brain tumor prognosis comprising PCNT (pericentrin) protein or a gene encoding the protein as an active ingredient.
A biomarker composition for predicting brain tumor prognosis containing, as an active ingredient, an agent capable of detecting PCNT (pericentrin) protein expression or activity level, or the expression level of a gene encoding the protein.
The method of claim 7,

The agent is a biomarker composition for predicting brain tumor prognosis, characterized in that selected from the group consisting of primers, probes, antibodies, peptides and aptamers.
A method for providing information for prognosis of brain tumors, comprising comparing the expression or activity level of PCNT (pericentrin) protein or the expression level of a gene encoding the protein from a sample isolated from a sample and a normal control group.
(1) measuring the expression level of PCNT (pericentrin) protein or the expression level of a gene encoding the protein from a sample isolated from a brain tumor patient;

(2) comparing the expression level of PCNT (pericentrin) protein or the expression level of the gene encoding the protein in step (1) with a control sample; and

(3) A method for providing information for combination therapy of temozolomide and glyceofulvin for the treatment of brain tumors, comprising the step of determining the dose of glyceofulvin to be used in combination with temozolomide (temozolomide) treatment.
The method of claim 10,

When the PCNT (pericentrin) protein or gene expression level in step (1) is overexpressed compared to the control group, the dose of griseofulvin to be used is determined to be 0.0176 mg / kg to 0.352 mg / kg. , Methods to provide information for temozolomide and glyceofulvin combination therapy for the treatment of brain tumors.
The method of claim 10,

When the level of PCNT (pericentrin) protein or gene expression in step (1) is lower than that of the control group, the dose of griseofulvin to be used is 0.0176 mg / kg to 0.352 mg / kg. How to provide information for temozolomide and glyceofulvin combination therapy for the treatment of brain tumors.