JP6967763B2 - Therapeutic agent - Google Patents

Description

The present invention particularly relates to a therapeutic agent for poorly vascular solid tumors, a method for predicting tumor prognosis, and a method for treating.

Brain tumors are tumors that occur in intracranial tissues and occur in about 12 out of 100,000 people every year. Brain tumors have already grown to a certain size by the time symptoms appear, and are refractory to treatment and have a poor prognosis, especially if they are malignant. About 30% of these primary brain tumors are particularly high-grade gliomas.

Here, referring to Non-Patent Document 1, in recent years, cancers caused by mutations in isocitrate dehydrogenase (hereinafter referred to as “IDH”) have been reported in the brain, blood, large intestine, cartilage and the like. ing.
Normal (mutant-free) IDH (hereinafter referred to as "normal IDH") is an oxidation that mutually converts isocitrate and 2-oxoglutaric acid (2-oxoglutalic acid, hereinafter referred to as "2OG"). It is a reductase.
However, IDH having a mutation (hereinafter referred to as "mutant IDH") is different from normal IDH in that 2-oxoglutarate to 2-hydroxyglutarate (2-hydroxyglutarate, hereinafter referred to as "2HG") is used. Generate. 2HG imparts tumorigenicity to cells as a cancer metabolite.

Specifically, the mutant IDH, unlike the normal IDH, produces 2HG from 2OG. Under homeostasis, 2HG does not have a known metabolic function in mammals, and D-2HG usually has errors in catalysis of phosphoglycerate dehydrogenase or hydroxyloxoacetic transferase reaction, and malate dehydrogenase reaction. Is produced only at low levels (<300 μM). Low levels of 2HG are usually converted to 2OG by chirality specific dehydrogenase (D-2HGDH, or L-2HGDH) and rapidly removed. However, 2HG production by mutant IDH overwhelms the normal clearance mechanism and accumulates 2HG as the most abundant metabolite in these cells at concentrations in mM units.
Therefore, in recent years, development of a specific inhibitor of mutant IDH has been promoted as an anticancer agent that suppresses the production of 2HG metabolites.

On the other hand, with reference to Patent Document 1, the conventional use of the composition, which has not been known at all in relation to tumor treatment, will be described.
In Patent Document 1, a structural analog (analog) of γ-butyrobetaine (hereinafter referred to as “GBB”) as a free base for the production of a drug for normalizing and stimulating sexual activity and sexual intercourse in mammals. ) Or the use of a pharmaceutically acceptable salt thereof is disclosed. The disclosed composition, when orally administered to unanesthetized male rats for 6 weeks, substantially enhances their sexual activity, reduces stimulation time, reduces the number of matings and the effectiveness of back riding attempts. Increase.
The new uses of this composition not disclosed in Patent Document 1 will be described in detail in the embodiments described later.

Japanese Patent Publication No. 2005-504071

L. Dang, K.K. Yen, E.I. C. Attar, "IDH mutations in cancer and progress toward developed of targeted therapies", Anals of Oncology, 2016, 27, 4, p. 599-608 Yan. H et al., "IDH1 and IDH2 mutations in gliomas", J Engl J Med, 2009, 360, p. 765-773 S. Miyata et al., "AnR132H Mutation in Isocitrate Dehydrogenase 1 Enhances p21 Expression and Inhibits Phosphorylation of Retinoblastoma Protein (10) Cell 645-654

Here, it was found that the prognosis of leukemia was not affected by the presence or absence of the IDH mutation. However, in glioma, the mutant IDH tumor had a significantly higher 5-year survival rate and a better prognosis than the normal IDH tumor without mutation.
However, there was no effective treatment for glioma with a poor prognosis of normal IDH. That is, there was almost no effective therapeutic composition for poor vascular solid tumors such as glioma with a poor prognosis of normal IDH.

The present invention has been made in view of such a situation, and an object of the present invention is to solve the above-mentioned problems.

The therapeutic agent of the present invention is a depletion vascular solid tumors of therapeutic agents, see containing a fatty acid oxidation inhibitor, wherein the solid tumor is a glioma, the fatty acid oxidation inhibitor, and characterized by a Meldonium do.

It is a conceptual diagram which shows the biochemical pathway which concerns on the fatty acid oxidation inhibitor of the therapeutic agent which concerns on embodiment of this invention. It is a photograph which shows the preoperative MRI FLAIR image of the brain tumor patient of the mutant type IDH or the normal type IDH which concerns on Example of this invention. It is a graph which shows the heat map of the metabolome analysis and the result of the principal component analysis in the brain tumor sample and the cell line of the mutant type IDH or the normal type IDH which concerns on an Example of this invention. It is a graph which shows the amount of 2OG and 2HG in the brain tumor sample and the cell line which concerns on Example of this invention. It is a graph which shows each product in the purine metabolism pathway of the brain tumor sample of the mutant type IDH or the normal type IDH which concerns on Example of this invention. It is a graph which shows AEC and total adenilate in the purine metabolism pathway of the brain tumor sample of the mutant IDH or the normal IDH which concerns on Example of this invention. It is a graph which shows the amino acid amount of the brain tumor sample of the mutant type IDH or the normal type IDH which concerns on Example of this invention. It is a graph which shows the fatty acid metabolism of the brain tumor sample of the mutant type IDH or the normal type IDH which concerns on Example of this invention. It is a graph which shows the fatty acid metabolism in the cell line of the mutant type IDH or the normal type IDH which concerns on Example of this invention. It is a conceptual diagram which shows the carnitine synthesis pathway which concerns on Example of this invention. It is a graph which shows the proliferation of the cell line which was cultured by adding carnitine which concerns on Example of this invention. It is a graph which shows the proliferation of the cell line cultured by adding the carnitine inhibitor which concerns on Example of this invention. It is a graph which shows the amount of carnitine and 2HG of the tissue sample of the glioma which concerns on Example of this invention.

<Embodiment>
In recent years, cancers caused by mutations in isocitrate dehydrogenase (IDH) have been reported in the brain, blood, large intestine, cartilage and the like. For leukemia, the presence or absence of IDH mutation has no effect on prognosis. However, in glioma, tumors with mutant IDH with the IDH mutation had a better prognosis than those without the mutation, and the reason was unknown. In addition, there were not many effective therapeutic agents for glioma with a poor prognosis without IDH mutation.
Therefore, the inventors of the present invention earnestly aim to develop a therapeutic agent for a glioma having a poor prognosis, and to clarify the starting point of action in which a normal IDH glioma having no IDH mutation has a poor prognosis. An experiment was conducted. Specifically, a comprehensive metabolomics analysis was performed using a clinical brain tumor sample of mutant IDH and normal IDH glioma and a cell line of glioma expressing normal IDH and mutant IDH, and compared. ..
As a result, it was clarified that the fatty acid metabolism pathway was inhibited in the glioma having a mutant IDH with a good prognosis, and the decrease in carnitine was particularly remarkable, and the present invention was completed.

More specifically with reference to FIG. 1, 2HG, which is a cancer metabolite synthesized from 2OG by mutant IDH, has a tumor-forming action by hypoxia-inducing transcriptional regulation and demethylation via HIF1α, JHDM, TET2 and the like. Causes. In addition, as a tumor suppressive action, it suppresses ATP5B (ATP synchronize subunit beta) and suppresses the synthesis of ATP.
Here, the present inventors supply acetyl-CoA and ATP by oxidation of fatty acids using carnitine up to the decrease of BBOX1, 2 to ATP shown by the thick black line in FIG. 1, and the supply of acetyl-CoA and ATP is a poor vascular solid such as a brain tumor. It was found to be important for tumor growth. That is, the present inventors have clarified that the glioma of mutant IDH had a good prognosis by reducing carnitine and inhibiting the fatty acid metabolism pathway.
Carnitine maintains homeostasis by absorption from food in normal tissues. However, during poor vascular tumor growth, carnitine is exhausted inside the tumor, so the intracellular carnitine synthesis system and / or the fatty acid β-oxidation system using carnitine, that is, the pathway of energy generation by fatty acid oxidation. However, it was important for tumor growth.

Therefore, the therapeutic agent according to the embodiment of the present invention is a therapeutic agent for a poor vascular solid tumor, and is characterized by containing a fatty acid oxidation inhibitor.
Further, the therapeutic method according to the embodiment of the present invention is a method for treating a poor vascular solid tumor, and is characterized by administering a fatty acid oxidation inhibitor.
Further, as the fatty acid oxidation inhibitor of the present embodiment, various medical compositions such as small molecule compounds, antibody drugs, nucleic acids, and PNAs targeting enzymes related to fatty acid oxidation as described below can be used. Is. In addition, the fatty acid oxidation inhibitor of the present embodiment also includes a medical composition or the like that realizes a method related to gene modification such as genome editing.

Further, by using the therapeutic agent containing the fatty acid oxidation inhibitor of the present embodiment as a therapeutic method in combination with surgery, irradiation, anticancer drug treatment, etc., it is effective for highly malignant poor vascular solid tumors. It will be a good treatment.
By constructing in this manner and using the fatty acid oxidation inhibitor of the present embodiment as a therapeutic agent or as a therapeutic method, energy generation due to fatty acid oxidation is suppressed, and vascularity such as a brain tumor having a poor prognosis of normal IDH is impaired. It is possible to suppress the growth of solid tumors. That is, the therapeutic agent of the present embodiment can be provided as a means for treating a poor vascular solid tumor such as a glioma having a poor prognosis of normal IDH.
As the fatty acid oxidation inhibitor, it is also possible to use a regulator such as an activator that promotes fatty acid oxidation and conversely depletes carnitine, 2OG and the like described below.

Further, the therapeutic agent according to the embodiment of the present invention comprises a group consisting of a fatty acid oxidation inhibitor, a carnitine synthesis inhibitor, an absorption inhibitor, a decomposition promoter, and a fatty acid β-oxidation inhibitor. It is a feature.
With this configuration, the therapeutic targets for vascular poor vascular solid tumors containing glioma are the carnitine synthesis system and the fatty acid β-oxidation system using carnitine, and the synthesis inhibitor, which is a substance that inhibits carnitine synthesis, inhibits absorption. Growth of brain tumors by including at least one of a group consisting of an absorption inhibitor, which is a substance that promotes decomposition, a decomposition promoter, which is a substance that promotes decomposition, and a fatty acid β-oxidation inhibitor that inhibits β-oxidation of fatty acids themselves. Can be suppressed.

Specifically, as described in the Examples below, carnitine, acylcarnitine, acetylcarnitine, GBB, and N ⁶ , N ⁶ , N ⁶ -trimethyl-L-lysine in tissue samples of mutant IDH glioma. Due to the low concentration of (N ⁶ , N ⁶ , N ^6- trimethylly-L-lysine, N ^ε- trimethyllysine, hereinafter referred to as "TML"), β-oxidation is used for the tumor-suppressing effect of the mutant IDH glioma. Suppression is considered important. That is, it is provided as a therapeutic method for suppressing the growth of avascular tumors including brain tumors by using an inhibitor for the biosynthetic pathways related to carnitine synthesis, absorption and degradation, and / or the biosynthetic pathways related to β-oxidation of fatty acids. It becomes possible to do. In addition, a composition containing at least one of the group consisting of a carnitine synthesis inhibitor, an absorption inhibitor, a degradation promoter, and a fatty acid β-oxidation inhibitor is provided as a therapeutic agent for avascular solid tumor including a brain tumor. can.
Moreover, in the case of normal cells in the body, carnitine is absorbed from food, so that even if the carnitine synthesis system is inhibited, there are few side effects. On the other hand, since the carnitine synthesis system is important inside a poorly vascular tumor, inhibition of this can suppress the growth of malignant cells inside the tumor, and it has a selective effect only on the tumor. It becomes possible to provide a therapeutic agent that occurs.

Further, the fatty acid oxidation inhibitor according to the embodiment of the present invention is characterized by being a BBOX1 and / or a BBOX2 inhibitor.
Here, as shown in FIG. 10 of the examples described later, the mechanism of carnitine lowering by 2HG is that 2HG produced by the mutant IDH or a derivative thereof is a carnitine synthetic enzyme BBOX1 and / or BBOX2. This is thought to be due to the inhibition of enzyme activity. Since BBOX1 and / or BBOX2, which are low in expression in normal brain tissue, are highly expressed in glioma, it is highly possible that energy supply by β-oxidation is working for tumor hyperplasia.
Therefore, by targeting each enzyme of BBOX1 and BBOX2 (TMLHE) of the carnitine synthesis system pathway using 2OG as a substrate as a therapeutic target, the same effect as the depletion of 2OG due to the accumulation of 2HG in the tumor of mutant IDH occurs. It is possible to make it. That is, the fatty acid oxidation inhibitor of the present embodiment can be used as a therapeutic agent that suppresses the growth of poor vascular solid tumors by inhibiting the fatty acid oxidation, thereby reducing the supply of acetyl-CoA and ATP.
By inhibiting BBOX1 and 2 or lowering the carnitine concentration in this way, it becomes possible to suppress the tumor even in a glioma having a poor prognosis of normal IDH having no mutation.
Moreover, even if a specific inhibitor of mutant IDH is developed, only an effect on a tumor having mutant IDH can be expected. On the other hand, by therapeutically targeting BBOX1 and 2 as in the present embodiment, the types and types of tumors that can be treated increase.

Further, the therapeutic agent according to the embodiment of the present invention is characterized in that the fatty acid oxidation inhibitor is Meldonium.
Meldonium, which is a structural analog of GBB described in Patent Document 1, is a small molecule compound that inhibits the action of BBOX1. Meldonium functions as a GBB hydrolase-dependent L-carnitine biosynthesis inhibitor as an action of inhibiting carnitine synthesis. It also has the effect of inhibiting the absorption of carnitine. From these actions, Meldonium inhibits fatty acid β-oxidation. In addition, Meldonium has anti-ischemic and neuroprotective effects and can be orally administered. As described above, conventionally, Patent Document 1 has disclosed only the sexual enhancing effect of Meldonium.
In contrast, the present embodiment discloses the use of Meldonium as a therapeutic agent for poorly vascular solid tumors with few side effects. As shown in Examples described later, by using Meldonium, it is possible to inhibit the carnitine synthesis system inside a poorly vascular tumor in which carnitine is exhausted, and it is possible to inhibit fatty acid oxidation and suppress tumor growth. Will be. This allows Meldonium to be used as a therapeutic agent for poorly vascular solid tumors. As described above, Meldonium is commercially available for another purpose and can be expected to have few side effects.
As the fatty acid oxidation inhibitor of the present embodiment, it is possible to use a BBOX1 inhibitor having a similar action, for example, 3,5-Pyridinedicarboxylic Acid, or the like, in addition to Meldonium. In addition, BBOX2 may also be used as an inhibitor by synthesizing a target small molecule compound or the like.

The therapeutic agent according to the embodiment of the present invention is characterized in that the solid tumor is a brain tumor or chondrosarcoma.
The therapeutic agent according to the embodiment of the present invention has, as a target solid tumor, a brain tumor such as a glioma having a normal IDH without mutation and a poor prognosis, and a poor vascularity such as chondrosarcoma. Includes solid tumors.
The therapeutic agent according to the embodiment of the present invention can also be applied to cancers of other solid tumors. For example, the therapeutic agent of the present embodiment includes other brain tumors such as neuroblastoma, other sarcoma, malignant lymphoma, thyroid cancer, head and neck cancer, esophageal cancer, breast cancer, gastric cancer, colon cancer, ovarian cancer, lung cancer, and pancreas. It can also be applied to various solid tumors such as cancer, liver cancer, bile sac cancer, skin cancer, renal cancer, bladder cancer, uterine cancer, testis cancer, and prostate cancer. It is also possible to suppress the infiltration and metastasis of these solid tumors. In addition, the therapeutic agent of the present embodiment may be enhanced by using various tumor angiogenesis inhibitors and the like in combination to suppress angiogenesis around or inside the tumor.

Further, the tumor prognosis prediction method according to the embodiment of the present invention is a tumor prognosis prediction method for an avascular solid tumor, and is a carnitine, acylcarnitine, acetylcarnitine, GBB in a tumor sample of a collected solid tumor. , TML, and a composition comprising a member of the group consisting of metabolites corresponding to fatty acid oxidation, and the prognosis is predicted.
Carnitine, acylcarnitine, acetylcarnitine, GBB, TML, and other fatty acid oxidation-corresponding metabolites (hereinafter, "carnitine and the like") in the biological sample of the poorly vascular solid tumor thus constructed and obtained. By measuring the concentration of the composition of any one of ()) or any combination, it is possible to predict the prognosis and determine the therapeutic effect. That is, it becomes possible to use the measurement of carnitine or the like in a poor vascular solid tumor such as a brain tumor as a prognosis prediction marker. Specifically, the prognosis of the tumor can be identified by measuring the concentration of carnitine or the like in the tumor sample obtained from the brain tumor at the time of surgery. This makes it possible to provide a diagnostic method capable of predicting the prognosis of a tumor and selecting a treatment method by measuring the concentration of carnitine or the like. This concentration may be an absolute concentration, a relative concentration, or a concentration based on a "level" of a specific concentration range.
With this configuration and combination of prognosis prediction methods, it is possible to effectively treat highly malignant tumors and predict the prognosis. In addition, after administration of the drug, the therapeutic effect can be determined using the concentration of carnitine or the like as a guide.

The method for predicting the prognosis of a tumor according to an embodiment of the present invention is characterized in that when the concentration of carnitine in a tumor sample is equal to or higher than a predetermined concentration, it is determined that the prognosis is poor.
Further, in tumor prognosis method according to the embodiment of the present invention, a predetermined concentration, characterized in that it is a 100~190 n mol / g.
As will be described in Examples described later, there is a large difference in the amount of 2HG contained even in a tumor having a good prognosis of mutant IDH, and the lower limit of a tumor having a good prognosis and the upper limit of 2HG of a tumor having a poor prognosis are There may not be a significant difference.
On the other hand, in the tumor prognosis prediction method of the present embodiment, it is possible to easily determine whether or not the prognosis is poor by using a predetermined concentration of carnitine or the like as a threshold value. This predetermined concentration is about 100 to 190 n mol / g, more preferably, it is possible to set a threshold value of about 100~150nmol / g. Thereby, measurement of metabolites showing decreased or activated carnitine and fatty acid oxidation in poor vascular solid tumors can be provided as usable for predicting prognosis.

One of the group consisting of a carnitine synthesis inhibitor, an absorption inhibitor, a decomposition promoter, and a fatty acid β-oxidation inhibitor according to the embodiment of the present invention may be a gene expression regulator.
This gene regulator is intended for proteins (hereinafter referred to as "target proteins") such as BBOX1, BBOX2 (TMLHE), SHMT1, ALDH9A1 of the carnitine synthesis system pathway, and enzymes of the pathway related to fatty acid β-oxidation. Nucleic acids (nucleotides) such as DNA and RNA that regulate transcription and translation, peptides containing antibodies, PNA, nucleic acids and proteins related to genome editing, and the like may be used. In addition to this, various gene regulators for pathways related to carnitine synthesis, absorption and degradation, and pathways related to fatty acid oxidation can be used.
Of these, the nucleic acid may be antisense DNA or RNA, siRNA, shRNA, ribozyme, or the like.
In addition, the gene expression regulator of the present embodiment can be identified from the amino acid sequence, mRNA sequence, genomic DNA sequence, etc. of the target protein and can be produced by a method that is easy for those skilled in the art.

Further, when one of the group consisting of a carnitine synthesis inhibitor, an absorption inhibitor, a decomposition promoter, and a fatty acid β-oxidation inhibitor according to the embodiment of the present invention is a small molecule compound, it is provided as various salts. May be. These salts may be produced by reacting a small molecule compound with an acid or base that can be used in the production of pharmaceutical products.

The therapeutic agent according to the embodiment of the present invention also includes a prodrug. Here, the prodrug means a derivative that is decomposed and converted under specific pH conditions, the action of an enzyme, or the like after administration under physiological conditions. In addition, the prodrug may be inactive when administered to a patient, but is converted into an active therapeutic agent in vivo.

Further, the therapeutic agent according to the embodiment of the present invention can be used as an acceptable carrier in any formulation, for example, physiological saline, glucose, D-sorbitol, D-mannose, D-mannitol, sodium chloride, ethanol, polyalcohol. , For example, it can be administered together with propylene glycol, polyethylene glycol, nonionic surfactant and the like. In addition, the therapeutic agent of the present embodiment may contain an appropriate excipient or the like.

In addition, the therapeutic agent according to the embodiment of the present invention may contain an appropriate pharmaceutically acceptable carrier in order to prepare a pharmaceutically acceptable carrier. The carrier may include biocompatible materials such as silicone, collagen and gelatin.
Further, for example, pharmaceutical additives such as diluents, fragrances, preservatives, excipients, disintegrants, lubricants, binders, emulsifiers and plasticizers may be contained.

In addition, the therapeutic agent according to the embodiment of the present invention may be formulated using a pharmaceutically acceptable carrier known in the art in a dosage form suitable for administration for oral administration.
The route of administration of the pharmaceutical composition according to the embodiment of the present invention is not particularly limited, but it can be administered orally or parenterally.
Parenteral administration includes, for example, intravenous, intraarterial, subcutaneous, intradermal, intramuscular or intraperitoneal administration.
It is also possible to administer parenterally using a drug delivery system such as liposomes or nanocapsules or a viral vector. At this time, depending on the site of the tumor, for example, in the case of a brain tumor, it can be configured to penetrate the blood-brain barrier. Further, as the virus vector, various medical vectors such as AAV (adeno-associated virus vector), adenovirus vector, lentivirus vector, retrovirus vector and the like can be used. Of these, by using AAV, it is possible to realize genome editing and the like specific to the tumor to be treated such as a brain tumor.

In addition, the therapeutic agent according to the embodiment of the present invention can be a therapeutic target for an organism or a part of the body of the organism. This organism is not particularly limited, and includes, for example, animals such as humans, livestock animal species, and wild animals.
At this time, the therapeutic agent according to the embodiment of the present invention can be mainly used for humans and various vertebrates.

Further, in order to use the therapeutic agent according to the embodiment of the present invention for the above-mentioned treatment, the administration interval and the dose should be appropriately selected and changed according to various conditions such as the condition of the tumor and the condition of the subject. Is possible.
Here, in the case of treatment, a dose that slows down the growth of the tumor can be used. In addition, when used in combination with other therapeutic agents, it can be effectively used by suppressing nausea and serious side effects.

The single dose and frequency of administration of the therapeutic agent according to the embodiment of the present invention are appropriately selected according to the purpose of administration and various conditions such as the age and weight of the patient, the symptoms and the severity of the disease. And can be changed.
The number and duration of administration shall be monitored for the condition, and administration may be repeated or repeated depending on the condition.

The therapeutic agent according to the embodiment of the present invention can also be used in combination with other therapeutic compositions such as an anticancer agent. Further, in addition to the therapeutic agent of the present embodiment, a therapeutic composition that relieves pain or improves QOL may be added for treatment. Further, the composition of the present invention may be administered at the same time as other compositions, or may be administered at intervals, but the order of administration is not particularly limited.

Further, in the embodiment of the present invention, the period for which the disease is improved or alleviated is not particularly limited, but may be temporary improvement or alleviation, or may be improvement or alleviation for a certain period.

In the present embodiment, "one or more" means either one or a combination of a plurality of each configuration. That is, any one may be used, and any combination of two to all of these may be included.

Next, the present invention will be further described by way of examples based on the drawings, but the following specific examples do not limit the present invention.

〔experimental method〕
(Cell line)
A cell line U87MG (hereinafter, simply referred to as "U87 strain") of glioblastoma, which is a kind of human glioblastoma, was obtained from ATCC (Virginia, USA) and maintained by a culture method common to those skilled in the art. The U87 strain is a cell having normal IDH1 and IDH2.

(Human tumor tissue)
Tumor tissues of 10 patients who underwent glioma surgery at Jichi Medical University from 2006 to 2012 were obtained. Regarding the use as a clinical sample, it was obtained after informed consent was given to the patient, and it was used with the permission of the Ethics Committee of Jichi Medical University (license number 11-31). In addition, the experiments of this example were carried out in accordance with the GCP guidelines, the ethical code of the Declaration of Helsinki in 1964. Brain tumor samples were obtained at the time of surgery, immediately frozen in liquid nitrogen in the operating room, and stored at -80 ° C until metabolome analysis. Anti-cancer agents that may affect metabolism, such as mercaptopurine and fluorouracil, were not used prior to surgery. After the acquisition, histopathological diagnosis was performed in the Department of Pathology of the University. All tumors were classified according to the WHO classification for tumors of the central nervous system.
FIG. 2 shows an image of MRI FLAIR (Fluid-Attenuated Inversion Recovery) of each tumor tissue before surgery. By the analysis of the detection of the mutation shown below, ^{the brain tumor samples of 5 patients in which IDH1 R132H} , which is a kind of mutant IDH, was detected, and the brain tumor samples of 5 patients of normal IDH (IDH ^WT ) without mutation were analyzed. Used.

(Detection of mutations in IDH1 and IDH2)
DNA was extracted from cells and tumor tissue samples of the U87 strain using the DNeasy Blood & Tissue Kit (manufactured by Qiagen). The DNA fragment of IDH1 or IDH2 was amplified using the existing primers described in Non-Patent Document 2, and the base mutation was confirmed by direct sequencing.

(Plasmid and transfection)
A plasmid of normal IDH1 expressed by the control of the CMV promoter described in Non-Patent Document 3 or IDH1 ^{R132H, which is a kind of mutant IDH1, was used.} These plasmids were transfected into the U87 strain with Lipofectamine 2000 (manufactured by Invitrogen). Expression of IDH1 was confirmed by Western blotting by a method common to those skilled in the art.

(Metabolome analysis)
The cells of the U87 strain cultured in the triple wells were used to inactivate the enzyme 24 hours after transfection, so that a standard solution containing methanol (solution ID: H3304-1002, manufactured by Human Metabolome Technologies, Yamagata Prefecture) was used. It was quenched in Tsuruoka City, Japan). The cells were then scraped and stored at -80 ° C until analyzed.
The cell numbers of the U87 strain transfected for analysis are shown in Table 1 below.

Brain tumor samples were also quenched with 1500 μL acetonitrile and 20 μL D-campohr-10-sulfonic acid as standard solutions and homogenized in ice.
The amount of brain tumor sample used for the analysis is shown in Table 2 below.

(Mass analysis)
(Capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS))
Metabolome measurements and data processing were performed using an Agilent CE-TOFMS (manufactured by Agilent) and a fused silica capillary with an inner diameter of 50 μm x 80 cm. Measurements of the extracted metabolites in positive and negative modes (positive ion mode, negative ion mode) were performed using commercially available electrophoresis buffer.
The alignment of the detected peaks was performed by the mass-to-charge ratio (m / z) values shown in Table 3 below and the standardized migration time.
Glycolysis, pentotholic acid circuit, TCA circuit, purine metabolic pathway, pyrimidine metabolic pathway, nicotinic acid, nicotinamide metabolic pathway, and various amino acid metabolic pathways were analyzed. After that, the present inventors made a relative ratio of these data by a standard amount. Furthermore, the present inventors made measurements to determine the absolute amount of 116 metabolites.

(Liquid Chromatography Time-of-Flight Mass Spectrometry (LC-TOFMS))
Measurements were performed in positive and negative modes using an Agilem 1200 series, Rapid Resolution LC System SL (manufactured by Agilent Technologies) equipped with an Octa Decile Silk analysis column with a diameter of 2 mm × 50 mm and a particle size of 2 μm. Metabolome measurements were performed by the equipment and services of Human Metabolome Technology, Inc. (Tsuruoka, Japan).
This condition is shown in Table 4 below.

(Data processing and analysis)
The detected peaks were automatically extracted using the automatic integration software "MasterHands", version 2.9.0.9 (developed by Keio University). In addition, values of m / z, peak area as peak information, migration time (MT) of CE-TOFMS, and retention time (RT) of LC-TOFMS were obtained. The obtained peak area was converted into a relative peak area by the following equation (1):

Relative peak area = target, peak area / (internal standard standard peak area x sample volume) …… Equation (1)

The detected peaks were compared to all metabolites registered on the Human Metabolome Technology Metabolite Database based on m / z and MT or RT values. Margin of error for search is ± 0 in MT or RT. It was ± 10 ppm at 5 min and m / z. Metabolites in each pathway are shown as a bar graph with the obtained peaks.

(Culture test by adding carnitine to U87 strain)
Cell number, U87 strain 2 × 10 ⁵ (manufactured by ATCC) were wrapped in 21.29cm ² (60-mm diameter) culture dishes. Cells were cultured in D-MEM medium containing 10% dialysis fetal bovine serum (D-FBS) or D-FBS supplemented with 100 μM carnitine.

(Culture test of U87 strain with carnitine synthesis inhibitor added)
Cell number, U87 strain 2 × 10 ⁵ (manufactured by ATCC) were wrapped in 21.29cm ² (60-mm diameter) culture dishes. Cells were cultured in D-MEM medium containing FBS with 10% fetal bovine serum (FBS) or 2 mM Meldonium (trade name: Mildronate®, SIGMA-ALDRICH, Missouri, USA).

〔result〕
(Metabolism profile of mutant IDH and normal IDH glioma)
We used two methods, CE-TOFMS and LC-TOFMS, to transfect and express mutant IDH or normal IDH, a U87 cell line, and a glioma brain tumor sample with or without an IDH mutation. Was analyzed by metabolome.
As a result, we found by CE-TOFMS analysis 82 different metabolites in cation mode and 105 in anion mode, ie a total of 187 different metabolites in cells of the U87 strain. We also found a total of 254 different metabolites in the patient's brain tumor sample, 139 in cation mode and 115 in anion mode.
Furthermore, the present inventors found 41 substance peaks in the cationic mode and 33 in the anionic mode, that is, a total of 74 substance peaks in the U87 cell line by LC-TOFMS analysis. In the patient's brain tumor sample, 70 substance peaks were found in the cation mode and 72 in the anion mode, that is, a total of 142 substance peaks were found.

According to FIG. 3, the inventors then used these metabolome data to draw a heat map.
The heat map of FIG. 3 (a) shows the average of 316 metabolites in the lysates of 5 glioma mutant IDH (IDH mutation) brain tumor samples and normal IDH (IDH normal) brain tumor samples, respectively. show.
The heatmap for the U87 cell line of FIG. 3 (b) shows the metabolism of 216 in lysates of mutant IDH (IDH mutation), normal IDH (IDH normal), and vector-only controls, each performed three times. Shows the average of things.
FIG. 3 (c) is a graph in which PC1 and PC2 are plotted by principal component analysis for a brain tumor sample and FIG. 3 (d) is a U87 cell line. It can be seen that each is separated.

(Metabolism of the TCA cycle is suppressed in cells and tissues with IDH mutations)
According to FIG. 4A, metabolic analysis of the TCA cycle in mutant IDH (IDH mutation) and normal IDH (IDH normal) brain tumor samples shows that 2OG and its downstream intermediate metabolites in the TCA cycle. Production was curtailed. The U87 cell line also showed the same tendency by expressing ^{IDH1 R132H} , which is a kind of mutant IDH.
According to FIG. 4 (b), the amount of 2HG was significantly increased in the mutant IDH brain tumor sample and the U87 cell line expressing the mutant IDH1. IDH1 ^R132H is known to produce 2HG from 2OG in the cytoplasm. Therefore, in a glioma with an IDH1 mutation, 2HG is actually accumulated. 2HG is expected to competitively suppress α-keto acid transaminase, which reduces the amount of 2OG and results in suppression of the TCA cycle.
These results clearly show that the sample preparation method and metabolome data from the clinical brain tumor samples in this example are very reliable.

(Mutant IDH glioma reduces nucleic acid and ATP production)
According to FIG. 5, in a brain tumor sample of a patient with mutant IDH in a purine production pathway corresponding to nucleic acid production such as ATP, ADP, and AMP, which is closely related to energy production, the amount of ATP is higher than that of normal IDH. It was significantly reduced. In brain tumor samples of patients with mutant IDH, the amount of ATP was significantly reduced compared to normal IDH in the purine production pathway corresponding to nucleic acid production such as ATP, ADP, and AMP, which are closely related to energy production. rice field. In addition, GTP and PRPP (phosphoribosyl pyrophosphate, phosphoribosyl diphosphate) were also significantly reduced in the mutant IDH1 brain tumor sample. Further, even in the U87 cell line expressing the mutant IDH1, a similar decreasing tendency was shown. This indicates that reduced ATP production actually occurs in both spontaneous tumors and cultured cells expressing mutant IDH.
According to FIG. 6, the adenilate energy sufficiency rate (AEC) and the total adenylate, which are indicators of the energy state in the cell, were calculated by the following equations (2) and (3):

AEC = {(ATP) +0.5 * (ADP)} / {(ATP) + (ADP) + (AMP)} …… Equation (2)
Total adenirate = {(ATP) + (ADP) + (AMP)} …… Equation (3)

AEC and total adenirate were reduced in brain tumor samples of patients with mutant IDH.
However, there was no significant difference in AEC in the cultured cells of the U87 strain. However, total adenilate was slightly reduced in U87 cell lines transfected with mutant IDH.

(Amino acid metabolism is suppressed in mutant IDH brain tumor samples and cells)
Interestingly, according to FIG. 7, all 20 amino acids were reduced in the mutant IDH1 brain tumor sample. This decrease was similar in U87 cell lines transfected with mutant IDH1. Also, these results were more pronounced in brain tumor samples than in the U87 cell line.
Non-essential amino acids are produced by intermediate metabolites of glycolysis and the TCA cycle, including 2OG, pyruvate, acetyl-CoA, fumaric acid, succinyl-CoA, and oxaloacetate. That is, the production of non-essential amino acids is significantly affected by metabolism in the TCA cycle. In addition, essential amino acids are considered to have decreased due to the promotion of consumption. Therefore, it is considered that the amounts of all amino acids were reduced in the mutant IDH1 brain tumor sample and the U87 cell line.

(Fatty acid metabolism is reduced in mutant IDH brain tumor samples)
Β-oxidation will be described with reference to the conceptual diagram of FIG. 8 (a). Beta-oxidation is an important metabolic pathway that produces acetyl-CoA by oxidizing fatty acids and extracts energy. In β-oxidation, first, fatty acids in the cytoplasm bind to CoA to produce acyl-CoA. Acyl-CoA binds to carnitine and is converted to acyl-carnitine and CoA. After that, acylcarnitine passes through the mitochondrial membrane, becomes acyl-CoA again in the mitochondria, and is decomposed to acetyl-CoA by β-oxidation. Acetyl-CoA is then used as a substrate in the TCA cycle and other metabolic reactions.
FIG. 8 (b) shows the amount of carnitine in a brain tumor sample. The vertical axis shows the absolute amount (nmol / g). Interestingly, the amount of carnitine in the mutant IDH brain tumor sample was significantly reduced.
FIG. 8 (c) shows the amounts of acylcarnitines having different carbon numbers and double bond numbers. In this table, the horizontal axis indicates the number of double chains and the vertical axis indicates the number of carbon atoms. In each graph, the left side shows the relative amount of mutant IDH1 and the right side shows the relative amount of normal IDH1 in a brain tumor sample. As shown in each graph, as a result of the decrease in the amount of carnitine, the amount of acylcarnitine was all decreased in the brain tumor sample of mutant IDH1.
FIG. 8D is a graph showing the amount of fatty acid. The vertical axis shows the absolute amount (nmol / g). Also in this table, the horizontal axis shows the number of double chains and the vertical axis shows the number of carbon atoms. In each graph, the left side shows the relative amount of the mutant IDH1 and the right side shows the relative amount of the normal IDH1. The amount of fatty acid itself was not significantly different between the mutant IDH and normal IDH brain tumor samples.
As a result, in brain tumor samples, carnitine and acylcarnitine are significantly reduced in the group of mutant IDHs, indicating that β-oxidation is suppressed. Compared to other tissues, beta-oxidation was generally thought to play a less important role in the brain. However, in mutant IDH1 in which β-oxidation is greatly suppressed, the ATP level is significantly lower than that in normal IDH1, so that β-oxidation functions efficiently for ATP supply during tumor growth. Was suggested.

(Fatty acid metabolism does not change in cultured cells of mutant IDH1)
FIG. 9 (a) shows the amount of carnitine in a U87 cell line transfected with mutant IDH1, normal IDH1, and vector alone. In the U87 cell line expressing the mutant IDH1, the carnitine concentration tended to decrease, but no significant decrease was observed.
FIG. 9B shows the amounts of acylcarnitine in the U87 cell line with different carbon numbers and double bond numbers. The amount of acylcarnitine was not significantly different between the mutant IDH1 and the normal IDH1, but rather increased in the mutant IDH1.
FIG. 9 (c) is a graph showing the amount of fatty acid in the U87 cell line. There was also no significant difference in the amount of each fatty acid.
It is considered that these results are due to the fact that carnitine is originally contained in the serum of the culture medium and is supplied intracellularly from the outside, as will be described later.

(Conclusion of metabolome analysis)
Comprehensive metabolome analysis was performed using the above-mentioned mutant IDH1 brain tumor sample and normal IDH1 brain tumor sample, and a comparative study revealed that the fatty acid metabolism pathway was inhibited in the glioma of mutant IDH with a good prognosis. It was revealed that the decrease in carnitine was particularly remarkable.
In general, carnitine maintains homeostasis when absorbed from food. However, the carnitine synthesis system is important for the growth of poor vascular tumors such as glioma, and it is presumed that carnitine in the glioma is not supplemented by diet. Therefore, in the tumor tissue of glioma in the living body, it was important to supply acetyl-CoA and ATP by oxidation of fatty acid using carnitine synthesized in the tissue.

In the carnitine synthesis pathway shown in FIG. 10, the present inventors are affected by the accumulation of 2HG by the mutant IDH, which are carnitine synthases using 2OG as a substrate, BBOX1 (gamma-butyrobetaine dioxygenase), and BBOX2 (BBOX2). It was presumed to be TMLHE (trimethyllysine dioxygenase). It is known that these proteins are highly expressed in glioma with respect to normal brain tissue.

(Therapeutic effect of brain tumor)
According to FIG. 11, using the cells of the U87 strain, it was first confirmed that carnitine is important for cell proliferation. The boxes in the figure indicate the average, and the bars indicate the standard error (n = 3). The symbol (*) indicates a significant difference (p <0.05).
As shown in FIG. 11, the cell proliferation rate was low under the culture conditions using carnitine-free dialysis serum (D-FBS). On the other hand, it was revealed that the cell proliferation rate was increased in the medium (D-FBS + Car) in which 100 μM carnitine was added to the dialysis serum.

According to FIG. 12, Meldonium, which is an inhibitor of BBOX1 which is a carnitine synthase and an inhibitor of intracellular transport of carnitine, inhibited the growth of the U87 cell line. The boxes in the figure indicate the average, and the bars indicate the standard error (n = 3). The symbol (*) indicates a significant difference (p <0.05).
The cells proliferated well under the culture conditions using the normal serum (FBS) containing carnitine, whereas the cell growth was suppressed under the culture conditions in which 2 mM Meldonium was added to the normal serum (FBS + Mildr).

In conclusion, it was shown that carnitine is required even for the proliferation of normal IDH glioma-derived cells, and that treatment with carnitine synthesis or transport inhibitors suppresses the proliferation of tumor cells.

(Prediction of prognosis of brain tumor and judgment of therapeutic effect)
According to FIG. 13, the clinical prognosis can be predicted by measuring the carnitine concentration from the tissue sample at the time of tumor removal surgery.
FIG. 13 (a) shows the results of measuring the carnitine concentration in the brain tumor sample. The unit is the concentration of nmol per tumor tissue weight (g). In the brain tumor sample (n = 5) of mutant IDH1 (MT) having a good prognosis, the carnitine concentration was 19.8 to 61.2 nmol / g, and the average was 36 nmol / g. On the other hand, in the brain tumor sample (n = 5) of normal IDH1 (NT) having a poor prognosis, it was 137.2 to 329.2 nmol / g, and the average was 267 nmol / g. That is, it was shown that the tumor tissue having a poor prognosis had a higher carnitine concentration than the tumor tissue having a good prognosis. The significant difference in t-test between mutant IDH1 and normal IDH1 was p <0.003. As a result, if the carnitine concentration greatly exceeds 130 nmol / g, it can be presumed that the prognosis is poor.
On the other hand, FIG. 13 (b) shows the results of measuring the 2HG concentration in the same brain tumor sample. Thus, in the mutant IDH1 (MT) brain tumor samples, they were 68.8, 555.9, 890.0, 62.6, 1390.0 nmol / g, respectively, and the average was 593 nmol / g. .. In the normal IDH1 (NT) brain tumor samples, the values were 20.5, 17.8, 14.8, 31.5, and 46.9 nmol / g, respectively, and the average was 26.3 nmol / g. As described above, 2HG may have a large variation in the tumor tissue of the mutant IDH1 and a small difference from the normal type. In fact, the tests of these samples showed no significant difference at p = 0.088494.

Here, in Table 5 below, CE- in the brain tumor samples of mutant IDH1 (MT) (“AMT” to “EMT”) and the brain tumor samples of normal IDH1 (NT) (“FNT” to “JNT”). The results of carnitine, acetylcarnitine (O-Acetylcarnitine), and GBB (γ-Butyrobetaine), which were subjected to relative quantification by TOFMS, are shown.

"Standardized Reactive Area" in the table indicates the place where there was a change, and the value between "EMT" and "FNT" among the places "AMT" to "JNT" of the relative area value of the set peak. Was converting. That is, when the brain tumor sample of mutant IDH1 (MT) and the brain tumor sample of normal IDH1 (NT) were compared by relative quantification, the opposite correlation was shown for all the compounds. In addition, all the compounds had p <0.01 or less in the t-test.

From the above results, it was shown that the quantification of carnitine in the glioma tissue is an index for determining the prognosis. It was also shown that acetylcarnitine and GBB are also candidates for prognostic indicators. This made it possible to predict the prognosis of brain tumors, or to treat gliomas with poor prognosis without mutations in isocitrate dehydrogenase (IDH), for which there was no effective means, and to determine the therapeutic effect.
Preliminary experiments have shown that acylcarnitine and TML are also indicators of prognosis.

It is needless to say that the configuration and operation of the above-described embodiment are examples and can be appropriately modified and executed without departing from the spirit of the present invention.

The therapeutic agent of the present invention can provide a therapeutic agent having a new mechanism for a poor vascular poor vascular solid tumor, and can be used industrially.

Claims (1)

A therapeutic agent for poorly vascular solid tumors
Only contains the fatty acid oxidation inhibitors,
The solid tumor is a glioma and
The fatty acid oxidation inhibitor is a therapeutic agent characterized by being Meldonium.

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