Inhibitors of branched-chain-aminotransferase-1 (BCAT1) for the treatment of brain tumors

A branched chain amino, brain tumor technology, applied in the field of compounds in one method, capable of solving problems ranging from months to 16 months

Inactive Publication Date: 2013-10-30
DEUTES KREBSFORSCHUNGSZENT STIFTUNG DES OFFENTLICHEN RECHTS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This means that despite these treatments, the average lifespan after a cancer diagnosis is only 12 to 16 months

Method used

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  • Inhibitors of branched-chain-aminotransferase-1 (BCAT1) for the treatment of brain tumors
  • Inhibitors of branched-chain-aminotransferase-1 (BCAT1) for the treatment of brain tumors
  • Inhibitors of branched-chain-aminotransferase-1 (BCAT1) for the treatment of brain tumors

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] general method

[0082] (a) RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)

[0083] Total RNA was extracted using the AllPrep DNA / RNA / Protein Mini Kit (Qiagen) according to the manufacturer's instructions. from Ambion The human brain was referenced to total RNA as a pool of normal brain RNA molecules (n=23 donors). Total RNA (500 ng) was reverse transcribed using random primers and superscript II (reverse transcriptase) (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. Each complementary protein was analyzed in triplicate using the Applied Biosystems Prism7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with Absolute SYBR Green ROX Mix (ABgene, Epsom, UK). DNA samples. Relative amounts of specific mRNAs were normalized to ARF, B2M and TBP. Primer sequences are given in Table 1A below.

[0084] Table 1A

[0085]

[0086] (b) Western blot analysis

[0087] All experiments invo...

Embodiment 2

[0131] Inhibition of cell proliferation by gabapentin

[0132] Human glioblastoma cell lines U87-MG (HTB-14), HS683 (HTB-138) and U373-MG (HTB-17; LGC standard, Teddington TW110LY, UK) were cultured in supplemented with 10% fetal Dulbecco's minimum essential medium with bovine serum and 1% penicillin / streptomycin mixture. Seed at 5x104 cells / well in a total volume of 500 µL of cell culture medium in a 24-well plate. At 4 hours after seeding the cells, the medium was removed, and then 500 μL of 1-(aminomethyl)-cyclohexaneacetic acid (cyclohexylessigsaure) (gabapentin, dissolved at 500 mM in 200 mM HEPES buffer) for medium replacement. A corresponding volume of 200 mM HEPES was added to each control well. At 5 h after the medium exchange, 5-ethynyl-2'deoxyuridine (EdU; Carlsbad, CA92008, USA) was added to utilize Invitrogens (Carlsbad, CA92008, USA) The proliferation kit measures cell proliferation. Sixteen hours after EdU application, cells were harvested and proliferatio...

Embodiment 3

[0134] Inhibition of cell proliferation by BCAT1 antisense oligonucleotides

[0135] Lentivirus was produced by co-transfection of HEK293T cells with psPAX2 (Addgene12260, Didier Trono, packaging vector), pMD2.G (Addgene12259, Didier Trono, envelope plasmid) and pLKO.1shRNA construct (Sigma-Aldrich, St. Louis, MO63178, USA) carrier. use -LT1 (Mirus Bio LLC, Madison, WI 53711, USA) was transfected, and virus was obtained 48 hours and 72 hours after transfection. Two separate shRNA constructs targeting different regions of the BCAT1 mRNA transcript were used: MISSION shRNA TRCN0000005907NM_005504.3-1064slcl (shRNA1) and TRCN0000005909NM_005504.3-751slcl (shRNA2); both Sigma-Aldrich, St. Louis, MO63178, USA. Non-targeting shRNA was used as a control.

[0136] Human glioblastoma cell lines U87-MG (HTB-14), HS683 (HTB-138) and U373-MG (HTB-17; LGC standard, Teddington TW110LY, UK) were seeded in 24-well plates (5x10 ^4 cells / well) in a total volume of 500 μL of cell culture me...

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Abstract

Described is a compound capable of reducing or inhibiting (a) the biological activity of branched-chain-aminotransferase-1 (BCAT1) or (b) the expression of the gene encoding BCAT1 for use in a method of treating a neoplasia. A preferred compound is 1-(aminomethyl)cyclohexaneacetic acid (gabapentin).

Description

technical field [0001] The present invention provides a compound capable of reducing or inhibiting (a) the biological activity of branched chain aminotransferase-1 (BCAT1) or (b) the expression of a gene encoding BCAT1 for use in a method of treating brain tumors. Background technique [0002] Among human malignant brain tumors, malignant human glioblastoma accounts for the largest number. To date, treatment of gliomas has included neurosurgical techniques (resection or stereotaxic surgery), radiation therapy, and chemotherapy. For example, the current standard of care for astrocytic tumors includes surgical tumor resection, which can be followed by chemotherapy and radiation therapy with the oral alkylating agent temozolomide (TMZ). However, glioblastoma is considered incurable because glioblastoma does not respond to ionizing radiation, chemotherapy, and surgical resection. In other words, the use of these therapies can only prolong the patient's life only very limitedly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/7088A61K31/00A61P35/00
CPCC12Y206/01042A61K31/00C12N2310/14C12N15/1137A61K31/7088A61K39/39558A61K31/713C12N2310/531C12N15/113C12N2310/11A61K31/196A61K31/195A61P25/00A61P35/00A61P43/00A61K38/45C07K16/40
Inventor B·雷德尔维默M·图尼杰斯S·巴布斯P·利希特尔
Owner DEUTES KREBSFORSCHUNGSZENT STIFTUNG DES OFFENTLICHEN RECHTS
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