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Markers associated with wnt inhibitors

一种抑制剂、生物标记物的技术，应用在药物组合、生物材料分析、生物化学设备和方法等方向，能够解决靶向Wnt信号传导成功受限制、缺乏有效治疗剂、缺乏敏感的确定的患者群体等问题

Active Publication Date: 2015-12-23
NOVARTIS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far success in targeting Wnt signaling therapeutically has been limited
This is largely due to the lack of effective therapeutics for targets in the Wnt pathway and the lack of established patient populations that would be sensitive to Wnt inhibitors

Method used

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  • Markers associated with wnt inhibitors
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  • Markers associated with wnt inhibitors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0248] Example 1: Compound A and Compound B (respectively figure 1 A and 1B) are effective PORCN inhibitors in biochemical and cellular testing.

[0249] Radioligand binding detection: Membrane preparation: Fugene6 (Roche), using pcDNA3.1 structure with human PORCN (Invitrogen) to transfect about 10 8 293 cells. After 48 hours, cells were harvested by scraping in PBS and centrifuged at 1,000×g for 10 minutes. Aspirate the buffer. The cell pellets were frozen in a dry ice bath, and then gently resuspended in 10 ml of 50 mM Tris pH 7.5, 250 mM sucrose buffer (Sigma) containing an EDTA-free protease inhibitor mixture. Cells were lysed using polytron (Brinkman). The lysed cells were centrifuged at 1,600×g for 20 minutes at 4°C, the supernatant was transferred and centrifuged at 20,000 rpm in an SS34 rotor at 4°C for 20 minutes. Discard the supernatant and use three 10-second pulses using Polytron to resuspend these pellets in 10% sucrose, 50mM TrispH7.5, 5mM MgCl 2 , 1mMEDTA solut...

Embodiment 2

[0255] Example 2: Inhibition of PORCN by compound A blocks Wnt signaling in vitro

[0256] Reporter gene detection: at 37°C and 5% CO 2 In an air atmosphere, mouse Leydig cells TM3 cells (obtained from American Type Culture Collection, ATCC, Manassas, Virginia) were cultured in 2.5% FBS ( Gibco / Invitrogen, Carlsbad, California) and 5% horse serum (Gibco / Invitrogen, Carlsbad, California), 50 units / mL penicillin and 50μg / mL streptomycin (Gibco / Invitrogen, Carlsbad, California) Ham's F12 medium and Dulbecco's modified Eagle's medium (Gibco / Invitrogen, Carlsbad, California) in a 1:1 mixture. The TM3 cells in a 10cm culture dish are made with 8μg STF-reporter plasmid containing luciferase gene driven by Wnt responsive elements and 2μgpcDNA3.1-Neo (Gibco / Invitrogen, Cal Spard) and 30μLFuGENE6 (RocheDiagnostics, Indianapolis, Indiana) co-transfected. The stable cell line (TM3Wnt-Luc) was screened by 400 μg / mL G418 (Gibco / Invitrogen, Carlsbad, California). At 37°C with 5% CO 2 In an a...

Embodiment 3

[0258] Example 3: Cellular functional effects of Wnt inhibitors in human head and neck cancer cell lines

[0259] In order to identify human cancer cell lines that respond to porcupine suppression, we used the mRNA expression level of AXIN2 as a read-out to characterize more than 300 cell lines. At 37°C with 5% CO 2 Cultivate all cell lines in the humidified incubator. HN30 cells (Wayne State University) and UMSCC cells (University of Michigan) are derived from human head and neck squamous cell carcinoma (HNSCC) patient tumor samples. Separately, using QiagenRNeasy and DNeasy blood and tissue kits to isolate total RNA or DNA in accordance with the manufacturer's instructions. In short, the cells were disrupted by adding buffer RLT and homogenized using a QIAshredder rotating column. After adding a volume of 70% ethanol to homogenize the lysate and mixing thoroughly, the samples were transferred to the RNeasy spin column. Discard the overflow after centrifugation. After washin...

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Abstract

The invention provides methods of monitoring differential gene expression of biomarkers to determine patient sensitivity to Wnt inhibitor, methods of determining the sensitivity of a cell to an Wnt inhibitor by measuring biomarkers, methods of screening for candidate Wnt inhibitor, Wnt inhibitor for use in head and neck squamous cell carcinoma.

Description

Technical field [0001] The present invention relates to the field of pharmacogenomics, and can be used to determine patient sensitivity before treatment, subsequent patient response after treatment, cancer sensitivity, screening compounds, treatment methods, and biomarkers of pharmaceutical compositions used in the treatment use. Background technique [0002] Wnt signaling is an important oncogenic pathway in many cancers 1、2 . When binding to its receptors (low density lipoprotein receptor related protein 5 / 6 (LRP5 / 6) at the plasma membrane (LRP5 / 6) and frizzled protein (FZD) (both are single-channel transmembrane receptors required for Wnt signaling) After the body)), the Wnt ligand can trigger the interruption of the β-catenin degradation mechanism composed of Axin2, GSK3β, APC and other proteins, which leads to the accumulation of β-catenin in the cytoplasm 3 . The increased concentration of β-catenin will eventually lead to its translocation into the nucleus to form a comp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574A61P35/00
CPCA61K31/444A61K31/497A61K31/501G01N33/57407G01N33/57484G01N2800/52A61P35/00C12Q1/6804C12Q1/6844C12Q1/6858C12Q1/686C12Q1/6874C12Q1/6881C12Q1/6886G01N33/574C12Q2600/106C12Q2600/156C12Q2600/158
Inventor J·车J·哈里斯H·谢李杰刘峻N·吴
Owner NOVARTIS AG
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