Interrogatory cell-based assays for indentifying drug-induced toxicity markers

A technology of biomarkers and drugs, which can be used in drug combination, microbe measurement/testing, antiviral agents, etc., and can solve complex problems

Inactive Publication Date: 2015-04-01
BERG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

QTc prolongation is a very powerful but complex marker
However, it is difficult to make stop or continue decisions in early drug development based on QTc alone

Method used

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  • Interrogatory cell-based assays for indentifying drug-induced toxicity markers
  • Interrogatory cell-based assays for indentifying drug-induced toxicity markers
  • Interrogatory cell-based assays for indentifying drug-induced toxicity markers

Examples

Experimental program
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Effect test

preparation example Construction

[0264] Preparation of secretome samples: 1) In one embodiment, the cells can be cultured in a serum-free medium: the conditioned medium can be concentrated by a lyophilizer, reduced (10 mM dithiothreitol (DTT), 55° C., 1 hour), alkylation (25 mM iodoacetamide, at room temperature, incubation for 30 minutes) and then desalted by acetone precipitation. Equal amounts of protein from concentrated conditioned media can be digested with trypsin (1:25 w / w, 200 mM triethylammonium bicarbonate (TEAB), 37°C, 16 hours).

[0265] In one embodiment, cells can be cultured in serum-containing medium: the volume of the medium can be reduced using 3kMWCO Vivaspin columns (GE Healthcare Life Sciences) and then can be reconstituted with IxPBS (Invitrogen). Serum albumin can be depleted from all samples using AlbuVoid columns (Biotech Support Group, LLC) with buffer exchange modified to optimize conditioned medium application following the manufacturer's instructions.

[0266] iTRAQ 8-plex label...

Embodiment 1

[0595] Example 1: Establishment of a drug-induced cardiotoxicity model using platform technology

[0596] In this example, the platform technology described in detail in International PCT Application No. PCT / US2012 / 027615 was employed to integrate data obtained from custom drug-induced cardiotoxicity models and to identify novel mechanisms driving pathogenesis / drug cardiotoxicity. protein / pathway. The relationship map resulting from this analysis has provided biomarkers of drug-induced cardiotoxicity.

[0597] In a healthy heart, systolic function depends on the balance of fatty acid and carbohydrate oxidation. Chronic dysregulation of uptake, utilization, organelle biosynthesis and secretion in non-adipose tissues (heart and liver) is thought to be central to mitochondrial damage and dysfunction and a key player in drug-induced cardiotoxicity. Applicants describe here a systematic approach combining protein and lipid imprinting with functional endpoint analysis focused spec...

Embodiment 2

[0615] Example 2: Identification of additional cardiotoxicity markers using a drug-induced cardiotoxicity model

[0616] Further data obtained from the same custom cardiotoxicity model were similarly integrated using the platform technology described in Example 1 above. Cardiotoxicity was modeled using five patient cardiomyocyte lines, as explained in the detailed description above. These five cardiomyocyte lines were then subjected to mitochondrial ATP analysis to analyze mitochondrial dysfunction induced by drug treatment or the absence of drug treatment (indicated as + and −) in diabetic state (hyperglycemia) and normoglycemia (normoglycemia). A decrease in mitochondrial ATP was observed in the diabetic state upon drug treatment only in 2 of 5 cardiotoxicity models (see Figure 30 ). The results of these further experiments led to the identification of additional novel proteins / pathways driving the pathology of drug cardiotoxicity, such as Figure 26-34 summarized in.

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Abstract

Described herein is a discovery Platform Technology for analyzing a drug-induced toxicity condition, such as cardiotoxicity via model building.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application Serial No. 61 / 650462, filed May 22, 2012, which is hereby incorporated by reference in its entirety. Background of the invention [0003] The pharmaceutical industry currently indicates a wastage of 90% of potential compounds entering clinical development, 30% of which is due to poor clinical safety (Kola et al., (2004) Nat Rev Drug Discovery: 3711-715). Fatal adverse drug reactions (ADRs) are the 4th to 6th leading cause of death in the United States. Costs directly attributable to ADRs can result in an additional $1.56 billion to $4 billion in direct hospital costs annually in the United States (Lazarou J et al., (1998) JAMA; 279(15):1200-1225). The cost of drug discovery and development has risen to about $1 billion, partly due to increased compound and NME depletion late in clinical development (Adams CP, Brantner VV (2010) "Spending on New Drug Developm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/48G01N33/53G06F19/12G16B5/00G16B20/20G16B20/50
CPCG01N33/68G16B5/00G16B20/20G16B20/50C12Q1/6883C12Q2600/142C12Q2600/158G16B20/00A61P39/00A61P39/02A61P9/00A61P9/04A61P9/06C12Q1/6837
Inventor N·R·纳莱恩R·萨兰加拉詹V·K·维施努达斯
Owner BERG
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