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Methods for detecting lp-pla2 activity and inhibition of lp-pla2 activity

a technology of lppla2 activity and inhibition, applied in the field of methods for detecting lppla2 activity and inhibition of lppla2 activity, can solve the problems of reducing the risk of heart disease and morbid coronary events, limiting blood flow, and blocking the artery

Inactive Publication Date: 2007-07-19
GLAXO GRP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides methods for determining the inhibition of Lp-PLA2 enzyme activity in tissue samples from animals that have been administered with Lp-PLA2 inhibitor. The methods involve preparing a solution containing a substrate for Lp-PLA2, contacting the tissue sample with the solution, and detecting Lp-PLA2 activity. The invention allows for the reliable and accurate measurement of Lp-PLA2 activity in tissue samples, which can aid in the development of effective treatments for various diseases associated with Lp-PLA2 activity.

Problems solved by technology

This build-up is associated with an increased risk of heart disease and morbid coronary events.
The growth of such lesions can eventually block the artery and restrict blood flow.
However, the methods provided with the Auto PAF AH assay are insensitive to measuring inhibition of Lp-PLA2 activity when an inhibitor of Lp-PLA2 has been administered to an animal prior to tissue sample collection.

Method used

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  • Methods for detecting lp-pla2 activity and inhibition of lp-pla2 activity
  • Methods for detecting lp-pla2 activity and inhibition of lp-pla2 activity
  • Methods for detecting lp-pla2 activity and inhibition of lp-pla2 activity

Examples

Experimental program
Comparison scheme
Effect test

example 1

The Auto PAF AH Assay Kit

[0070] The Auto PAF AH assay kit, manufactured by Azwell (Osaka, Japan), is commercially available in the United States through Karlan Research Products Corporation (Santa Rosa, Calif.). This assay was evaluated on an Olympus Au640 clinical chemistry analyzer and is described in this Example 1.

Materials

Azwell Auto PAF-AH Assay Kit:

[0071] R1: 200 mM HEPES, 200 mM NaCl, 5 mM EDTA, 10 mM CHAPS, 10 mM sodium 1-nonanesulfonate, pH 7.6

[0072] R2A: 20 mM citric acid monohydrate, 10 mM sodium 1-nonanesulfonate, pH 4.5

[0073] R2B: 90 mM 1-myristoryl-2-(4-nitrophenylsuccinyl) phosphatidylcholine

Assay Procedure

[0074] 1. Enter assay parameters from the following table into the Olympus Au640 analyzer, and create a PAF AH assay program: [0075] Sample volume: 2 μL [0076] Reagent 1: 240 μL [0077] Reagent 2: 80 μL [0078] Wavelength (main): 410 nm [0079] Wavelength (sub): 480 nm [0080] Method: Rate [0081] Point 1 (FST): 14 [0082] Point 1 (LST): 21 [0083] Calibration...

example 2

High Throughput Radiometric Assay for Measurement of Lp-PLA2 Activity

[0097] A high throughput radiometric assay was developed for measuring Lp-PLA2 activity in a sample. This assay is fully described in WO2005 / 001416. A summary of a high throughput radiometric activity assay is provided in this Example 2.

[0098] Equipment

Scintillation CounterTopCount Microplate Scintillation andLuminescence Counter, Perkin-Elmer (formerlyPackard), CACentrifugeAllegra 25R benchtop centrifuge, BeckmanCoulter, CAPlate shakerLab-Line Titer Plate Shaker (VWR cat #57019-600)OvenBarnstead / Thermolyne, series 9000, temperaturerange 10-250° C. (VWR cat#52205-065)12-channel PipettorsBRAND Transferpette ®-12, BrandTechScientific, Inc., Essex, CT

[0099] Material

PolypropyleneCostar* Brand 96-Well Plates, Polypropylene,PlatesNonsterile, Without Lids, Costar 3365, Corning, Inc.,Corning, NY (VWR cat #29444-104)PicoPlate96-Well white solvent-resistant microplates, PerkinPlatesElmer Life Sciences, Inc, Boston, MA...

example 3

Correlation of Auto PAF AH assay and High Throughput Radiometric Assay

[0133] A panel of 120 plasma samples from healthy human volunteers was assayed for Lp-PLA2 activity at three clinics using the high-throughput radiometric assay described in Example 2. The same sample panel was assayed using Azwell's Auto PAF AH assay described in Example 1 on the Olympus Au640 analyzer. Correlation was obtained against data generated on the same panel of samples by the high throughput radiometric assay. Correlation coefficients (r) were 0.96, 0.94, and 0.95 for Auto PAF AH vs. the radiometric activity assay at the three clinics, respectively. The average CV between duplicates was 2.14% for the Auto PAF AH assay.

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Abstract

This invention relates to methods for determining the activity of Lp-PLA2 in at least one sample from an animal. The invention also relates to methods for determining the inhibition of Lp-PLA2 activity in samples from animals that are administered an Lp-PLA2 inhibitor.

Description

CROSS-REFERENCE TO PREVIOUS APPLICATION [0001] This application claims the benefit of U.S. Provisional Application No. 60 / 563,078, filed Apr. 16, 2004.FIELD OF THE INVENTION [0002] This invention relates generally to methods and materials for determining lipoprotein-associated phospholipase A2 (herein “Lp-PLA2”) enzyme activity and inhibition of activity in tissue samples from animals. BACKGROUND OF THE INVENTION [0003] Coronary heart disease (herein “CHD”) is the leading cause of death in many industrial countries. Atherosclerosis is a form of arteriosclerosis or hardening of the arteries in which there is the progressive build-up of plaque containing cholesterol and lipids in blood arteries. This build-up is associated with an increased risk of heart disease and morbid coronary events. The build-up of plaque in the arteries is associated with an immune response that is triggered by damage to the endothelium. Initially, monocyte-derived macrophages accumulate at the damaged site, d...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/48C12Q1/34C12Q1/44C12Q1/61
CPCC12Q1/34C12Q1/44G01N2333/918C12Q2334/10C12Q1/61
Inventor SHOU, YAPINGSIU, YIN-FAIWALKER, GEORGE
Owner GLAXO GRP LTD
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