WO2013045429A1

WO2013045429A1 - Epr method for free radicals detection using at least two different spin trapping compounds

Info

Publication number: WO2013045429A1
Application number: PCT/EP2012/068840
Authority: WO
Inventors: Bernard Gallez; Philippe Leveque; Valérie MARCHAND
Original assignee: Universite Catholique de Louvain UCL
Current assignee: Universite Catholique de Louvain UCL
Priority date: 2011-09-26
Filing date: 2012-09-25
Publication date: 2013-04-04
Anticipated expiration: 2014-03-26

Abstract

The present invention provides a method, a kit and their use for free radicals detection and identification in a sample. The method comprising the steps of: reacting said sample with a spin trapping composition, to produce spin adducts of said spin trapping composition and free radicals and detecting said spin adducts by electron paramagnetic resonance (EPR) spectroscopy or electron spin resonance (ESR), thereby obtaining an EPR signal. The method is characterized in that said spin trapping composition comprises at least two different spin trapping compounds.

Description

EPR METHOD FOR FREE RADICALS DETECTION USING AT LEAST TWO DIFFERENT SPIN TRAPPING COMPOUNDS

FIELD OF THE INVENTION

The present invention relates to the field of free radicals detection. More in particular, the present invention relates to the field of free radicals detection and identification in biological systems. BACKGROUND

Reactive oxygen species (ROS), including free radicals, are considered as deleterious agents involved in the genesis of pathologies, but are also important mediators in a range of important biological and physiological processes. It is well established that many chemicals exert their effect on living cells and tissues through a mechanism involving, at least at one step, the formation of radical species centered on various atoms (O, C, N, S).

The research field on oxidative stress, oxidative damage, redox biology and antioxidants, is the subject of many controversies. Generally, it has been considered that the shortcomings published in the literature are coming either from the misidentification of the ROS involved in a process, either from the debatable causal link with the biological consequence. Hence, the claim for the occurrence of an oxidative stress in tissues is sometimes based on the use of end products of a sequence of events or poorly validated biomarkers, rendering any conclusion debatable. Different techniques are available to detect radicals in biological media. Fluorescence based methods are highly popular, widely available as commercial kits, but also widely criticized so that results should be used with caution. This has stimulated recent research for new probes specific for a particular species or accumulating in particular cell compartment. The term "ROS" encompass a different range of compounds including both free radicals and other non-radical reactive species. To identify unambiguously the presence of a free radical, the method of choice is Electron Paramagnetic Resonance (EPR) spectroscopy using spin-trapping experiments. The trapping of a short-lived free radical with a spin trap to generate a persistent spin adduct which could be characterized by its EPR spectrum constitutes the well-known spin trapping technique. EPR is a magnetic resonance based method that detects only species with unpaired electrons. To detect a short-lived free radical, a spin-trap (generally, a nitrone) is added to a system in order to react with the free radical to form a spin-adduct, a nitroxide radical that is longer-lived than the original radicals. The identity of free radicals can be inferred from the particular EPR spectrum using EPR constants such as g values and hyperfine splitting constants. While the method has been extended to simple sensitive immunoassays to detect large radical molecules, such as DNA-radical or protein- radical, EPR remains the gold standard for identifying the presence of radicals in small molecules.

Methods of spin trapping using nitrone compounds that react with a target free radical to form a persistent and distinguishable spin adduct that can be detected by EPR spectroscopy has been described for instance in Frejaville C, et al., J.

Chem. Soc, Chem. Commun. 1793-1794 (1994); Frejaville C, et al., J. Med.

Chem. 38: 258-265 (1995); Olive G, et al., Free Rad. Biol. Med. 28 :403-408

(2000); Ouari O, et al., J. Org. Chem. 64: 3554-3556 (1999); Zeghdaoui A, et al., J. Chem. Soc. Perkin Trans. 2 :2087-2089 (1995); Bade et al.,

Spectrochimica acta part A 69: 1354-1366 (2008); Shi et al., Archives of

Biochemistery and Biophysics 437 : 59-68 (2005) and Villamena and Zweier, antioxidants & redox signaling, 6 : 619-629 (2004). A disadvantage of the classical EPR spin-trapping technique is the fact that the technique is a "hypothesis-driven" approach rather than a "data-driven" approach, because the design of the experiment requires an a priori assumption regarding the nature of the free radical. Hence, the choice of the spin-trap depends on its ability to react with the expected free radical or the expected localization of the site of production (intracellular or extracellular). Overall, the experimental conditions are generally tuned to fit these a priori assumptions and many experimental spin traps and conditions have to be performed sequentially. This renders the traditional spin-trapping experiments time and energy consuming. A rapid screening to seek for a possible free radical involvement in a biological or toxicological process is not available using the traditional spin trapping technique. Moreover, the chances of not detecting the presence of a free radical are rather high.

The aim of the present invention is to provide a solution to overcome at least part of the above mentioned disadvantages. The invention thereto aims to provide a novel approach called "radicalomics". In particular, the invention provides a method for screening and detecting the presence of free radicals in a sample without an a priori assumption regarding the nature of the free radicals. The invention thereto provides a method as described in claim 1. Further details of the invention are provided by the dependent claims.

SUMMARY In a first aspect, the present invention provides a method for detecting free radicals in a sample comprising the steps of reacting said sample with a spin trapping composition, to produce spin adducts of said spin trapping composition and free radicals and detecting said spin adducts. The method is characterized in that said spin trapping composition comprises at least two different spin trapping compounds.

In a preferred embodiment, the present invention provides a method wherein said spin adducts are detected by electron paramagnetic resonance (EPR) spectroscopy or electron spin resonance (ESR).

In another preferred embodiment, the present invention provides a method wherein said different spin trapping compounds have different lipophilicity. This is advantageous as a large range of hydrophilicity and lipophilicity is covered by the method of the present invention.

In another different embodiment, the present invention provides a method wherein said different spin trapping compounds have different affinity for free radicals. In a preferred embodiment, the present invention provides a method wherein said spin adducts provides an EPR signal. In a preferred embodiment, the present invention provides a method further comprising the step of deconvolving said EPR signal, thereby identifying the free radicals. In another preferred embodiment, the present invention provides a method wherein the spin trapping composition comprises spin trapping compounds selected from the group of N-tert-butyl-a-phenylnitrone (PBN), a(4-pyridil-l- oxide)-N-tert-butylnitrone (POBN), 5-diethoxyphosphoryl-5-methyl-l-pyrroline- N-oxide (DEPMPO), 5-ethoxycarbonyl-5-methyl-pyrroline-N-oxide (EMPO), 5- tert-Butoxycarbonyl-5-methyl-l-pyrroline-N-oxide (BMPO), 5,5-Dimethyl-l- pyrroline-N-oxide (DMPO), 2,2-Dimethyl-4-phenyl-2H-imidazole-l-oxide (DMPIO), 2,2,4-Trimethyl-2H-imidazole-l-oxide (TMIO), 2-(2-Carboxyethyl)-2- methyl-4-phenyl-2H-imidazole-l-oxide (MCPIO), 5- (Diisopropoxyphosphoryl)-5-methyl-l-pyrroline-N-oxide;2- Diisopropylphosphono-2-methyl-3,4-dihydro-2H-pyrrole-l-oxide (DIPPMPO), 3,3,5,5-Tetramethyl-pyrroline-N-oxide; M4PO (TMPO), Phosphonium, [2- [[[[(2R,3S)-2-(diethoxyphosphinyl)-3,4-dihydro-2-methyl-l-oxido-2H-pyrrol-3- yl]methoxy]carbonyl]amino]ethyl]triphenyl-, bromide(mito-DEPMPO), 5- Carbamoyl-5-methyl-l-pyrroline N-oxide (AMPO), 5 (diphenylphosphinoyl)-5- methyl-l-pyrroline N-oxide (DPPMPO),5-(2,2-Dimethyl-l,3-propoxy cyclophosphoryl)-5-methyl-l-pyrroline N-oxide (CYPMPO),

Cyclohexylmethoxycarbonyl-5-methyl-l-pyrroline N-oxide(CMMPO), 5- diethoxyphosphoryl-4-succinimidyloxycarbonyloxymethyl-5-methyl-l-pyrroline- N-oxide (NHS-DEPMPO), 5(diphenylphosphinoyl)-5-methyl-4,5-dihydro-3H- pyrrole N-oxide (DPPMDPO), N-(2,4-dodecyloxybenzylidene)-N-tert-butylamine N-oxide (DIDOD), N-(4-hydroxybenzyliene)-l-diethoxyphosphoryl-l- methylethylamine N-oxide (4-HOPPN), 5-carbamoyl-3,5-dimethyl-pyrroline N- oxide (CADMPO), 3,5-dimethyl-5-methylcarbamoyl-pyrroline N-oxide (DMMCAPO), 5-carbamoyl-3-ethyl-5-methyl-pyrroline N-oxide (CAEMPO), 3- ethyl-5-methyl-5-methylcarbamoyl-pyrroline N-oxide (EMMCAPO), 5- (secbutoxycarbonyl)-5-methyl-l-pyrroline N-oxide"(sBuMPO), 5-

(benzyloxycarbonyl)-5-methyl-l-pyrroline N-oxide (BnMPO), furoxanyl nitrone (FxBN). In a further preferred embodiment, the present invention provides a method wherein the spin trapping composition comprises N-tert-butyl-a-phenylnitrone (PBN), a(4-pyridil-l-oxide)-N-tert-butylnitrone (POBN), 5-diethoxyphosphoryl-5- methyl-l-pyrroline-N-oxide (DEPMPO) and 5-ethoxycarbonyl-5-methyl-pyrroline- N-oxide (EMPO). In a preferred embodiment, the present invention provides a method wherein said free radicals are selected from the group comprising oxygen-centered radicals, carbon-centered radicals, nitrogen-centered radicals and sulfur- centered radical. In another preferred embodiment, the present invention provides a method wherein the sample comprises a biological system selected from the group of cells, cell cultures, tissue homogenate, a biological fluid, an organ and a living system. In a second aspect, the present invention provides a kit for the detection of free radicals comprising the above described spin trapping compounds.

In a preferred embodiment, the present invention provides a kit wherein the concentration of said spin trapping compounds is comprised between 20 and lOOmM, preferably between 30 and 80mM, more preferably between 40 and 70mM, most preferably around 50mM. Said kit is provided for the use in a method for the identification of free radicals according to the present invention.

The present invention provides an approach that the inventor called "radicalomics", which provides without a priori a signature of free radical production in a sample.

The present invention uses a cocktail of spin traps, differing in their reactivity and lipophilicity, which is an invaluable screening tool that for the unambiguous detection of a free radical process in biological media. Moreover, the unique signature of the EPR signal recorded is a fingerprint of the free radical produced .

The invention provides a screening test without a priori that will rapidly give the indication regarding the radical species involved and present in a given sample.

Very importantly, the method is compatible with high-throughput screening in different biomedical disciplines (i.e. toxicology, biomaterial and drug development, or fundamental studies on oxidative stress). DESCRIPTION OF THE FIGURES

Further features, advantages and objects of the present invention will become apparent for the skilled person when reading the following detailed description of embodiments of the present invention, when taken in conjunction with the enclosed figures.

FIG.l shows EPR spectra of a spin trap compounds cocktail in the presence of different free radicals generating systems and controls experiments without generating systems. I : methyl radical, II: hydroxyl radical, III : azidyl radical, IV: sulfite radical, V: superoxide anion radical.

FIG.2 shows EPR spectra of K562 cells exposed to intoxicants. I : Tert- butylhydroperoxide, II : Menadione, III : Hydrogen peroxide, IV: Acetaldehyde, V : Phenylhydrazine. VI : control experiment (K562 cells in the presence of cocktail without toxic agent).

FIG.3 shows EPR spectra of K562 cells after exposure to menadione using spin trapping agents. I: PBN, II : POBN, III : DEPMPO, IV: EMPO, V: Cocktail of spin traps, VI: Cocktail of spin traps without or with SOD incubation.

FIG.4 shows an overlay (A) of the experimental spectrum (B) and the simulated spectra (C). The experimental spectrum (B) and the spectral simulation (C) of methyl adducts produced by Fenton reaction in a medium containing DMSO.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method, a kit and their use for screening and detecting free radicals in a sample. The inventor provides herein a novel and inventive approach that he called "radicalomics".

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention. As used herein, the following terms have the following meanings:

"A", "an", and "the" as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more than one compartment.

"About" as used herein referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of +/-20% or less, preferably +/-10% or less, more preferably +/-5% or less, even more preferably +/-1% or less, and still more preferably +/-0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.

"Comprise," "comprising," and "comprises" and "comprised of" as used herein are synonymous with "include", "including", "includes" or "contain", "containing", "contains" and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

The expression "% by weight" (weight percent), here and throughout the description unless otherwise defined, refers to the relative weight of the respective component based on the overall weight of the formulation.

The terms "EPR signal" and "EPR spectra" are used herein as synonyms. "Coefficient of partition (D)" is the ratio of concentrations of a spin trap molecule in two phases of a mixture of two immiscible solvents at equilibrium. In the present invention, the first solvent is water or an aqueous buffer while the second solvent is hydrophobic, preferably octanol. The expression "cocktail of spin traps" is used herein as synonym of "combination of spin traps" and "association of spin traps" and refers to at least two different spin traps. The term "deconvolution" used herein refers to the mathematical analysis and the simulation of the spectrum to be obtained.

The present invention provides the "radicalomics" approach which detects all free radicals, whatever their chemical nature; measures the free radical production in different compartments (hydrophilic or lipophilic) and provides a signature (fingerprint) that is unique to the type of free radical detected.

In a first aspect, the present invention provides a method for detecting free radicals in a sample comprising the steps of reacting said sample with a spin trapping composition, to produce spin adducts of said spin trapping composition and free radicals and detecting said spin adducts. The method is characterized in that said spin trapping composition comprises at least two different spin trapping compounds. The cocktail of spin trap covers the full range of all free radicals, covers a large range of hydophilicity and lipophilicity. Moreover the cocktail of spin traps gives different patterns of spectrum of the spin adducts obtained after trapping of different radicals.

In a preferred embodiment, the present invention provides a method wherein said different spin trapping compounds have different lipophilicity in order to be localized in different compartments of a complex medium (extracellular, intracellular, intra-organite). Said spin trapping compounds have also different capabilities to trap free radicals so that any biologically relevant free radical can be trapped by the spin trapping composition and offer a large variety in the EPR spectra of the spin adducts, without mutual interference in order to discriminate as much as possible the free radicals involved.

In a preferred embodiment, the present invention provides a method, wherein said different spin trapping compounds have different hydrophilicity and different lipophilicity. Said spin trapping compounds are selected such as their (D) values is comprised between 0 and 350, preferably between 0.01 and 300, more preferably between 0.03 and 250, even more preferably between 0.05 and 200, most preferably between 0.08 and 150, even most preferably between 0.1 and 100. In another preferred embodiment, the present invention provides a method wherein the cocktail of spin traps comprises at least 2 different spin traps wherein one has an extra-cellular localization and the other have an intracellular localization. In a further preferred embodiment, said cocktail of spin traps comprises at least 2 different spin traps wherein spin trap have a (D) value lower than 1, more preferably lower than 0.5 and the other spin trap have a (D) value higher than 0.5, preferably higher than 2, more preferably higher than 4, most preferably higher than 5. In another preferred embodiment, the present invention provides a method wherein said different spin trapping compounds have different affinity for free radicals. The affinity also called capacity herein, is the ability of a spin trap to provide an EPR spectrum after reaction with a free radical. This "capacity" is depending on several factors, such as the reactivity of the spin trap with a free radical to give a long-lived spin adduct (depending on the rate constant for the addition of radicals to spin traps), the rates of decay of adduct radicals once formed (depending on the presence of metal ions, reductants, pH, temperature,...)- A person having ordinary skill in the art generally chooses a spin trap according to the nature of the hypothetical free radical.

In a preferred embodiment, the present invention provides a method wherein the spin trapping composition comprises any combination of at least two spin trapping compounds selected from the group of N-tert-butyl-a-phenylnitrone (PBN), a(4-pyridil-l-oxide)-N-tert-butylnitrone (POBN), 5-diethoxyphosphoryl-5- methyl-l-pyrroline-N-oxide (DEPMPO), 5-ethoxycarbonyl-5-methyl-pyrroline-N- oxide (EMPO), 5-tert-Butoxycarbonyl-5-methyl-l-pyrroline-N-oxide (BMPO), 5,5- Dimethyl-l-pyrroline-N-oxide (DMPO), 2,2-Dimethyl-4-phenyl-2H-imidazole-l- oxide (DMPIO), 2,2,4-Trimethyl-2H-imidazole-l-oxide (TMIO), 2-(2- Carboxyethyl)-2-methyl-4-phenyl-2H-imidazole-l-oxide (MCPIO), 5-(Diisopropoxyphosphoryl)-5-methyl-l-pyrroline-N-oxide;2-

Diisopropylphosphono-2-methyl-3,4-dihydro-2H-pyrrole-l-oxide (DIPPMPO), 3,3,5,5-Tetramethyl-pyrroline-N-oxide; M4PO (TMPO), Phosphonium, [2- [[[[(2R,3S)-2-(diethoxyphosphinyl)-3,4-dihydro-2-methyl-l-oxido-2H-pyrrol-3- yl]methoxy]carbonyl]amino]ethyl]triphenyl-, bromide(mito-DEPMPO), 5- Carbamoyl-5-methyl-l-pyrroline N-oxide (AMPO), 5 (diphenylphosphinoyl)-5- methyl-l-pyrroline N-oxide (DPPMPO),5-(2,2-Dimethyl-l,3-propoxy cyclophosphoryl)-5-methyl-l-pyrroline N-oxide (CYPMPO),

(benzyloxycarbonyl)-5-methyl-l-pyrroline N-oxide (BnMPO), furoxanyl nitrone (FxBN). In another preferred embodiment, the present invention provides a method wherein the spin trapping composition comprises N-tert-butyl-a-phenylnitrone (PBN) and at least one spin trapping compound selected from the group of a(4- pyridil-l-oxide)-N-tert-butylnitrone (POBN), 5-diethoxyphosphoryl-5-methyl-l- pyrroline-N-oxide (DEPMPO), and 5-ethoxycarbonyl-5-methyl-pyrroline-N-oxide (EMPO), 5-tert-Butoxycarbonyl-5-methyl-l-pyrroline-N-oxide (BMPO), 5,5- Dimethyl-l-pyrroline-N-oxide (DMPO), 2,2-Dimethyl-4-phenyl-2H-imidazole-l- oxide (DMPIO), 2,2,4-Trimethyl-2H-imidazole-l-oxide (TMIO), 2-(2- Carboxyethyl)-2-methyl-4-phenyl-2H-imidazole-l-oxide (MCPIO), 5-

(Diisopropoxyphosphoryl)-5-methyl-l-pyrroline-N-oxide;2- Diisopropylphosphono-2-methyl-3,4-dihydro-2H-pyrrole-l-oxide (DIPPMPO), ), 3,3,5,5-Tetramethyl-pyrroline-N-oxide; M4PO (TMPO), Phosphonium, [2- [[[[(2 ,3S)-2-(diethoxyphosphinyl)-3,4-dihydro-2-methyl-l-oxido-2H-pyrrol-3- yl]methoxy]carbonyl]amino]ethyl]triphenyl-, bromide(mito-DEPMPO), 5- Carbamoyl-5-methyl-l-pyrroline N-oxide (AMPO), 5 (diphenylphosphinoyl)-5- methyl-l-pyrroline N-oxide (DPPMPO),5-(2,2-Dimethyl-l,3-propoxy cyclophosphoryl)-5-methyl-l-pyrroline N-oxide (CYPMPO),

(benzyloxycarbonyl)-5-methyl-l-pyrroline N-oxide (BnMPO),Furoxanyl nitrone (FxBN).

In another preferred embodiment, the present invention provides a method wherein the spin trapping composition comprises a(4-pyridil-l-oxide)-N-tert- butylnitrone (POBN) and at least one spin trapping compound selected from the group of N-tert-butyl-a-phenylnitrone (PBN), 5-diethoxyphosphoryl-5-methyl-l- pyrroline-N-oxide (DEPMPO), and 5-ethoxycarbonyl-5-methyl-pyrroline-N-oxide (EMPO), 5-tert-Butoxycarbonyl-5-methyl-l-pyrroline-N-oxide (BMPO), 5,5- Dimethyl-l-pyrroline-N-oxide (DMPO), 2,2-Dimethyl-4-phenyl-2H-imidazole-l- oxide (DMPIO), 2,2,4-Trimethyl-2H-imidazole-l-oxide (TMIO), 2-(2- Carboxyethyl)-2-methyl-4-phenyl-2H-imidazole-l-oxide (MCPIO), 5-

Cyclohexylmethoxycarbonyl-5-methyl-l-pyrroline N-oxide(CMMPO), 5- diethoxyphosphoryl-4-succinimidyloxycarbonyloxymethyl-5-methyl-l-pyrroline- N-oxide (NHS-DEPMPO), 5(diphenylphosphinoyl)-5-methyl-4,5-dihydro-3H- pyrrole N-oxide (DPPMDPO), N-(2,4-dodecyloxybenzylidene)-N-tert-butylamine N-oxide (DIDOD), N-(4-hydroxybenzyliene)-l-diethoxyphosphoryl-l- methylethylamine N-oxide (4-HOPPN), 5-carbamoyl-3,5-dimethyl-pyrroline N- oxide (CADMPO), 3,5-dimethyl-5-methylcarbamoyl-pyrroline N-oxide (DMMCAPO), 5-carbamoyl-3-ethyl-5-methyl-pyrroline N-oxide (CAEMPO), 3- ethyl-5-methyl-5-methylcarbamoyl-pyrroline N-oxide (EMMCAPO), 5- (secbutoxycarbonyl)-5-methyl-l-pyrroline N-oxide"(sBuMPO), 5- (benzyloxycarbonyl)-5-methyl-l-pyrroline N-oxide (BnMPO),Furoxanyl nitrone (FxBN).

In another preferred embodiment, the present invention provides a method wherein the spin trapping composition comprises 5-diethoxyphosphoryl-5- methyl-l-pyrroline-N-oxide (DEPMPO) and at least one spin trapping compound selected from the group of N-tert-butyl-a-phenylnitrone (PBN), a(4-pyridil-l- oxide)-N-tert-butylnitrone (POBN), and 5-ethoxycarbonyl-5-methyl-pyrroline-N- oxide (EMPO), 5-tert-Butoxycarbonyl-5-methyl-l-pyrroline-N-oxide (BMPO), 5,5- Dimethyl-l-pyrroline-N-oxide (DMPO), 2,2-Dimethyl-4-phenyl-2H-imidazole-l- oxide (DMPIO), 2,2,4-Trimethyl-2H-imidazole-l-oxide (TMIO), 2-(2- Carboxyethyl)-2-methyl-4-phenyl-2H-imidazole-l-oxide (MCPIO), 5-

(Diisopropoxyphosphoryl)-5-methyl-l-pyrroline-N-oxide;2- Diisopropylphosphono-2-methyl-3,4-dihydro-2H-pyrrole-l-oxide (DIPPMPO), 3,3,5,5-Tetramethyl-pyrroline-N-oxide; M4PO (TMPO), Phosphonium, [2- [[[[(2 ,3S)-2-(diethoxyphosphinyl)-3,4-dihydro-2-methyl-l-oxido-2H-pyrrol-3- yl]methoxy]carbonyl]amino]ethyl]triphenyl-, bromide(mito-DEPMPO), 5- Carbamoyl-5-methyl-l-pyrroline N-oxide (AMPO), 5 (diphenylphosphinoyl)-5- methyl-l-pyrroline N-oxide (DPPMPO),5-(2,2-Dimethyl-l,3-propoxy cyclophosphoryl)-5-methyl-l-pyrroline N-oxide (CYPMPO),

The method according to any of claims 1-3, wherein the spin trapping composition comprises 5-ethoxycarbonyl-5-methyl-pyrroline-N-oxide (EMPO) and at least one spin trapping compound selected from the group of N-tert- butyl-a-phenylnitrone (PBN), 5-diethoxyphosphoryl-5-methyl-l-pyrroline-N- oxide (DEPMPO), a(4-pyridil-l-oxide)-N-tert-butylnitrone (POBN), 5-tert- Butoxycarbonyl-5-methyl-l-pyrroline-N-oxide (BMPO), 5,5-Dimethyl-l-pyrroline- N-oxide (DMPO), 2,2-Dimethyl-4-phenyl-2H-imidazole-l-oxide (DMPIO), 2,2,4- Trimethyl-2H-imidazole-l-oxide (TMIO), 2-(2-Carboxyethyl)-2-methyl-4-phenyl- 2H-imidazole-l-oxide (MCPIO), 5-(Diisopropoxyphosphoryl)-5-methyl-l- pyrroline-N-oxide;2-Diisopropylphosphono-2-methyl-3,4-dihydro-2H-pyrrole-l- oxide (DIPPMPO), 3,3,5,5-Tetramethyl-pyrroline-N-oxide; M4PO (TMPO), Phosphonium, [2-[[[[(2R,3S)-2-(diethoxyphosphinyl)-3,4-dihydro-2-methyl-l- oxido-2H-pyrrol-3-yl]methoxy]carbonyl]amino]ethyl]triphenyl-, bromide(mito- DEPMPO), 5-Carbamoyl-5-methyl-l-pyrroline N-oxide (AMPO), 5 (diphenylphosphinoyl)-5-methyl-l-pyrroline N-oxide (DPPMPO),5-(2,2-Dimethyl- 1,3-propoxy cyclophosphoryl)-5-methyl-l-pyrroline N-oxide (CYPMPO), Cyclohexylmethoxycarbonyl-5-methyl-l-pyrroline N-oxide(CMMPO), 5- diethoxyphosphoryl-4-succinimidyloxycarbonyloxymethyl-5-methyl-l-pyrroline- N-oxide (NHS-DEPMPO), 5(diphenylphosphinoyl)-5-methyl-4,5-dihydro-3H- pyrrole N-oxide (DPPMDPO), N-(2,4-dodecyloxybenzylidene)-N-tert-butylamine N-oxide (DIDOD), N-(4-hydroxybenzyliene)-l-diethoxyphosphoryl-l- methylethylamine N-oxide (4-HOPPN), 5-carbamoyl-3,5-dimethyl-pyrroline N- oxide (CADMPO), 3,5-dimethyl-5-methylcarbamoyl-pyrroline N-oxide (DMMCAPO), 5-carbamoyl-3-ethyl-5-methyl-pyrroline N-oxide (CAEMPO), 3- ethyl-5-methyl-5-methylcarbamoyl-pyrroline N-oxide (EMMCAPO), 5- (secbutoxycarbonyl)-5-methyl-l-pyrroline N-oxide"(sBuMPO), 5-

In a further preferred embodiment, the present invention provides a method wherein the spin trapping composition comprises N-tert-butyl-a-phenylnitrone (PBN), a(4-pyridil-l-oxide)-N-tert-butylnitrone (POBN), 5-diethoxyphosphoryl-5- methyl-l-pyrroline-N-oxide (DEPMPO) and 5-ethoxycarbonyl-5-methyl-pyrroline- N-oxide (EMPO).

In a preferred embodiment, the present invention provides a method wherein said spin adducts provides an EPR signal which is detected by electron paramagnetic resonance (EPR) spectroscopy or electron spin resonance (ESR). In a preferred embodiment of the method according to the present invention, the cocktail of spin traps reveals a particularly complex EPR signature for all spin adducts, thereby revealing the presence of any biologically relevant free radical. In a further preferred embodiment of the method, the obtained EPR signal is a complex EPR signal which is then deconvolved to recover the individual components and thereby identify the free radicals. This will restrict the number of experiments to carry out to further characterize the free radical produced in a biological medium. Said complex EPR signature is deconvolved to identify the free radicals produced after reaction with the spin trapping composition

In a preferred embodiment of the method of the present invention, the free radicals are selected from the group comprising oxygen-centered radicals such as but not limited to hydroxyl, hydroperoxyl, alkoxyl and acyloxyl, superoxide anion, nitric oxide; carbon-centered radicals such as but not limited to methyl, alkyl, aryl; nitrogen-centered radicals such as but not limited to azidyl, sulfur- centered radical such as but not limited to thy il, sulfite.

In another preferred embodiment of the method according to the present invention, the sample comprises a biological system selected from the group of cells, cell cultures, tissue homogenate, a biological fluid, an organ and a living system such as but not limited to a plant or an animal. In a further preferred embodiment of the method according to the present invention, the sample comprises any system wherein free radicals are and/or or can be present and/or produced.

In a preferred embodiment, the present invention provides a method of a spin trapping procedure which is based on the use of a spin trapping composition (cocktail) for the assessment of the toxicity induced in animals. In a further preferred embodiment, said toxicity is induced by tested chemicals and the assessment comprises the comparison of the induced toxicity with the toxicity produced by a known toxic.

In another preferred embodiment, the cocktail of spin traps is used to detect, both in vivo or ex-vivo, the formation of free radicals in samples such as but not limited to body fluids, organ homogenates, or organs, after the administration of the chemical. Control vehicle and/or reference drugs are used to induce a controlled level of toxicity which can be compared to the signal observed with the tested chemicals. EPR is used either in the high frequency mode (9 GHz or higher), for the ex-vivo detection, or in the low frequency L-band mode for the in vivo detection. Said control vehicle and/or reference drugs include, but are not limited to, H₂0₂, menadione, tert-butylhydroperoxide, phenylhydrazine, acetaldehyde, carbon tetrachloride, acetaminophen, alpha-naphtyl isothiocyanate and l, l'-dimethyl-4,4'-bipyridinium.

In a further preferred embodiment, the present invention provides a method for in vivo detection and identification of free radicals, comprising the steps of: - administering said chemical and/or vehicle and/or control drug by the appropriate route of administration. Said administration can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, subcutaneous, intramuscular, transdermal, intratracheal, intravenous, etc., administration; - incubating in-vivo to test the drug inducement of free radical production; - administering the spin trapping composition. Said administration can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, subcutaneous, intramuscular, transdermal, intratracheal, intravenous, etc., administration; - measuring, in vivo, the EPR signal in a selected organ, such as but not limited to liver, in L band mode; - measuring, ex vivo, the signal in organ or tissue homogenates, or in body fluids such as but not limited to urine, blood, bile; - comparing the signal obtained with the tested chemical with the signal obtained with a vehicle and/or a reference drug.

In a second aspect, the present invention provides a kit for the detection of free radicals. Said kit comprises at least two spin trapping compounds selected from the group of N-tert-butyl-a-phenylnitrone (PBN), a(4-pyridil-l-oxide)-N-tert- butylnitrone (POBN), 5-diethoxyphosphoryl-5-methyl-l-pyrroline-N-oxide (DEPMPO), 5-ethoxycarbonyl-5-methyl-pyrroline-N-oxide (EMPO), 5-tert- Butoxycarbonyl-5-methyl-l-pyrroline-N-oxide (BMPO), 5,5-Dimethyl-l-pyrroline- N-oxide (DMPO), 2,2-Dimethyl-4-phenyl-2H-imidazole-l-oxide (DMPIO), 2,2,4- Trimethyl-2H-imidazole-l-oxide (TMIO), 2-(2-Carboxyethyl)-2-methyl-4-phenyl- 2H-imidazole-l-oxide (MCPIO), 5- (Diisopropoxyphosphoryl)-5-methyl-l-pyrroline-N-oxide;2-

Diisopropylphosphono-2-methyl-3,4-dihydro-2H-pyrrole-l-oxide (DIPPMPO), 3,3,5,5-Tetramethyl-pyrroline-N-oxide; M4PO (TMPO), Phosphonium, [2- [[[[(2 ,3S)-2-(diethoxyphosphinyl)-3,4-dihydro-2-methyl-l-oxido-2H-pyrrol-3- yl]methoxy]carbonyl]amino]ethyl]tnphenyl-, bromide(mito-DEPMPO), 5- Carbamoyl-5-nnethyl-l-pyrroline N-oxide (AMPO), 5 (diphenylphosphinoyl)-5- methyl-l-pyrroline N-oxide (DPPMPO),5-(2,2-Dimethyl-l,3-propoxy cyclophosphoryl)-5-methyl-l-pyrroline N-oxide (CYPMPO),

(benzyloxycarbonyl)-5-methyl-l-pyrroline N-oxide (BnMPO), furoxanyl nitrone (FxBN). In a further preferred embodiment, the kit according to the present invention comprises spin trapping compounds having a final concentration comprised between 20 and lOOmM, preferably between 30 and 80mM, more preferably between 40 and 70mM, most preferably around 50mM. In a further preferred embodiment, the kit according to the present invention provides an EPR spectrum that is analysed to provide the identification of the trapped free radical and/or free radicals.

In a preferred embodiment, the kit according to the present invention further comprises a cellular system to be used as an alternative way to demonstrate a free-radical mediated process of cellular origin. Said cellular systems include non-adherent cells, semi-adherent cells, adherent cells, isolated cells, slice of tissues, tissues, and organs. In another aspect the present invention provides for the use of a kit and/or a method according to the present invention for the identification of free radicals. Examples

Example 1 : measure of a large variety of free radicals in characterized conditions

A cocktail of different spin traps: N-tert-butyl-a-phenylnitrone (PBN), a(4-pyridil- l-oxide)-N-tert-butylnitrone (POBN), 5-diethoxyphosphoryl-5-methyl-l- pyrroline-N-oxide (DEPMPO), and 5-ethoxycarbonyl-5-methyl-pyrroline-N-oxide (EMPO) was used.

The ability of the cocktail of spin traps to give an EPR signature of biologically relevant free radicals produced by well-defined enzymatic or chemical reactions was tested. Oxygen-centered radicals, hydroxyl radical and superoxide anion, were produced by Fenton reaction and a combination of xanthine and xanthine oxidase, respectively; nitrogen-centered radical (azidyl radical) was produced by using azide, H₂0₂ and peroxidase; carbon-centered radical (methyl radical) was produced using a Fenton system in dimethyl sulfoxide; sulphur-centered radical (sulfite radical) was produced using sulfite in dichromate. Four spin traps were selected to cover a large range of lipophilicity and reactivity against the main family of radicals. DEPMPO (5-diethoxyphosphoryl-5methyl-l- pyrroline-N-oxide), EMPO (5-ethoxycarbonyl-5-methyl-pyrroline-N-oxide) were obtained from Radical Vision (Marseille, France). POBN (a (4-pyridil-l-oxide)-N- tert-butylnitrone), PBN (N-tert-butyl- a- phenylnitrone) were purchased from Enzo-Alexis Biochemicals (Lausanne, Switzerland). Spin traps were dissolved in water except for PBN which was dissolved in DMSO. Stock solution (2 M) were stored at -80°C.

Chemicals for radicals generating system : xanthine, xanthine oxidase, diethylenetriaminepentaacetic acid (DTPA), ferrous ammonium sulfate (Fe (NH₄)₂(S0₄)2), hydrogen peroxide (H₂0₂), sodium sulfite, sodium dichromate, sodium azide and horsedarish peroxidase were purchased from Sigma Aldrich (St Louis, MO). To generate superoxide anion radical, xanthine oxidase (lU/ml) was incubated with xanthine (1 mM) in PBS (pH 7.4, 100 mM) containing DTPA (500 μΜ) and 25 mM spin trap. Hydroxyl radicals were generated from a Fenton system : Fe(NH₄)₂(S0₄)2 (2mM) was added to a solution of 25 mM spin trap, 2 mM H₂0₂, and 1 mM DTPA in PBS (100 mM).

The sulfite radicals were generated and spin trapped by adding sodium sulfite (20 mM) and sodium dichromate (10 mM) into PBS containing 25 mM of the spin trap- To generate methyl radicals, 10% v/v of DMSO was added to the Fenton system.

Azidyl radicals were generated by addition of sodium azide (30 mM) to a solution of 20 mM spin trap, DPTA (10 μΜ), H₂0₂ (350 μΜ) and horsedarish peroxidase (lmg/ml) in PBS.

EPR spectra were recorded at room temperature using a Bruker EMX EPR spectrometer (Bruker, Germany) operating at 9.8 GHz. The magnetic field centre was 348.0 mT, with a Sweep Width of 15.0 mT (2048 pts). The microwave power was 3.19 mW; modulation frequency used was 100 kHz, modulation amplitude was 0.1 mT; the conversion time was 10.24 ms and the time constant was 10.24 ms. Spectra were the mean of 15 scans. The results showed that the cocktail of spin traps was able to react with all types of free radicals providing an EPR signature (Fig.l, references 6 to 10) that is totally different from the controls (Fig.l, references 1 to 5) comprising the cocktail of spin traps without the generating system. Fig.l (I to V wherein I : methyl radical, II : hydroxyl radical, III : azidyl radical, IV: sulfite radical, V: superoxide anion radical), shows the obtained EPR signature which is unique and selective to the free radical produced.

Example 2: cell culture conditions The human leukemic cell line K562 was purchased from the European Collection of Cell Cultures (ECACC, Salisbury, United Kingdom). These cells were selected because they are non-adherent and are commonly used to test the toxicity of different chemicals. The cells were maintained in RPMI-1640 medium supplemented with 10 % of fetal calf serum, streptomycin ΙΟΟμς/ηηΙ, penicillin 100 IU/ml (Gibco, Paisley, United Kingdom) at 37°C in a 95% air/5% C0₂ atmosphere with 100% humidity.

Example3: Spin trapping in cell cultures

Cells were incubated in RPMI-1640 medium at a concentration of 2.10 cells/ml in a final volume of 0.5 ml. Cell suspensions were pre-incubated with individual spin traps (final concentration 50mM), or the cocktail of ST (50mM for each ST) during ten minutes at 37°C under an atmosphere 5% C0₂.

Toxic was then added directly to the medium and incubated for 10 more minutes to generate radicals in situ, with the exception of acetaldehyde which was incubated for 30 minutes. Based on literature data, hydrogen peroxide (5mM) was used to produce ΟΗ·, tert-butylhydroperoxide (15mM) for alkoxyl radicals, phenylhydrazine (5mM) for N centered radical, acetaldehyde (80mM) for the generation of CH₃». Menadione (ImM final concentration) was used to produce the superoxide anion 0₂ ^~· and ΟΗ·.

In control experiments, when applicable, specific enzymes such Super Oxide Dismutase SOD (100 U/ml) or catalase CAT (100 U/ml) were used to degrade the toxic or the radical formed by the toxic and consequently suppress the EPR signal. Enzymes were added to the medium just prior the addition of the ST.

For the EPR experiments in cellular systems, four hundred microliters of the medium were transferred into a quartz flat cell for aqueous sample (ER 160 FC- Q, Bruker, Rheinstatten, Germany). The cell was positioned in a X-band EPR Super High Q cylindrical resonator (ER4122SHQE, 10mm diameter, Bruker, Germany) with its flat side perpendicular to the direction of the magnetic field. EPR spectra were measured at room temperature using a Bruker Elexys 540 spectrometer (Bruker, Germany) operating at 9.8 GHz. The magnetic field centre was 348.0 mT, with a Sweep Width of 20.0 mT (512pts). The microwave power was 13 mW; modulation frequency used was 100 kHz, modulation amplitude was 0.1 mT; the conversion time was 20.48 ms and the time constant was 81.92 ms. Spectra were the mean of 30 scans.

Example 4: screening for radical-mediated toxicity

To test whether our method could be applied to a screening for radical-mediated toxicity which is an application of the radicalomics apprach, we exposed nonadherent K562 cells to several known free-radical mediated intoxicants: H₂0₂, menadione, tert-butylhydroperoxide, phenylhydrazine, and acetaldehyde.

Toxics were chosen to produce radicals representative of the main types commonly observed (C, O, N or S-centered radicals). Menadione bisulfite, hydrogen peroxide, tert-butylhydroperoxide, phenylhydrazine, acetaldehyde, catalase (CAT) and polyethylene glycol, SOD were obtained from Sigma Aldrich (St Louis, MO).

For each compound, an EPR signature was recorded confirming the involvement of a free radical reaction (Fig.2, I : Tert-butylhydroperoxide, II : Menadione, III : Hydrogen peroxide, IV: Acetaldehyde, V: Phenylhydrazine. VI : control experiment).

Example 5: free radicals degradation in the presence of an enzyme

A major achievement of the radicalomic approach, when applied in cells, is illustrated with the particular case of menadione: an EPR fingerprint is recorded using the cocktail of spin traps while some spin traps, when used alone (here, PBN and POBN), were unable to detect a free radical involvement after menadione exposure (Fig.3). This illustrates that false negative can be registered when using traditional spin trapping using inadequate individual spin traps, and the interest to use together multiple spin traps with different properties and lipophilicities to increase the probability to detect free radicals in a complex biological medium. Indeed, the signal Fig.3 V recorded after menadione exposure is the sum of the EPR signal recorded using DEPMPO Fig.3 III and EMPO Fig.3 IV, with no particular distortion nor alteration of the resultant spectrum (Fig.3). It is known that the final proof for the involvement of a particular free radical relies on the capacity to use specific enzymes to degrade the radical formed and abolish the EPR signature. This was obtained as we observed a decrease in the EPR signal of the cells exposed to menadione in the presence of SOD (Fig.3 VI, 12) compared to the cells exposed to menadione in the absence of SOD (Fig.3 VI, 11). Comparable results were obtained using catalase after intoxication by hydrogen peroxide.

Example 6: spectrum deconvolution and analysis for free radicals identification

Methyl radical (CH₃») was generated by a Fenton reaction in a medium containing DMSO and the following spin trap composition : DEPMPO, EMPO, POBN and PBN. The EPR spectrum was recorded and analysed using WINSIM (EPR simulation software available on the website of the National Institute for Health, USA).

First, the theoretical spectrum of each species was generated using the simulation function of the software. Parameters used for the simulation are presented in table 1.

The measured complex spectrum of the methyl adducts was fitted to the theoretical spectra. FIG. 4 shows the experimental spectrum (B) and the fitted function also called spectral simulation (C). A very close overlap (A) was obtained with the following relative concentrations: DEPMPO- CH₃» 17.98%, EMPO- ΟΗ₃· : 40.86% and PBN/POBN- CH₃» : 41.16%.

Table 1 : parameters used for the simulation using WINSIM

Species 1 LW : 0.63

Nuclei Coupling Spin Number

1 14.96 1.0 1

2 22.30 0.5 1

3 47.50 0.5 1

Species 2 LW : 0.61

Nuclei Coupling Spin Number

1 15.21 1.0 1

2 21.80 0.5 1

Species 3 LW : 0.69

Nuclei Coupling Spin Number

1 15.71 1.0 1

2 2.74 0.5 1 The results showed that mathematical analysis of the experimental spectrum obtained with the composition of spin traps can demonstrate the identity of the radical, and the proportion of the relative spin trap-adducts.

To conclude, the radicalomic procedure provides for the measurement of a large variety of free radicals and for the unambiguous detection of free radical production in cells exposed to chemicals known as radical-mediated intoxicants. Although the present invention has been described with reference to preferred embodiments thereof, many modifications and alternations may be made by a person having ordinary skill in the art without departing from the scope of this invention which is defined by the appended claims.

Claims

1. A method for detecting free radicals in a sample comprising the steps of:

- reacting said sample with a spin trapping composition, to produce spin adducts of said spin trapping composition and free radicals and

- detecting said spin adducts by electron paramagnetic resonance (EPR) spectroscopy, thereby obtaining an EPR signal,

characterized in that,

said spin trapping composition comprises at least two different spin trapping compounds.

2. The method according to claim 1, wherein said different spin trapping compounds have different lipophilicity.

3. The method according to claim 1, wherein said different spin trapping compounds have different hydrophilicity.

4. The method according to any of claims 1 to 3, wherein said different spin trapping compounds have different affinity for free radicals.

5. The method according to any of claims 1 to 4, further comprising the step of deconvolving said EPR signal, thereby identifying the free radicals.

6. The method according to any of claims 1 to 5, wherein the spin trapping composition comprises spin trapping compounds selected from the group of N-tert-butyl-a-phenylnitrone (PBN), a(4-pyridil-l-oxide)-N-tert-butylnitrone (POBN), 5-diethoxyphosphoryl-5-methyl-l-pyrroline-N-oxide (DEPMPO), 5- ethoxycarbonyl-5-methyl-pyrroline-N-oxide (EMPO), 5-tert-Butoxycarbonyl- 5-methyl-l-pyrroline-N-oxide (BMPO), 5,5-Dimethyl-l-pyrroline-N-oxide (DMPO), 2,2-Dimethyl-4-phenyl-2H-imidazole-l-oxide (DMPIO), 2,2,4- Trimethyl-2H-imidazole-l-oxide (TMIO), 2-(2-Carboxyethyl)-2-methyl-4- phenyl-2H-imidazole-l-oxide (MCPIO), 5- (Diisopropoxyphosphoryl)-5-methyl-l-pyrroline-N-oxide;2- Diisopropylphosphono-2-methyl-3,4-dihydro-2H-pyrrole-l-oxide

(DIPPMPO), 3,3,5,5-Tetramethyl-pyrroline-N-oxide; M4PO (TMPO), Phosphonium, [2-[[[[(2R,3S)-2-(diethoxyphosphinyl)-3,4-dihydro-2- methyl-l-oxido-2H-pyrrol-3-yl]methoxy]carbonyl]amino]ethyl]triphenyl-, bromide(mito-DEPMPO), 5-Carbamoyl-5-methyl-l-pyrroline N-oxide (AMPO), 5 (diphenylphosphinoyl)-5-methyl-l-pyrroline N-oxide (DPPMPO),5-(2,2-Dimethyl-l,3-propoxy cyclophosphoryl)-5-methyl-l- pyrroline N-oxide (CYPMPO), Cyclohexylmethoxycarbonyl-5-methyl-l- pyrroline N-oxide(CMMPO), 5-diethoxyphosphoryl-4- succinirnidyloxycarbonyloxymethyl-5-nnethyl-l-pyrroline-N-oxide (NHS- DEPMPO), 5(diphenylphosphinoyl)-5-methyl-4,5-dihydro-3H-pyrrole N- oxide (DPPMDPO), N-(2,4-dodecyloxybenzylidene)-N-tert-butylamine N- oxide (DIDOD), N-(4-hydroxybenzyliene)-l-diethoxyphosphoryl-l- methylethylamine N-oxide (4-HOPPN), 5-carbamoyl-3,5-dimethyl-pyrroline N-oxide (CADMPO), 3,5-dimethyl-5-methylcarbamoyl-pyrroline N-oxide (DMMCAPO), 5-carbamoyl-3-ethyl-5-methyl-pyrroline N-oxide (CAEMPO), 3-ethyl-5-methyl-5-methylcarbamoyl-pyrroline N-oxide (EMMCAPO), 5- (secbutoxycarbonyl)-5-methyl-l-pyrroline N-oxide"(sBuMPO), 5- (benzyloxycarbonyl)-5-methyl-l-pyrroline N-oxide (BnMPO), furoxanyl nitrone (FxBN).

7. The method according to any of claims 1-5, wherein the spin trapping composition comprises N-tert-butyl-a-phenylnitrone (PBN), a(4-pyridil-l- oxide)-N-tert-butylnitrone (POBN), 5-diethoxyphosphoryl-5-methyl-l- pyrroline-N-oxide (DEPMPO) and 5-ethoxycarbonyl-5-methyl-pyrroline-N- oxide (EMPO).

8. The method according to any of claims 1 to 7, wherein said free radicals are selected from the group comprising oxygen-centered radicals, carbon- centered radicals, nitrogen-centered radicals and sulfur-centered radical.

9. The method according to any of claims 1 to 8, wherein the sample comprises a biological system selected from the group of cells, cell cultures, tissue homogenate, a biological fluid, an organ and a living system.

10. A kit for the detection of free radicals comprising spin trapping compounds as described in claims 1 to 9.

11. A kit according to claim 10, wherein the concentration of said spin trapping compounds is comprised between 20 and lOOmM, preferably between 30 and 80mM, more preferably between 40 and 70mM, most preferably around 50mM.

12. Use of a kit according to claims 10 and 11, in a method as described in claims 1 to 9 for the identification of free radicals.