OXIDIZED LIPIDS AS BIOMARKERS FOR NEUROPATHIC PAIN

Abstract

Diagnostic methods are useful for diagnosing neuropathic pain in a subject, for predicting whether a subject is at risk of developing neuropathic pain, or for determining whether a neuropathic pain therapy is successful. Tools for carrying out the aforementioned methods, include diagnostic devices, and oxidized lipids, for example, an epoxylipid, for use in the aforementioned methods.

Claims

1. A method for diagnosing neuropathic pain in a subject comprising: (a) determining in a plasma sample of a subject suspected to suffer from neuropathic pain the amount of at least one oxidized lipid; (b) comparing the said amount of the at least one oxidized lipid with a reference amount, whereby neuropathic pain is to be diagnosed.

2. The method of claim 1, wherein said at least one oxidized lipid is an epoxylipid.

3. The method of claim 1, wherein said at least one expoxylipid is selected from the group consisting of: 9,10-EpOME (()9(10)-epoxy-12Z-octadecaenoic acid), 9-HODE (()9-hydroxy-10(E),12(Z)-octadecadienoic acid) and 13-HODE (()13-hydroxy-9(Z),11(E)-octadecadienoic acid).

4. The method of claim 1, wherein said neuropathic pain is chemotherapy-induced neuropathic pain (CIPN).

5. The method of claim 1, wherein said CIPN is induced by paclitaxel and/or oxaliplatin.

6. The method of claim 4, wherein the amount of the at least one oxidized lipid is determined 24 h after the start of chemotherapy.

7. The method of claim 4, wherein said reference amount corresponds to the amount of said at least one oxidized lipid before the start of chemotherapy.

8. A method for predicting whether a subject is at risk of developing neuropathic pain comprising: (a) determining in a plasma sample of the subject the amount of at least one oxidized lipid; (b) comparing the amount of the said at least one oxidized lipid to a reference amount, whereby it is predicted whether a subject is at risk of developing neuropathic pain.

9. The method of claim 8, wherein said at least one oxidized lipid is an epoxylipid.

10. The method of claim 8, wherein said neuropathic pain is chemotherapy-induced neuropathic pain (CIPN).

11. The method of claim 8, wherein the amount of the at least one oxidized lipid is determined 24 h after the start of chemotherapy.

12. A device for carrying out a method according to claim 1, comprising: a) an analysing unit comprising at least one detector for at least one oxidized lipid as predictive and/or diagnostic biomarker, wherein said analyzing unit is adapted for determining the amount of at least one oxidized lipid as predictive and/or diagnostic biomarker by the at least one detector; and, operatively linked thereto b) an evaluation unit comprising a computer comprising tangibly embedded a computer program code for carrying out a comparison of the determined amount of the at least one oxidized lipid as predictive and/or diagnostic biomarker, with a reference and a data base comprising said reference for said at least one oxidized lipid as predictive and/or diagnostic biomarker, whereby it is predicted and/or diagnosed whether a subject suffers from neuropathic pain.

13. A method for determining whether a neuropathic pain therapy is successful, the method comprising: a) determining at least one oxidized lipid in a first and a second sample of the subject wherein said first sample has been taken prior to or at the onset of the neuropathic pain therapy and said second sample has been taken after the onset of the said therapy; and b) comparing the amount of the said at least one oxidized lipid in the first sample to the amount in the second sample, whereby a change in the amount determined in the second sample in comparison to the first sample is indicative for neuropathic pain therapy being successful.

14. The method of claim 13, wherein said neuropathic pain therapy comprises administering a cytochrome P450 expoygenase (CYP)-antagonist.

15. (canceled)

16. The method of claim 3, wherein said at least one expoxylipid is 9,10-EpOME (()9(10)-epoxy-12Z-octadecaenoic acid).

17. The method of claim 9, wherein said epoxylipid is selected from the group consisting of: 9,10-EpOME (()9(10)-epoxy-12Z-octadecaenoic acid), 9-HODE (()9-hydroxy-10(E),12(Z)-octadecadienoic acid) and 13-HODE (()13-hydroxy-9(Z),11(E)-octadecadienoic acid).

18. The method of claim 9, wherein said at least one expoxylipid is 9,10-EpOME (()9(10)-epoxy-12Z-octadecaenoic acid).

19. The method of claim 10, wherein said chemotherapy-induced neuropathic pain (CIPN) is induced by paclitaxel and/or oxaliplatin.

20. The device of claim 12, wherein the at least one oxidized lipid as predictive and/or diagnostic biomarker is 9,10-EpOME.

Description

FIGURES

[0095] FIG. 1: (A) Concentrations of 9,10-EpOME in nervous tissue (sciatic nerve, lumbar dorsal root ganglia (DRGs) and dorsal horn of the spinal cord 24 h after i.p.-injection of paclitaxel (6 mg/kg) or oxaliplatin (3mg/kg). Data are shown as meanSEM from five mice per group; one-way ANOVA, *p<0.05, ***p<0.001. (B) time-course of mechanical allodynia after paclitaxel-injection in mice.

[0096] FIG. 2: (A) Concentrations of 9,10-EpOME in nervous tissue (sciatic nerve, lumbar dorsal root ganglia (DRGs) and dorsal horn of the spinal cord 8 d after multiple i.p.-injection of paclitaxel (42 mg/kg, injection every other day). (B) Concentrations of 6-keto-PGF.sub.1 in nervous tissue (sciatic nerve, lumbar dorsal root ganglia and dorsal horn of the spinal cord 8 d after multiple i.p.-injection of paclitaxel (42 mg/kg, injection every other day). Concentrations of 12S-(C) and 15S-HETE (D) 8 d after i.p.-injection of paclitaxel (6 mg/kg) in nervous tissue. Data are shown as meanSEM from five mice per group; one-way ANOVA, *p<0.05, **p<0.01, n.d: not determined.

[0097] FIG. 3: Concentrations of 13-HODE (A)and 9-HODE (B) in nervous tissue (sciatic nerve, lumbar dorsal root ganglia (DRGs) and dorsal horn of the spinal cord 10 d after i.p.-injection of oxaliplatin (3 mg/kg, injection every other day). Data are shown as meanSEM from five mice per group; one-way ANOVA, *p<0.05.

[0098] FIG. 4: Plasma-concentrations of 9,10-EpoME and its Metabolite 9,10-DiHOME 8d after paclitaxel-treatment (6 mg/kg) can be reduced by administration of telmisartan (10 mg/kg, 2h). Data are shown as meanSEM from five mice per group; one-way ANOVA, *p<0.05.

[0099] The invention will now be described by way of Examples. However, the Examples shall not be construed, whatsoever, as limiting the scope of the invention.

EXAMPLES

Example 1: Models of Chemotherapy-Induced Peripheral Neuropathic Pain

[0100] All animal experiments were performed according to the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the local Ethics Committees for Animal Research (Darmstadt) with the permit number F95/42. For all behavioral experiments, only 6-12 weeks old male C57BL/6N mice were used and purchased from commercial breeding companies (Charles River, Sulzfeld, Germany, Janvier, Le Geneset-Saint-Isle, FR). To compare mechanical thresholds, age and sex matched littermates were used as control.

[0101] Paclitaxel was dissolved in Cremophor EL/Ethanol 1:1 and diluted in saline. The dose for intraperitoneal injection was set to 6 mg/kg as described previously [4]. Oxaliplatin was dissolved in saline. The dose for intraperitoneal injection was set to 3 mg/kg as described previously [5].

Example 2: Measurement of Oxidized Lipids From Plasma Using LC-MS/MS

Standards and Internal Standards

[0102] For the measurement of epoxy lipids and HETEs stock solutions of the analytes 9,10-EpOME, 9-HODE, 13-HODE, 12-HETE and 15-HETE and internal standards 9,10-EpOME-d4, 9-HODE-d4, 13-HODE-d4, 12-HETE-d4 and 15-HETE-d4 are generated with concentrations of 2500 ng/ml in ethanol. Working standards were obtained by further dilution with a concentration range of 0.1-250 ng/ml for all analytes.

[0103] For prostanoids, stock solutions with 50,000 ng/ml of all analytes (6-keto-PGF.sub.1) and internal standards (6-keto-PGF.sub.1-d4) were prepared in methanol. Working standards were obtained by further dilution with a concentration range of 0.1-1,250 ng/ml

Lipid-Extraction From Plasma

[0104] Lipids are extracted twice with 600 l of ethyl acetate using liquid-liquid extraction. The combined organic phases were removed at a temperature of 45 C. under a gentle stream of nitrogen. The residues were reconstituted with 50 l of methanol/water/butylated hydroxytoluene (BHT) (50:50:10.sup.3, v/v/v) (EpOMEs, HODEs and HETEs), or 50 l of acetonitrile/water/formic acid (20:80:0.0025, v/v/v) (6-keto-PGF.sub.1) and then centrifuged for 2 min at 10,000 g, and transferred to glass vials waiting for analysis.

Instrumentation For Lipid Measurement

[0105] The LC-MS/MS system consists of a QTrap 5500 (AB Sciex, Darmstadt, Germany) equipped with a Turbo-V source operating in negative electrospray ionization mode, an Agilent 1200 binary HPLC pump and degasser (Agilent, Waldbronn, Germany), and an HTC Pal autosampler (CTC analytics, Zwingen, Switzerland). High-purity nitrogen for the mass spectrometer was produced by a NGM 22-LC-MS nitrogen generator (cmc Instruments, Eschborn, Germany).

[0106] For the chromatographic separation of EpOMEs, HODEs and HETEs, a Gemini NX C18 column and precolumn were used (1502 mm inner diameter, 5 m particle size, and 110 pore size; Phenomenex, Aschaffenburg, Germany). For the chromatographic separation of prostanoids, a Synergi 4 u Hydro-RP column (1502 mm inner diameter, 4 m, Phenomenex, Aschaffenburg, Germany) and a precolumn of same material were used.

LC-Gradient and Data Analysis

[0107] For measurements of EpOMEs, HODEs und HETEs a linear gradient was used at a flow rate of 0.5 ml/min with a total run time of 17.5 min. Mobile phase A consist of water: ammonia (100:0.05, v/v), and mobile phase B of acetonitrile ammonia (100:0.05, v/v). The gradient changed from 85% A to 10% within 12 min. These conditions were held for 1 min. Then, the mobile phase shifted back to 85% A within 0.5 min and it was maintained for 4 min to re-equilibrate the column.

[0108] For the chromatographic separation of prostanoids, a Synergi 4 u Hydro-RP column (1502 mm inner diameter, 4 m, Phenomenex, Aschaffenburg, Germany) and a precolumn of same material were used. Chromatographic separation was carried out in gradient elution mode at a flow rate of 0.3 ml/min, Total run time was 16 min Mobile phase A consisted of water/formic acid (100:0.0025, v/v), and mobile phase B of acetonitrile/formic acid (100:0.0025, v/v). The linear gradient started with 90% A for 1 min and then changed to 60% A within 1 min, It was held for 1 min at 60% in phase A. Within 1 min, the mobile phase shifted to 50% in phase A and was held for 2 min. Within 2 min, the mobile phase shifted to 10% A and was held for 1 min. Composition of the gradient shifted back to 90% A in one min and it was maintained for 6 min to re-equilibrate the column.

[0109] A volume of 20 l (EpOMEs, HODEs und HETEs) or 45 l (prostanoids) of the extracted samples was injected into the LC-MS/MS system. Quantification was performed with Analyst software version 1.6 (Applied Biosystems) using the internal standard method (isotope-dilution mass spectrometry). Ratios of analyte peak area and internal standard area. (y-axis) were plotted against concentration (x-axis), and calibration curves were calculated by least-squares regression with 1/square concentration weighting.

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