Lysophosphatidic acid detection

Patent metadata

Field	Value
Publication number	US-9952231-B2
Application number	US-201414776273-A
Country	US
Kind code	B2
Filing date	Mar 14, 2014
Priority date	Mar 15, 2013
Publication date	Apr 24, 2018
Grant date	Apr 24, 2018

Abstract

Embodiments of methods and compounds for isolating and detecting lysophosphatidic acids (LPAs) are disclosed. Kits for performing the methods also are disclosed. LPAs are isolated from biological samples by liquid-liquid extraction followed by solid phase extraction. LPA species may be separated by HPLC, and the separated species may be identified and quantified. Also disclosed are embodiments of compounds capable of universally detecting a plurality of LPA species with substantially equivalent sensitivity. Embodiments of the disclosed compounds are useful for determination of total LPA concentration in a sample comprising a plurality of LPA species without separation of individual LPA species.

First claim

We claim:

1. A compound according to
(a) general formula I

wherein:
R 1 -R 6 independently are hydrogen, hydroxyl, thiol, C 1 -C 10 alkyl, carboxyalkyl, amino, C 1 -C 10 alkoxy, or halogen,
R 7 -R 10 independently are hydrogen, alkyl, acyl, carboxyl, nitro, amino, alkylamino, or —SO 3 H,
R 11 is N—C(═R 13 )—NH 2 , N—C(NH 2 )═N—C(═R 13 )—NH 2 , or N—NH—C(NH 2 )═N—C(═R 13 )—NH 2 , where R 13 is O, S, or NH, or R 11 is N—NH—C(═R 13 )—NH 2 , where R 13 is O or NH,
each R 12 independently is hydrogen or lower alkyl, or each of R 1 , R 2 , R 5 , and R 6 may together with an adjacent R 12 and N atom form a 6-membered heterocyclic ring, and
X is O, S, CH 2 , NH, or SiR 14 where R 14 is H or C 1 -C 10 alkyl; or
(b) general formula II

wherein:
R 1 -R 5 , R 15 , R 17 , and R 18 independently are hydrogen, hydroxyl, thiol, C 1 -C 10 alkyl, carboxyalkyl, amino, C 1 -C 10 alkoxy, or halogen,
R 16 is —N(R 12 ) 2 ,
R 7 -R 10 independently are hydrogen, alkyl, acyl, carboxyl, nitro, amino, alkylamino, or —SO 3 H,
R 11 is N—C(═R 13 )—NH 2 , N—NH—C(═R 13 )—NH 2 , N—C(NH 2 )═N—C(═R 13 )—NH 2 , or N—NH—C(NH 2 )═N—C(═R 13 )—NH 2 , where R 13 is O, S, or NH,
each R 12 independently is hydrogen or C 1 -C 10 alkyl, or each of R 1 , R 2 , R 15 , and R 17 may together with an adjacent R 12 and N atom form a 6-membered heterocyclic ring, and
X is O, S, CH 2 , NH, or SiR 14 where R 14 is H or C 1 -C 10 alkyl; or
(c) general formula III

wherein:
R 2 -R 5 , R 15 , R 17 , and R 19 -R 21 independently are hydrogen, hydroxyl, thiol, C 1 -C 10 alkyl, carboxyalkyl, amino, C 1 -C 10 alkoxy, or halogen,
one of R 16 and R 18 is hydrogen, hydroxyl, thiol, lower alkyl, carboxyalkyl, amino, C 1 -C 10 alkoxy, or halogen, and the other of R 16 and R 18 is —N(R 12 ) 2 ,
R 7 -R 10 independently are hydrogen, alkyl, acyl, carboxyl, nitro, amino, alkylamino, or —SO 3 H,
R 11 is N—C(═R 13 )—NH 2 , N—NH—C(═R 13 )—NH 2 , N—C(NH 2 )═N—C(═R 13 )—NH 2 , or N—NH—C(NH 2 )═N—C(═R 13 )—NH 2 , where R 13 is O, S, or NH,
each R 12 independently is hydrogen or C 1 -C 10 alkyl, or if R 16 is —N(R 12 ) 2 , each of R 15 , R 17 , R 20 , and R 21 may together with an adjacent R 12 and N form a 6-membered heterocyclic ring, and
X is O, S, CH 2 , NH, or SiR 14 where R 14 is H or C 1 -C 10 alkyl; or
(d) general formula IV

wherein:
R 1 -R 4 , R 6 , and R 22 -R 24 independently are hydrogen, hydroxyl, thiol, C 1 -C 10 alkyl, carboxyalkyl, amino, C 1 -C 10 alkoxy, or halogen,
R 7 -R 10 independently are hydrogen, alkyl, acyl, carboxyl, nitro, amino, alkylamino, or —SO 3 H,
R 11 is N—C(═R 13 )—NH 2 , N—NH—C(═R 13 )—NH 2 , N—C(NH 2 )═N—C(═R 13 )—NH 2 , or N—NH—C(NH 2 )═N—C(═R 13 )—NH 2 , where R 13 is O, S, or NH,
each R 12 independently is hydrogen or C 1 -C 10 alkyl, or each of R 1 , R 2 , R 23 , and R 24 may together with an adjacent R 12 and N form a 6-membered heterocyclic ring, and
X is O, S, CH 2 , NH, or SiR 14 where R 14 is H or C 1 -C 10 alkyl.

2. The compound of claim 1 , wherein X is O.

3. The compound of claim 1 according to:
general formula I, wherein R 1 -R 10 are H;
general formula II, wherein R 1 -R 5 ; R 7 -R 10 , R 15 , R 17 , and R 18 are hydrogen;
general formula III, wherein R 2 -R 5 ; R 7 -R 10 , R 15 , R 17 , and R 19 -R 21 are hydrogen, one of R 16 and R 18 is hydrogen, and the other of R 16 and R 18 is —N(R 12 ) 2 ; or
general formula IV, wherein R 1 -R 4 , R 6-10 , and R 22 -R 24 are hydrogen.

4. The compound of claim 1 , wherein the compound is

5. A method for quantifying lysophosphatidic acid species, comprising:
combining a sample that may include one or more lysophosphatidic acid species with a compound according to claim 1 in a solvent comprising dimethylsulfoxide in methanol to form a solution;
exposing the solution to a light source;
measuring fluorescence intensity of the solution; and
determining, based on the fluorescence intensity, a total concentration of lysophosphatidic acid species in the sample.

6. The method of claim 5 , further comprising obtaining the sample by extracting lysophosphatidic acid species from a sample comprising plasma or serum, wherein the sample comprising plasma or serum is obtained from a subject suspected of being at risk of a condition associated with an aberrant LPA level, the method further comprising determining a risk level for the condition, wherein the risk level is based at least in part on the total concentration of lysophosphatidic acid species.

7. The method of claim 5 , wherein the compound is

and fluorescence intensity is measured at 570 nm.

8. The method of claim 6 , wherein the condition is cancer, cardiovascular disease, platelet aggregation, ischemia perfusion injury, neuropathic pain, a neuropsychiatric disorder, a reproductive disorder, or fibrosis.

9. A kit for detecting and quantifying lysophosphatidic acid, comprising at least one compound according to claim 1 .

10. The kit of claim 9 , wherein the compound is

11. The method of claim 6 , wherein the condition is ovarian cancer.

12. The method of claim 6 , wherein extracting lysophosphatidic acid species from the sample comprising plasma or serum further comprises:
combining the sample comprising plasma or serum with a solvent comprising a C 1 -C 10 alkyl alcohol and chloroform to form a mixture;
separating organic and aqueous layers of the mixture;
extracting the aqueous layer with a buffer at neutral pH to form an extracted aqueous phase;
mixing the extracted aqueous phase with chloroform;
separating chloroform from the extracted aqueous phase to form a washed aqueous phase;
adding phosphoric acid to the washed aqueous phase to form an acidified aqueous phase;
loading the acidified aqueous phase onto a solid-phase extraction (SPE) cartridge including a stationary phase comprising silica derivatized with hydrocarbon chains;
flowing water and subsequently chloroform through the SPE cartridge;
drying the SPE cartridge; and
flowing a C 1 -C 10 alkyl alcohol through the SPE cartridge, thereby eluting lysophosphatidic acid species in the C 1 -C 10 alkyl alcohol from the SPE cartridge.

13. The compound of claim 1 , wherein R 13 is NH.

Assignees

• Univ Portland State

Inventors

• Strongin Robert
• Sibrian-Vazquez Martha
• Wang Jialu
• Wang Lei
• Escobedo-Cordova Jorge O

Classifications

• C09B11/24

  Phthaleins containing amino groups {; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes} · CPC title

• G01N33/92 Primary

  involving lipids, e.g. cholesterol {, lipoproteins, or their receptors (steroid hormones G01N33/743)} · CPC title

• C07D491/107 Primary

  with only one oxygen atom as ring hetero atom in the oxygen-containing ring · CPC title

• C07D491/22

  in which the condensed system contains four or more hetero rings · CPC title