Diagnostic methods for determining susceptibility to convulsive conditions

• US 20060008917A1
• Filed: 04/13/2005
• Published: 01/12/2006
• Est. Priority Date: 09/07/2001
• Status: Active Grant

• Current Assignee: D.F. Weaver Medical Inc.
• Original Assignee: Queen's University At Kingston
• Inventors: Weaver, Donald F., Carran, John R., Campbell, Allyson J., Lyon, Angela P.
• Granted Patent: US 7,153,692 B2
• Time in Patent Office: Days
• US Class Current: 436/90
• CPC Class Codes: G01N 2800/2857 Seizure disorders; Epilepsy
• CPC Class Codes: G01N 33/493 urine
• CPC Class Codes: G01N 33/6812 Assays for specific amino acids

Abstract

The present invention exploits the discovery that amounts of uracil and thymine metabolites, especially β-aminoisobutyric acid, in various bodily fluids, especially urine, are correlated with the occurrence of epilepsy when compared to matched control subjects. Analytical and diagnostic protocols, including a novel high performance liquid chromatography system, for use in the invention are disclosed.

Claims

1. A method of diagnosing a convulsive condition or susceptibility thereto in a subject comprising the steps of

a. analyzing a bodily fluid from a subject for the presence or amount of a neuro-active molecule, or the relative amounts of neuro-active molecules, associated with a convulsive condition;

and

b. diagnosing the subject as at risk of a convulsive condition or susceptibility thereto when the amount of said compound indicates a likelihood of same in said subject.

2. A method of modulating the onset of a convulsive condition in a subject comprising the steps of

analyzing a bodily fluid from a subject at risk of a convulsive condition for the presence or amount of a neuro-active molecule, or the relative amounts of neuroactive molecules, associated with a convulsive condition;

determining from the amount of said compound in said bodily fluid whether said subject is at risk of a convulsive condition; and

treating said subject, if at risk of a convulsive condition, to modulate the onset of said convulsive condition in said subject.

3. A method of diagnosing a convulsive condition or susceptibility thereto in a subject comprising the steps of

analyzing a bodily fluid from a subject for the presence or amount of a β

-amino acid, or the relative amount of β

-amino acid; and

diagnosing the subject as at risk of a convulsive condition when the amount of said β

-amino acid indicates a likelihood of same in said subject.

4. A method of modulating the onset of a convulsive condition in a subject comprising the steps of

analyzing a bodily fluid from a subject at risk of a convulsive condition for the presence or amount of a β

-amino acid, or the relative amount of β

-amino acid;

determining from the amount of said β

-amino acid in said bodily fluid whether said subject is at risk of a convulsive condition, and

treating said subject, if at risk of a convulsive condition, so as to modulate the onset of said convulsive condition in said subject.

5. The method of any claim herein, wherein said neuro-active molecule is a metabolite of uracil or thymine, or a derivative thereof.

7. The method of claim 5, wherein said metabolite is a β

-amino carboxylic acid.

8. The method of any one of claims 5, 6, or 7, wherein said β

-amino acid is selected from β

-aminoisobutyric acid and derivatives thereof.

6. The method of any claim herein, wherein said compound is an amino acid or a derivative thereof.

9. The method of any claim herein, wherein said convulsive condition is selected from the group consisting of epileptogenic associated disorders, epileptogenesis, and non-epileptic convulsions.

10. The method of any claim herein, wherein said convulsive condition is an epileptogenic-associated disorder selected from epilepsy, head trauma, stroke, multiple sclerosis, amyotrophic lateral sclerosis, psychoses, cerebral ischemia, motor neuron disease, Alzheimer'"'"'s disease, chicken-pox, measles, encephalitis, pertussis encephalitis, infections of the CNS, meningitis, encephalitis, subdural haematoma, brain tumour, birth defects, anoxic brain injury dementia, or other disorders in which altered activity of neuro-active molecules is a cause, at least in part, of the disorder.

11. The method of any claim herein, wherein said convulsive condition is selected from the group consisting of epilepsy and non-epileptic convulsions.

12. The method of any claim herein, wherein said subject has not yet developed seizures.

13. The method of any claim herein, wherein said treatment step comprises administering an effective amount of an anti-convulsive pharmaceutical composition.

23. The method of claim 13, wherein said anti-seizure pharmaceutical composition is carbamazepine, clobazam, diazepam, lamotrigine, lorazepam, oxazepam, phenobarbital, phenyloin, primidone, valproate, ethosuximide, topirimate, felbamate, clonazepam, clobazam, nitrazepam, vigabatrin, gabapentin, levetiracetam, zonasimide, or tiagabine, or combinations thereof.

14. The method of any claim herein, wherein said treatment step comprises administering an effective amount of an anti-epileptogenic pharmaceutical composition.

15. The method of any claim herein, further comprising the steps of deproteinizing said bodily fluid.

16. The method of claim 15, wherein said deproteinizing step is ultrafiltration, ultracentrifugation, or chemical precipitation.

17. The method of claim 16, wherein said chemical precipitation step employs sulfosalicylic acid, perchloric acid, trichloroacetic acid, picric acid, acetonitrile, ethanol, acetone, or methanol.

18. The method of any claim herein, further comprising derivatizing said amino acid prior to analyzing it.

19. The method of claim 18, wherein said derivatizing step covalently attaches a chromophore to said amino acid.

20. The method of claim 18, wherein said derivatizing step employs o-phthalaldehyde, 9-fluorenylmethylchloroformate, phenyl isothiocyanate, or 1-dimethylaminonaphthalene-5-sulphonyl chloride.

22. The method of claim 20, wherein said bodily fluid is urine.

21. The method of any claim herein, wherein said bodily fluid is urine, blood, plasma, blood serum, cerebrospinal fluid, sweat, lymph, amniotic fluid, synovial fluid, conjunctival fluid, salivary fluid, vaginal fluid, stool, seminal fluid, bile, tears, or mixtures thereof.

24. The method of any claim herein, wherein said subject is an animal or human.

25. The method of claim 24, wherein said subject has a family history of convulsive conditions, a history of cerebral hypoxia or ischemia, intracranial hemorrhage, central nervous system infection or disease, drug or alcohol withdrawal, fever, trauma, brain tumor, cerebrovascular disease, metabolic disorder, degenerative central nervous system disease, drug or alcohol addiction or use, uremia, hepatic dysfunction, hypoglycemia, epilepsy, or seizure;

or said patient has recently been administered an antibiotic, anesthetic, analgesic, immunomodulatory, psychotropic, sedative, antihistamine, radiographic contrast-enhancing, stimulant or hallucinogenic drug.

26. The method of any claim herein, wherein said subject has suffered a head trauma.

27. The method of claim 26, wherein said convulsive condition is post-traumatic epilepsy (PTE) or a susceptibility thereto.

28. The method of any claim herein, wherein said seizure is selected from the group consisting of complex partial, simple partial, absence, secondary generalized tonic clonic, primary generalized tonic clonic, myoclonic, and atonic.

32. The method of claim 28, wherein the amount of amino acid in the urine is statistically significantly higher than in a matched control population.

33. The method of claim 32, wherein said amino acid is β

-aminoisobutyric acid.

29. The method of any claim herein, wherein the analyzing step comprises chromatography, spectroscopy, spectrometry, or colorimetry.

30. The method of claim 29, wherein said chromatography is high-performance liquid chromatography, thin layer chromatography, or gas chromatography.

31. The method of any claim herein, wherein said treating is carried out when the amount of said amino acid is substantially different from the average amount present in a matched control population.

34. A method of quantifying β

-alanine or β

-aminoisobutyric acid comprising the steps of collecting a urine sample;

deproteinizing said urine sample;

derivatizing the amino acids present in said deproteinized urine sample; and

analyzing said derivatized amino acids by high performance liquid chromatography, said chromatography system comprising a reversed phase column, acetate buffer and methanol mobile phases, an internal standard, and a separation program which produces a resolution for each of β

-alanine and β

-aminoisobutyric acid with all other amino acids and molecules present in said urine of equal to or greater than one.

35. The method of claim 34, wherein said internal standard is D,L-ethionine.

36. The method of claim 35, wherein said reversed phase column is an C₈ or C₁₈ column.

37. A kit for use according to the invention comprising internal or external standards or derivatizing reagents, and instructions for use in the method of any claim herein.

38. The kit according to claim 37, further comprising a chromatography system.

39. The kit according to claim 38, wherein said chromatography system is a HPLC system.

40. The method of any claim herein, wherein said step of determining or diagnosing is positive when the urinary concentration of β

-alanine is greater than 0.8 μ

mol/mmol creatinine or β

-aminoisobutyric acid is greater than 10 μ

mol/mmol creatinine.