Methods for determining steroid responsiveness

• US 6,878,518 B2
• Filed: 01/22/2002
• Issued: 04/12/2005
• Est. Priority Date: 01/22/2002
• Status: Active Grant

• Current Assignee: University of Pennsylvania
• Original Assignee: Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Inventors: Whitehead, Alexander Steven
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US10/045,360
• Publication Number:
  US 20030138781A1
• Time in Patent Office: 1,176 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/24.3
• US Class Current: 506/14
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/743 Steroid hormones

Abstract

The invention provides a diagnostics assay for measuring the responsiveness to a drug by comparing the mRNA levels of a gene that responds to the drug, such as a steroid, to the mRNA levels of a gene that does not respond to the drug. Methods according to the invention are useful for predicting the ability of a patient (or a tissue, body fluid or cell sample in vitro) to respond to a drug or steroid at any stage of their treatment (i.e., before, during or after), and to monitor the patient (or a tissue, body fluid or cell) over time to assess continued responsiveness to the drug or steroid.

Claims

1. A method for determining steroid responsiveness in a subject, the method comprising the steps of:

(a) determining a first level of expression of RNA from a first gene known or suspected to be steroid responsive in a tissue, body fluid or cell from a subject undergoing steroid treatment;

(b) determining a second level of expression of RNA from a second gene known or suspected to be non-responsive to steroids in the tissue, body fluid or cell; and

(c) comparing the first and second levels of RNA to create a ratio;

(d) determining the steroid responsiveness of the subject based on the ratio, wherein the subject is steroid responsive if the ratio is higher than a predetermined control ratio for untreated or nonresponsive subjects, or similar to prior ratios for the subject when the subject was previously determined to be responsive.

8. The method of claim 1, wherein one or more of the determining steps requires amplification of the RNAs.

9. The method of claim 8, wherein one or more of the determining steps requires polymerase chain reaction (PCR) of the RNAs.

10. The method of claim 1, wherein one or more of the determining steps requires in situ detection of the first and second RNA.

11. The method of claim 1, wherein one or more of the determining steps requires direct probing of the RNAs.

12. The method of claim 1, further comprising monitoring or tracking steroid responsiveness over time to detect a change in steroid responsiveness.

13. The method of claim 1, further comprising the step of administering one or more pro-inflammatory and/or anti-inflammatory mediators to the tissue, body fluid or cell.

14. The method of claim 13, wherein the pro-inflammatory mediator is selected from the group consisting of interleukin 1α

(IL-1α

), interleukin-1β

(IL-1β

), interleukin 6 (IL-6), and tumor necrosis factor (TNF-α

).

15. The method of claim 13, wherein the anti-inflammatory mediator is selected from the group consisting of interleukin 1 receptor antagonist (IL-1RA), tumor necrosis factor receptor antagonist (TNF-RA) or derivatives thereof, soluble TNF receptors, anti-TNF antibodies, and anti-TNF-RA antibodies.

16. The method of claim 1, further comprising the step of administering one or more cytokines, chemokines, interferons or hormones to the tissue, body fluid or cell.

17. The method of claim 16, wherein the chemokine is selected from the group consisting of interleukin-8 (IL-8).

18. The method of claim 16, wherein the peptide hormone is selected from the group consisting of vasoactive intestinal peptide (VIP).

19. The method of claim 1, wherein the tissue comprises blood.

20. The method of claim 1, wherein the tissue comprises nucleated cells.

21. The method of claim 1, wherein the cell is selected from the group consisting of a monocyte, a macrophage, a neutrophil, a T-cell, B-cell, a basophil, a fibroblast, an endothelial cell and a epithelial cell.

22. The method of claim 1, wherein the tissue comprises buccal cells.

23. The method of claim 1, wherein the tissue comprises a biopsy sample.

24. The method of claim 1, wherein the tissue sample is stored in a stabilization solution prior to analysis.

25. The method of claim 1, wherein the tissue sample is stored frozen.

26. The method of claim 1, wherein the first gene encodes serum amyloid A1 (SAA1).

27. The method of claim 1, wherein the second gene encodes serum amyloid A2 (SAA2).

28. The method of claim 1, wherein the first gene or second gene encodes a chemokine, a cytokine agonist, a cytokine antagonist, or a complement component.

29. The method of claim 1, further comprising the step of quantifying the RNA level of a third gene and comparing the RNA level from the third gene to the RNA level from the first gene and the RNA level from the second gene.

30. The method of claim 29, wherein the third gene encodes an acute phase reactant.

31. The method of claim 29, wherein the third gene encodes a chemokines, cytokine agonist, a cytokine antagonist, or a complement component.

32. The method of claim 29, wherein the third gene is selected from the group consisting of C-reactive Protein (CRP), complement component 3 (C3), Factor B, and albumin.

33. The method of claim 1, wherein the subject suffers from an inflammatory condition, a disease with an inflammatory component, a disease with an inflammatory consequence, and/or a disease with inflammatory symptoms.

34. The method of claim 1, wherein the subject is being evaluated as a candidate for, is about to undergo, or has undergone a tissue or organ transplant.

35. The method of claim 1, wherein the subject may be refractory, less responsive, or more responsive to steroid treatment.

36. The method of claim 1, wherein the subject has cancer, is being treated for cancer, or is in remission for cancer.

37. The method of claim 1, wherein the cancer is selected from the group consisting of solid tumors, acute lymphocytic leukemia and lymphoma.

38. The method of claim 1, wherein the subject is steroid dependent.

39. The method of claim 1, wherein the subject suffers from an arthritic disease.

40. The method of claim 39, wherein the arthritic disease is selected from the group consisting of osteoarthritis, rheumatoid arthritis, psoriatic arthritis and idiopathic arthritis.

41. The method of claim 1, wherein the subject suffers from an autoimmune disease.

42. The method of claim 1, wherein the subject suffers from an inflammatory bowel disease.

43. The method of claim 42, wherein the subject suffers from Crohn'"'"'s disease or ulcerative colitis.

44. The method of claim 1, wherein the subject suffers from asthma, adult respiratory distress syndrome, systemic lupus erythematosus, inflammatory bowel disease, Crohn'"'"'s disease, ulcerative colitis, multiple sclerosis, insulin-dependent diabetes mellitus, autoimmune arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, inflammatory pulmonary syndrome, pemphigus vulgaris, idiopathic thrombocytopenic purpura, cerebral edema, autoimmune meningitis, myasthenia gravis, autoimmune thyroiditis, sarcoidosis, dermatitis, atopic dermatitis, eczematous dermatitis, psoriasis, Sjogren'"'"'s Syndrome, sarcoidosis, keratoconjunctivitis sicca secondary to Sjogren'"'"'s Syndrome, alopecia areata, allergic responses due to arthropod bite reactions, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, Stevens-Johnson syndrome, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, aplastic anemia, pure red cell anemia, autoimmune destruction of erythrocytes, idiopathic thrombocytopenia, polychondritis, Wegener'"'"'s granulomatosis, chronic active hepatitis, alcohol liver disease, Graves ophthalmopathy, primary biliary cirrhosis, uveitis posterior and interstitial lung fibrosis or a combination thereof.

45. The method of claim 1, wherein the subject suffers from a renal, allergic, infectious, ocular, skin, gastrointestinal, and/or endocrine disease.

46. The method of claim 1, wherein the subject suffers from stroke, coronary artery disease, vascular disease, atherothrombotic disease and/or spinal cord injury.

47. The method of claim 1, wherein the subject suffers from an endocrine disease.

48. The method of claim 1, wherein the subject is being evaluated as a candidate for, is about to undergo, or has undergone steroid replacement or substitution therapy.

49. The method of claim 1, wherein the subject suffers from acute adrenal insufficiency, chronic primary adrenal insufficiency, secondary adrenal insufficiency, and/or congenital adrenal hyperplasia.

50. The method of claim 1, wherein the first gene is controlled by a steroid responsive element.

51. The method of claim 50, wherein the steroid responsive element is a glucocorticoid responsive element (GRE).

52. The method of claim 51, wherein the GRE is a consensus GRE or a non-consensus GRE.

53. The method of claim 52, wherein the consensus GRE is GGTACAnnnTGTTCT or a variation thereof, where n is any nucleotide.

54. The method of claim 1, wherein the second gene is encoded by a gene which is not controlled by a steroid response element.

55. The method of claim 1, wherein the steroid is selected from the group consisting of a glucocorticoid, an estrogen, and an androgen.

56. The method of claim 1, further comprising the step of administering one or more glucocorticoid inhibitors, glucocorticoid antagonists or other steroid inhibitors or steroid antagonists.

57. The method of claim 56, wherein the glucocorticoid inhibitor is selected from the group consisting of mitotane, metyrapone, aminoglutethimide, ketoconazole, and trilostane.

58. The method of claim 1, wherein the steroid is selected from the group consisting of alclometasone diproprionate, amcinonide, beclomethasone diproprionate, betamethasone, betamethasone benzoate, betamethasone diproprionate, betamethasone sodium phosphate, betamethasone sodium phosphate and acetate, betamethasone valerate, clobetasol proprionate, clocortolone pivalate, cortisol (hydrocortisone), cortisol (hydrocortisone) acetate, cortisol (hydrocortisone) butyrate, cortisol (hydrocortisone) cypionate, cortisol (hydrocortisone) sodium phosphate, cortisol (hydrocortisone) sodium succinate, cortisol (hydrocortisone) valerate, cortisone acetate, desonide, desoximetasone, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, diflorasone diacetate, fludrocortisone acetate, flunisolide, fluocinolone acetonide, flucinonide, fluorometholone, flurandrenolide, halcinonide, medrysone, methylprednisolone, methylprendnisolone acetate, methylprednisolone sodium succinate, mometasone furoate, paramethasone acetate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, prednisone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate, and triamcinolone hexacetonide, or a synthetic analog thereof, or a combination thereof.

59. The method of claim 1, wherein the steroid is administered parenterally, orally or locally.

60. The method of claim 1, wherein the steroid is administered intravenously, intramuscularly, enterally, transdermally, nasally, transmucosally, via inhilation, and/or subcutaneously.

2. A method for determining steroid responsiveness in a tissue, body fluid or cell, the method comprising the steps of:

(a) exposing a tissue, body fluid or cell sample in vitro to a steroid;

(b) determining a first level of expression of RNA from a first gene known or suspected to be steroid responsive;

(c) determining a second level of expression of RNA from a second gene known or suspected to be non-responsive to steroids;

end

(d) comparing the first and second levels of RNA to create a ratio;

(e) determining the steroid responsiveness of the tissue, body fluid or cell based on the ratio, wherein the tissue, body fluid or cell sample is steroid responsive if the ratio is higher than a predetermined control ratio for untreated or nonresponsive subjects, or similar to prior ratios for the subject when the subject was previously determined to be responsive.

3. A method for determining steroid responsiveness in a subject, the method comprising the steps of:

(a) determining a level of expression of an RNA from a first gene known or suspected to be responsive to steroids in a pre-treatment tissue, body fluid, or cell from a subject;

(b) determining a level of expression of an RNA from a second gene known or suspected to be un-responsive to steroids in the pre-treatment tissue, body fluid, or cell;

(c) determining a post-treatment level of RNA expressed from the first gene;

(d) determining a post-treatment level of RNA expressed from the second gene;

(e) comparing the pre-treatment level of RNA expressed from the first gene to the pre-treatment level of RNA expressed from the second gene to create a first normalized value;

(f) comparing the post-treatment level of RNA expressed from the first gene to the post-treatment level of RNA expressed from the second gene to create a second normalized value;

(g) comparing the first normalized value to the second normalized value;

(h) determining steroid responsiveness of the subject based upon the comparison of the first normalized value to the second normalized value, wherein if the first normalized value is less than the second normalized value, it is indicative of steroid responsiveness in the tissue, body fluid or cell, and/or if the first normalized value is greater than or the same as the second normalized value, it is indicative of steroid non-responsiveness in the cell, and/or wherein the difference between the first normalized value and the second normalized value correlates to the ability of the subject to respond to the steroid.

4. A method for determining an effective dose of a steroid in a subject, the method comprising the steps of:

(a) administering to a subject a dose of a steroid;

(b) determining a first level of expression of RNA from a first gene known or suspected to be steroid responsive in a tissue, body fluid, or cell from the subject;

(c) determining a second level of expression of RNA from a second gene known or suspected to be non-responsive to steroids in the tissue, body fluid, or cell; and

(d) comparing the first and second levels of RNA to create a ratio;

(e) determining the effective dose of the steroid in the subject based on the ratio, wherein the difference between the first RNA level and the second RNA level is indicative of the effectiveness of the steroid dose in the subject.

5. A method for monitoring a subject'"'"'s ability to respond to a steroid, the method comprising the steps of:

(a) administering to a subject a dose of steroid;

(b) determining a first level of expression of RNA from a first gene known or suspected to be steroid responsive in a tissue, body fluid, or cell from the subject;

(c) determining a second level of expression of RNA from a second gene known or suspected to be non-responsive to steroids in the tissue, body fluid, or cell; and

(d) comparing the first and second levels of RNA to create a ratio;

(e) monitoring the subject'"'"'s ability to respond to the steroid based upon the ratio, wherein the subject is steroid responsive if the ratio is higher than a predetermined control ratio for untreated or nonresponsive subjects, or similar to prior ratios for the subject when the subject was previously determined to be responsive.

6. A method for determining drug responsiveness in a subject undergoing drug treatment, the method comprising the steps of:

(a) determining a first level of expression of RNA from a first gene known or suspected to be drug-responsive in a tissue, body fluid, or cell from a subject undergoing treatment with a drug;

(b) determining a second level of expression of RNA from a second gene known or suspected to be non-responsive to the drug in the tissue, body fluid, or cell; and

(c) comparing the first and second levels of RNA to create a ratio;

(d) determining drug responsiveness in the subject undergoing drug treatment based upon the ratio, wherein the subject is drug-responsive if the first level is higher than the second level and the subject is non-responsive to drug if the second level is higher than the first level.

7. A method for determining drug responsiveness in a tissue, body fluid or cell, the method comprising the steps of:

(a) exposing a tissue, body fluid or cell in vitro to a drug;

(b) determining a first level of expression of RNA from a first gene known or suspected to be drug-responsive;

(c) determining a second level of expression of RNA from a second gene known or suspected to be non-responsive to the drug; and

(d) comparing the first and second levels of RNA to create a ratio;

(e) determining drug responsiveness in the tissue, body fluid, or cell based upon the ratio, wherein the tissue, body fluid or cell is drug-responsive if the first level is higher than the second level and the tissue, body fluid or cell is non-responsive to the drug if the second level is higher than the first level.