COMPOSITIONS FOR DETECTING CELL DEATH AND METHODS OF USE THEREOF

• US 20100034800A1
• Filed: 07/29/2009
• Published: 02/11/2010
• Est. Priority Date: 07/30/2008
• Status: Active Grant

• Current Assignee: Dana-Farber Cancer Institute
• Original Assignee: Dana-Farber Cancer Institute
• Inventors: Banerjee, Pallab, Kung, Andrew
• Granted Patent: US 8,999,661 B2
• Time in Patent Office: Days
• US Class Current: 424/94.600
• CPC Class Codes: C12Q 1/32 involving dehydrogenase
• CPC Class Codes: G01N 2510/00 Detection of programmed cell death, i.e. apoptosis
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity

Abstract

The invention provides compounds and methods of their use in the detection of apoptosis and necrosis both in vitro and in vivo. Also provided are compounds and methods of their use in selective delivery of agents to cells undergoing apoptosis or necrosis. The compounds and methods are based on conjugates formed with a dehydrogenase such as lactate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, and malate dehydrogenase. The compounds and methods are useful in the diagnosis and treatment of conditions characterized by apoptosis, including cancer, cardiac disease, neurologic disease including stroke, and autoimmunity. The compounds and methods offer distinct advantages over corresponding compounds and methods based on Annexin V. Also provided are methods for screening for compounds that modulate, i.e., inhibit or promote, apoptosis.

Claims

1. An isolated conjugate of a dehydrogenase linked to a detectable label, wherein the detectable label is not an immunoglobulin or dehydrogenase-binding fragment thereof.

2-10. -10. (canceled)

11. An isolated conjugate of a dehydrogenase linked to at least one therapeutic agent.

12-17. -17. (canceled)

18. A polynucleotide encoding a dehydrogenase operably linked to a polynucleotide encoding a detectable label.

19-26. -26. (canceled)

27. A method for detecting cells undergoing cell death, the method comprising:

contacting a test population of cells with a conjugate of a dehydrogenase linked to a detectable label, under conditions suitable for uptake of the conjugate by the test population of cells; and

detecting the label in the test population of cells,

wherein presence of the label in the test population of cells indicates cells undergoing cell death.

28. The method of claim 27, wherein the dehydrogenase is a lactate dehydrogenase (LDH).

29. The method of claim 28, wherein the LDH is a human LDH.

30. The method of claim 27, wherein the dehydrogenase is selected from the group consisting of:

alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH) and malate dehydrogenase (MDH).

31. The method of claim 30, wherein the dehydrogenase is a human dehydrogenase.

32. The method of claim 27, wherein the cell death is apoptosis.

33. The method of claim 27, wherein the cell death is necrosis.

34. The method of claim 27, wherein the contacting and the detecting are carried out in vitro.

35. The method of claim 27, wherein the contacting and the detecting are carried out ex vivo.

36. The method of claim 27, wherein at least one of the contacting and the detecting is carried out in vivo.

37. The method of claim 36, wherein the contacting and the detecting are carried out in vivo.

38. The method of claim 27, wherein the test population of cells is obtained from a subject having or suspected of having a condition associated with apoptosis.

39. The method of claim 38, wherein the condition is selected from the group consisting of:

cancer;

chemotherapy-, radiation-, or hormone-induced apoptosis in solid and hematological tumors;

tumor resistance to therapy;

acute cardiac allograft rejection;

acute myocardial infarction;

anthracycline-induced cardiotoxicity;

arrhythmogenic right ventricle dysplasia;

skeletal muscle apoptosis;

congestive heart failure;

coronary artery disease;

atherosclerosis;

infectious endocarditis;

myocarditis;

myocardial dysfunction;

myocardial ischemia-reperfusion injury;

non-cardiac allograft rejection;

bacterial infection;

viral infection;

multiple organ dysfunction syndrome;

septic shock;

cerebral ischemia-reperfusion injury;

macular degeneration;

neurodegenerative disease;

central nervous system trauma;

autoimmune diabetes mellitus;

rheumatoid arthritis;

systemic lupus erythematosus;

inflammatory bowel disease;

multiple sclerosis;

other autoimmune diseases;

annexinopathies;

osteoarthritis;

renal failure;

chronic renal atrophy and renal fibrosis;

glomerular injury; and

polycystic renal disease.

40. The method of claim 27, further comprising:

measuring a test amount of label detected in the test population of cells;

comparing the test amount of label detected in the test population of cells to a reference amount of label detected in a reference population of cells; and

determining (a) there is an increased amount of cell death when the test amount is greater than the reference amount, or (b) there is a decreased amount of cell death when the test amount is less than the reference amount.

41-42. -42. (canceled)

43. A method for treating a condition associated with apoptosis, the method comprising:

administering to a subject having or suspected to have a condition associated with apoptosis a pharmaceutical composition comprising a conjugate of a dehydrogenase linked to at least one therapeutic agent in an amount effective to treat the condition.

44-49. -49. (canceled)

50. A method for selectively delivering an agent to an apoptotic cell, the method comprising:

contacting an apoptotic cell with a conjugate of a dehydrogenase linked to at least one agent.

51-56. -56. (canceled)

57. A method for identifying an anti-apoptotic agent, comprising:

contacting a test cell with a test agent and a conjugate of a dehydrogenase linked to a detectable label, under conditions that otherwise induce apoptosis in the test cell;

measuring a test amount of label in the test cell;

comparing the test amount of label to a reference amount of label in a reference cell contacted with the conjugate under the conditions that otherwise induce apoptosis in the test cell; and

identifying the test agent as an anti-apoptotic agent when the test amount of label is less than the reference amount of label.

58-65. -65. (canceled)