In vivo detection of apoptosis

Abstract

The invention provides methods and products, such as kits, useful for determining the apoptotic state of cells in an organism.

1 . A method for in vivo determination of the apoptotic state of one or more cells in an animal, comprising detecting the presence or abundance of at least one cell permeant caspase affinity labeling agent in the cells of an animal into which at least one cell permeant caspase affinity labeling agent has been introduced, wherein the presence or abundance of the cell permeant caspase affinity labeling agent correlates with the apoptotic state of the cells. 
     
     
         2 . A method for in vivo determination of whether a therapeutic agent modulates apoptosis in one or more cells in an animal, comprising detecting the presence of at least one cell permeant caspase affinity labeling agent in the cells of an animal that has been previously treated with the therapeutic agent and into which at least one cell permeant caspase affinity labeling agent has been introduced, wherein the presence of the cell permeant caspase affinity labeling agent correlates with the ability of the therapeutic agent to modulate apoptosis. 
     
     
         3 . The method of  claim 2 , wherein the method determines whether the therapeutic agent increases or decreases apoptosis. 
     
     
         4 . A method for in vivo determination of whether a radiation treatment modulates apoptosis in one or more cells in an animal, comprising detecting the presence of at least one cell permeant caspase affinity labeling agent in the cells of an animal into which at least one cell permeant caspase affinity labeling agent has been introduced, wherein the animal has been previously treated with radiation, wherein the presence of the cell permeant caspase affinity labeling agent correlates with the ability of the radiation to modulates apoptosis. 
     
     
         5 . The method of  claim 4 , wherein the method determines whether the radiation treatment increases or decreases apoptosis. 
     
     
         6 . An in vivo diagnostic method for determining the presence or absence of a disease characterized by the presence of apoptosis, comprising detecting the presence of at least one caspase affinity labeling agent in the cells of the animal into which at least one caspase affinity labeling agent has been introduced, wherein the presence or absence of at least one caspase affinity labeling agent correlates with the presence or absence of the disease. 
     
     
         7 . The method of  claim 6 , wherein the disease is a disease of the eye, a neurodegenerative disease, or a cardiac disease. 
     
     
         8 . The method of  claim 7 , wherein the disease is glaucoma, macular degeneration, proliferative retinopathy, neuropathy, Alzheimer's disease, multiple sclerosis, or Huntington's disease. 
     
     
         9 . An in vivo method for evaluating the sensitivity of a disease to at least one therapeutic agent or treatment, comprising detecting the presence or abundance of at least one caspase affinity labeling agent in the cells of an animal into which at least one caspase affinity labeling agent has been introduced, wherein the animal has previously received the therapeutic agent or treatment, wherein the presence or abundance of the caspase affinity labeling agent correlates with the sensitivity of the disease to at least one therapeutic agent or treatment. 
     
     
         10 . A method for the monitoring of cancer treatment in an animal, comprising detecting the presence or abundance of at least one caspase affinity labeling agent in the cells of the animal into which at least one caspase affinity labeling agent has been introduced that has also received a therapeutic agent or treatment, wherein the presence or abundance of at least one caspase affinity labeling agent correlates with the efficacy of the therapeutic agent or treatment. 
     
     
         11 . A method for the monitoring of leukemia treatment in an animal, comprising detecting the presence or abundance of at least one caspase affinity labeling agent in the cells of the animal into which at least one caspase affinity labeling agent has been introduced that has also received a therapeutic agent or treatment, wherein the presence or abundance of at least one caspase affinity labeling agent correlates with the efficacy of the therapeutic agent or treatment. 
     
     
         12 . A method for the monitoring of blood and bone marrow disease treatment in an animal, comprising detecting the presence or abundance of at least one caspase affinity labeling agent in the cells of an animal into which at least one caspase affinity labeling agent has been introduced that has also received a therapeutic agent or treatment, wherein the presence or abundance of at least one caspase affinity labeling agent correlates with the efficacy of the therapeutic agent or treatment. 
     
     
         13 . A method for determining if one or more compounds modulate caspase activity in an animal, comprising determining the level of at least one caspase affinity labeling agent in cells of an animal into which at least one caspase affinity labeling agent has been introduced, wherein the animal has also been contacted with one or more compounds, and determining whether the one or more compounds modulates the caspase activity. 
     
     
         14 . A method for determining if one or more compounds modulates apoptosis in an animal, comprising determining the level of at least one caspase affinity labeling agent in cells of an animal into which at least one caspase affinity labeling agent has been introduced, wherein the animal has been contacted with one or more compounds, and determining whether the one or more compounds modulates apoptosis. 
     
     
         15 . The method of  claim 14 , wherein the determination step comprises comparing the level of affinity labeling agent in the animal with a control animal not exposed to the compound. 
     
     
         16 . The method of  claim 14 , wherein the determination step is a longitudinal study that comprises comparing the level of affinity labeling agent in the animal before exposure to the compound and after the animal has been exposed to the compound. 
     
     
         17 . The method of  claim 1 , wherein detection is carried out using NMR, MRI, CT, CAT, PET scan, sctintigraphy, a flow cytometer, a laser scanning cytometer, a fluorescence microplate reader, a luminescence microplate reader, a chromogenic microplate reader, a fluorescence microscope, a confocal microscope, a luminescence microscope, a bright-field microscope, a whole animal fluorescence imaging system, a whole animal luminescence imaging system, or a combination thereof. 
     
     
         18 . The method of  claim 1 , wherein detection is carried out using a window chamber that has been inserted into the animal. 
     
     
         19 . The method of  claim 18 , wherein detection is carried out using a fluorescence microscope, a confocal microscope, a bright-field microscope, or a luminescence microscope. 
     
     
         20 . The method of  claim 1 , wherein detection is confirmed by removing a sample from the animal and detection is carried out on the sample that has been removed from the animal. 
     
     
         21 . The method of  claim 20 , wherein the detection is via flow cytometer; a laser scanning cytometer; a fluorescence microplate reader; a chromogenic microplate reader; a fluorescence microscope; a confocal microscope; a bright-field microscope; a luminescence microplate reader; or a luminescence microscope. 
     
     
         22 . The method of  claim 1 , wherein the presence or abundance of the affinity labeling agent is detected in the bone marrow, thymus, lymph nodes, spleen or circulating blood of the animal. 
     
     
         23 . The method of  claim 1 , wherein the presence or abundance of the affinity labeling agent is detected in peripheral blood monocytes. 
     
     
         24 . The method of  claim 1 , wherein the cells are comprised in tissues, organs or tumors of the animal. 
     
     
         25 . The method of  claim 1 , wherein the caspase affinity labeling agent is introduced into the animal by intravenous, intravascular, intraperitoneal, intravitreal, intraocular, intracranial, intrapleural, intrathoracic, intramuscular, intrapulmonary, injection, perfusion, or lavage administration. 
     
     
         26 . The method of  claim 1 , wherein the animal is a mammal. 
     
     
         27 . The method of  claim 26 , wherein the mammal is a human. 
     
     
         28 . The method of  claim 1 , wherein the caspase affinity labeling agent is a compound of formula I:
   L 1 -A 1 -X 1 —NH—CH(R 1 ′)C(═O)CH 2 F  (I)
   wherein:
 L 1  is a detectable group; 
 A 1  is a direct bond or a linker; 
 X 1  is absent, an amino acid, or a peptide; and 
 R 1 ′ comprises an aspartic acid side-chain, an ester of aspartic acid, an aza-peptide epoxide modification of the aspartic acid, an aza-peptide Michael acceptor, an aldehyde modification of the aspartic terminal carboxyl group, a chloromethyl ketone group, a fluoromethyl ketone group, OPh, or an acyloxy reactive group. 
   
     
     
         29 . The method of  claim 28 , wherein the detectable group comprises a Lanthanide series element. 
     
     
         30 . The method of  claim 28 , wherein the detectable group comprises a positron emitter. 
     
     
         31 . The method of  claim 28 , wherein the detectable group comprises a fluorescent label. 
     
     
         32 . The method of  claim 28 , wherein the detectable group comprises a radioisotope. 
     
     
         33 . The method of  claim 28 , wherein the detectable group comprises Gd, Tb, Eu, Ce, Pr, Nd, Pm, Sm, Dy, Ho, Er, Tm, Yb, Lu, Fe, Mn, Re, and Tc, I Ba,  11 C,  13 N,  15 O,  64 Cu, a fluorescein, a sulforhodamine, a Cy dye, a BODIPY, a coumarin,  3 H,  14 C, or  35 S. 
     
     
         34 . The method of  claim 28 , wherein X 1  is the amino acid or amino acid sequence V, VA, YVA, DEV, LEE, LEH, VDVA (SEQ ID NO:1), IET, WHE, AEV, A, V, or E. 
     
     
         35 . The method of  claim 28 , wherein R 1 ′ is CH 2 —COOH, or —CH 2 CO 2 R, wherein R is C 1 -C 6  alkyl or benzyl, CH 3 , C 2 H 5  or CH 2 C 6 H 5 . 
     
     
         36 . The method of  claim 1 , wherein the caspase affinity labeling agent is carboxyfluorescein-valanyl-alanyl-aspartyl(O-methyl)-fluoromethyl ketone. 
     
     
         37 . The method of  claim 1 , wherein the caspase affinity labeling agent is cell permeant. 
     
     
         38 . The method of  claim 1 , wherein the caspase affinity labeling agent binds covalently to the active catalytic site of a caspase and is retained within the cell. 
     
     
         39 . An assay kit comprising packaging materials, one or more caspase affinity labeling agents, injection buffer, and instructions for using the caspase affinity labeling agents to determine the level of apoptosis in vivo. 
     
     
         40 . The kit of  claim 39 , wherein the one or more caspase affinity labeling agents includes a compound as described in  claim 28 .