**2.1 Radiolabeled annexin V**

Annexin V (also known as annexin A5) is consisted of 319 amino acids and a 36-kD endogeneous human protein that is mainly distributed intracellularly with very high concentrations in placenta, and lower concentrations in endothelial cells, myocardium, skeletal muscle, kidneys, skin, red blood cells, platelets, and monocytes. Very low concentrations of annexin V (1-6 ng/ml) circulate in blood of healthy humans (Andree et al., 1992). Several advantages for the use of annexin V for apoptotic imaging have been described including very high affinity for PS with low nanomolar to subnanomolar dissociation constant values, ready production by recombinant DNA technology, and lack of toxicity of the protein.

In healthy cells, anionic phospholipid PS and phosphatidylethanolamine (PE) confined to the inner leaflet of the lipid bilayer plasma membrane by an ATP dependent enzyme called translocase. On the other hand, ATP dependent enzyme, floppase, pumps cationic phospholipids such as phosphatidylcholine (PC) and sphingomyelin to the cell surface. Therefore, an asymmetric distribution of different phospholipids between the inner and outer leaflet of the plasma membrane is maintained in normal cells. However, at the beginning of the execution phase of apoptosis, rapid redistribution of PS and PC across the cell membrane is facilitated by a calcium ion-dependent deactivation of translocase and floppase and activation of scramblase. Then, PS exposes on the cell surface and annexin V become accessible to PS. This PS exposure is attractive target for imaging apoptosis, since it is a near universal event in apoptosis, it occurs early after the apoptotic stimulus, and it presents millions of binding sites per cell on the cell surface.

Annexin V and its derivatives have been labeled with 123I, 124I, 125I, 99mTc, 18F, thereby providing a broad range of imaging applications in apoptosis research from autoradiography

Apoptosis Imaging in Diseased Myocardium 255

the analogs, WC-II-89 was labeled with 18F (Zhou et al., 2006). 18F- WC-II-89 showed a high uptake in the liver of chemically induced apoptosis. Although molecules that target caspase-3 is attractive, there are limited animal data and it is not yet known how sensitive this kind

Recently a novel family of low molecular mass amphipatic apoptosis markers (ApoSense) was developed targeting the cell membrane of apoptotic cells. 18F-labeled 5-fluoropentyl-2 methyl-malonic acid (18F-ML-10) belongs to this family of low-molecular-weight compounds used for the imaging of cell death in vivo. This compound responds to alterations in plasma membrane potential and phospholipid scrambling, which are hallmarks of apoptotic cells. To which cell membrane targets this probe binds is unknown. After systemic administration, the compound can detect apoptotic cells from the early stages of the death process, cross the intact plasma membrane, and accumulate in the cytoplasm. In animal model of cerebral infarction, selective uptake was observed in the region of ischemia at 24 hr

It has been believed that myocardium directly start to die via necrosis shortly after the onset of myocardial infarction that march of necrosis spread as a wavefront from endocardium to epicardium. However, recent animal experiments with permanent coronary artery occlusion revealed that cell death process starts as apoptosis and severe ATP depletion due to ischemia may preclude the execution of apoptosis and lead to plasma membrane permeability barrier breakdown and secondary necrosis. Therefore, apoptosis imaging might play important role in the assessment of myocardial cell death process in acute

In a rat model of permanent coronary artery ligation, Kajstura et al. investigated the contribution of the apoptosis and necrosis to cardiomyocyte death using TUNEL method and DNA laddering for apoptosis and antimyosin monoclonal antibody labeling (Kajstura, et a., 1996). After 2 h of the left main coronary artery ligation, TUNEL positive myocytes appeared in the central portion of the left ventricular free wall and peaked at 4.5 h. Myosin labeled cells also appeared at 2 h after ligation and significantly increased after 6 h to 2 days. However, at 2 h after coronary ligation, number of apoptotic cells was 2.8 x 106 and number of necrotic cells was 9 x 104. Therefore, early after myocardial infarction, apoptosis is the predominant form of cell death, and 1 to 2 days after infarction, necrosis is the dominant form of cell death followed by low levels of both apoptosis and necrosis at 7 days after infarction. If cells undergoing apoptosis are not cleared before they deplete their intracellular ATP stores that are necessary to maintain plasma membrane integrity, plasma membrane permeability barrier breakdown occurs and cells are converted from apoptosis to

In a mouse model of 15 and 30 min of ischemia and 90 min of reperfusion, percentage of cardiomyocytes staining positivity for biotinylated annexin V was far greater than the

of agents will be for actual imaging in humans.

after the middle cerebral artery occlusion (Reshef et al., 2008).

**3. Apoptosis imaging in acute myocardial ischemia** 

**3.1 Animal experiment in permanent occlusion of coronary artery** 

necrosis as known as secondary necrosis, results in inflammation.

**3.2 Animal experiment in acute ischemia and reperfusion** 

myoacrdial infarction, especially in its early stage.

**2.4 Uncategorized tracer** 

and single photon imaging including SPECT, and to PET. However, most of the radiolabeled annexin V used in clinical trials are 99mTc labeled annexin V, because 99mTc is characterized by the most suitable radionuclide properties for SPECT imaging in human and is inexpensive and easily available.

99mTc-BTAP-annexin V using a diamide dimercaptide N2S2 chelate for labeling (99mTc- N2S2 annexin V) was introduced in early 1990s and examined to detect left atrial thrombi in vivo in swine (Stratton et al., 1995). Biodistribution and dosimetry study of 99mTc-BTAP-annexin V in patients revealed that the radioactivity predominates in kidney, liver and urine bladder and fast and extensive bowel excretion of the tracer precludes the assessment of abdominal region (Kemerink, et al., 2001a). N2S2 labeling method is cumbersome but radiochemical yield is low and had a high degree of non specific tracer excretion into bowel via excretion to bile.

Accordingly, an improved labeling method using the bifunctional agent hydrazinonicotinamide (HYNIC) was introduced (Blankenberg, et al., 1998). 99mTc-labeling of reconstituted HYNIC-annexin V can be performed by simply reacting the conjugate with 99mTc-pertechnetate in the presence of stannous tricine for 5-10min in room temperature and it provides 99mTc-HYNIC-annexin V in high radiochemical yield (usually 92-95%) without requiring any additional purification step. Phase I clinical trial with 99mTc-HYNIC-annexin V also demonstrated strongest uptake in kidney, liver and urine bladder. However, in contrast to 99mTc-BTAP-annexin V no bowel excretion was observed in 99mTc-HYNIC-annexin V, having a favorable biodistribution for imaging of the abdominal as well as thoracic area.

As an alternative methods for radiolabeling annexin V, self chelating annexin V mutants had been introduced (Tait, et al., 2000, 2005). Annexin V mutants with endogeneous site for 99mTc chelation such as V-117 and V-128 have major advantages over the HYNIC chelatior in terms with lower renal retention. Many other kind of 99mTc-labeled annexin V have been introduced, however, only 99mTc-i-annexin V (Kemerink, et al., 2001b) was tested in clinical trial in addition to 99mTc-BTAP-annexin V and 99mTc-HYNIC-annexin V.

As a PET tracer, several approaches to label annexin V with 18F have been developed (Grierson, et al. 2004; Murakami et al., 2004).18F-annexin V has lower uptake in the liver, spleen, and kidneys than 99mTc-HYNIC-annexin V.
