• E-mail
• Print
• Comment
• Font Size
• Digg
• del.icio.us
• Discuss article

Structural Requirements for In Vivo Detection of Cell Death With ^Sup 99m^Tc-Annexin V

Posted on: Friday, 20 May 2005, 03:00 CDT

^sup 99m^Tc-Annexin V is used to image cell death in vivo via high-affinity binding to exposed phosphatidylserine. We investigated how changes in membrane-binding affinity, molecular charge, and method of labeling affected its biodistribution in normal mice and its uptake in apoptotic tissues. Methods: An endogenous Tc chelation site (Ala-Gly-GlyCys-Gly-His) was added to the N-terminus of annexin V to create annexin V-128. The membrane-binding affinity of annexin V-128 was then progressively reduced by 1-4 mutations in calcium- binding sites. In addition, mutations were made in other residues that altered molecular charge without altering membrane-binding affinity. All mutant proteins were labeled with ^sup 99m^Tc at the same N-terminal endogenous chelation site. Wild-type annexin V was also labeled with ^sup 99m^Tc after derivatization with hydrazinonicotinamide (HYNIC). Radiolabeled proteins were tested for biodistribution in normal mice and in mice treated to induce apoptosis of the liver. Results: Comparison of ^sup 99m^Tc-annexin V- 128 with ^sup 99m^Tc-HYNIC-annexin V showed that the protein labeled at the endogenous chelation site had the same or higher uptake in apoptotic tissues, while showing 88% lower renal uptake at 60 min after injection. The blood clearance of annexin V was unaffected by changes in either the membrane-binding affinity or the molecular charge. Kidney uptake was unaffected by changes in binding affinity. In marked contrast, uptake in normal liver and spleen decreased markedly as affinity decreased. The same pattern was observed in animals treated with cycloheximide to induce apoptosis. Control experiments with charge mutants showed that the effects seen with the affinity mutants were not due to the concomitant change in molecular charge that occurs in these mutants. Conclusion: (a) All four domains of annexin V are required for optimal uptake in apoptotic tissues; molecules with only 1 or 2 active domains are unlikely to be suitable for imaging of cell death in vivo, (b) Uptake in normal liver and spleen is specific (dependent on phosphatidylserine-binding affinity), whereas renal uptake is nonspecific, (c) ^sup 99m^Tc-Annexin V-128 detects cell death as well as ^sup 99m^Tc-HYNIC-annexin V, while showing 88% less renal retention of radioactivity due to much more rapid urinary excretion of radioactivity.

Key Words: annexin V; apoptosis; biodistribution; site-specific mutagenesis; phosphatidylserine

J Nucl Med 2005; 46:807-815

Apoptosis is important in normal physiology and numerous disease states. We have developed annexin V as a means to image cell death in vivo (1,2). This is based on the protein's high affinity for phosphatidylserine (PS) exposed on the extracellular face of cell membranes (3), which occurs when cells undergo apoptosis (4). Annexin V contains 4 similar domains of 70-80 amino acids arranged in a compact water-soluble structure (5). High-affinity membrane binding depends on multiple calcium-binding sites present in all 4 domains of the molecule (6). Clinical applications for annexin V imaging are being developed in oncology (7), organ transplantation (8), and cardiovascular disease (9).

Although ^sup 99m^Tc-HYNIC-annexin V (HYNIC is hydrazinonicotinamide) has been successfully used in many studies, it has certain limitations-most notably, its very high uptake in the kidney, which results in high radiation dose and may limit imaging in the abdomen (10). Alternative approaches for radiolabeling annexin V with technetium have been described (11-14). Annexin V has also been labeled with ^sup 18^F for PET (75). Although there is some variation in biodistribution and metabolism of these different radiolabeled annexins, many properties of the protein are the same regardless of the method of labeling. Initial blood clearance is relatively rapid (11,16,17). Organ uptake is almost always highest in the kidney, followed by the liver; there is moderate uptake in the spleen, lung, and bowel; uptake is very low in the brain, heart, and skeletal muscle. Recently, novel approaches based on modifications of the protein itself have also been described. There has been a report of a single-domain "mini-annexin" that may be useful for imaging (18). Annexin V has also been modified with polyethylene glycol to prolong its circulation time and, thereby, improve its availability over extended periods for uptake by tumors (19).

As part of an effort to develop improved annexin V-based imaging agents, we wanted to address several questions. First, why do some organs have much higher uptake of annexin V than others? Second, what structural features of annexin V control uptake in both normal and apoptotic tissues? Third, how much loss of membranebinding affinity can be tolerated before annexin V loses its ability to localize in apoptotic tissues? To address these questions, we prepared a series of site-directed mutants of annexin V that alter properties such as charge and membrane-binding affinity. We took advantage of the method we previously developed for ^sup 99m^Tc labeling of annexin V via an N-terminal endogenous chelation site consisting of the amino acid sequence Ala-Gly-Gly-Cys-Gly-His (12). All structural mutants of annexin V contained the same sequence in 1:1 stoichiometry; therefore, the site of ^sup 99m^Tc labeling was exactly the same for all mutant proteins. Thus, any differences observed could be attributed to the specific mutations introduced rather than differences in the site or degree of labeling. We show that the new annexin V-128 recombinant protein has substantially improved biodistribution properties compared with HYNIC-derivatized annexin V. We also show that alterations in membrane-binding affinity change biodistribution in normal animals and target uptake in an animal model of apoptosis. These results provide guidance in the development of improved annexin V-based apoptosis imaging agents radiolabeled with either ^sup 99m^Tc or positron emitters as well as annexin V-based agents used for fluorescence or MRI.

MATERIALS AND METHODS

Preparation and ^sup 99m^Tc Labeling of Mutant Proteins

Recombinant human annexin V-128 and derivative mutant proteins were prepared and purified as described (6). In preparation for technetium labeling, protein (0.6 mg) was diluted with phosphate- buffered saline ([PBS] containing 10 mmol/L sodium phosphate, pH 7.4, 140 mmol/L NaCl) to a final volume of 0.2 mL and reduced with 1 mmol/L dithiothreitol at 370C for 15 min under anaerobic conditions. The protein was applied to a Sephadex G-25 column (NAP-5; Pharmacia), which had been preequilibrated with deoxygenated buffer (20 mmol/L trisodium citrate, pH 5.4, 100 mmol/L NaCl), and the breakthrough (0.2 mL) was discarded. Elution was performed with the same buffer; the first 0.5 mL of eluate was discarded, and the subsequent 0.5 mL, containing the protein, was collected in argon- flushed glass vials. Aliquots of 0.1 mL (containing 0.1 mg protein, 0.59 mg trisodium citrate, and 0.58 mg NaCl) were stored frozen at - 20C in glass vials with Teflonsealed screw caps (DuPont). Tin reagent was prepared as described (12). Each vial of lyophilized reagent contained 5 mg of sodium glucoheptonate, 0.128 mg SnCl^sub 2^-2H^sub 2^O, and 0.128 mg of sodium gentisate. Just before use, the vial was reconstituted with 0.2 mL of deoxygenated water.

^sup 99m^Tc-Annexin V-128 was prepared as follows. ^sup 99m^TcO^sub 4^ (185370 GBq [5-10 mCi] in 0.1 mL of 0.9% NaCl) was added to a polypropylene microfuge tube, followed by 0.005 mL of tin reagent and 0.05 mL (0.05 mg) of reduced protein. After a 60-min anaerobic incubation at 37C, the reaction mixture was brought to a volume of 0.4 mL with PBS, pH 7.4, and applied to a Sephadex G-25 column (NAP-5; Pharmacia) equilibrated and eluted with PBS, pH 7.4. The labeled protein was collected in a volume of 1.4 mL (0.4 mL of breakthrough and the first 1.0 mL of eluate). The percentage incorporation of technetium determined by instant thinlayer chromatography (ITLC) was routinely 85%-97%, corresponding to a specific activity of about 3.7 GBq/mg (100 mCi/mg). The labeling reaction can also be performed at room temperature for 20-30 min with minimal loss of labeling efficiency (12). A dose of 1.85-7.4 MBq of tracer (0.05-0.2 mCi; 0.1-0.2 mL, 0.05-0.1 mg protein/kg body weight) was injected via tail vein for all animal studies. ^sup 99m^Tc-HYNIC-annexin V was prepared and labeled with the tin- tricine method as previously described (I).

Membrane-Binding Affinity Measurement by Quantitative Calcium Titration

The affinity of labeled protein for red blood cells (RBCs) with exposed PS was determined by the calcium titration assay previously described for fluorescently labeled annexin V (20). Annexin V-128 was labeled with ^sup 99m^Tc as described and diluted to a working concentration of 200 nmol/L in 50 mmol/L N-(2- hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES)-sodium, pH 7.4, 100 mmol/L NaCl, 3 mmol/L NaN^sub 3^, and 5 mg/mL bovine serum albumin (BSA). The final concentration of ^sup 99m^Tc-annexin V-128 in the assay was 1 nmol/L, RBCs were added at 1 10^sup 8^ per milliliter and calcium was varied from O to 5 mmol/L, in an assay buffer containing 50 mmol/L HEPES-sodium, pH 7.4, 100 mmol/L NaCl, 3 mmol/L NaN^sub 3^, with 1 mg/mL BSA as \carrier protein. After an 8- min incubation at room temperature, cells were pelleted by centrifugation, resuspended in 1 mL of assay buffer containing the same concentration of calcium used in the binding step, and centrifuged again. The supernatant was removed and the ""1Tcannexin V-128 bound to the RBCs was released with 0.7 mL of assay buffer containing 5 mmol/L ethylenediaminetetraacetic acid. The RBCs were pelleted by centrifugation and the amount of ^sup 99m^Tc-annexin V- 128 bound to the RBCs at each calcium concentration was determined by counting an aliquot (0.3-0.6 mL) of the supernatant on a γ- counter (RIASTAR Jr; Hewlett Packard). Calcium titration curves were analyzed to determine the binding affinity as described (20); results are expressed as a pK value, which is the logarithm of the equilibrium constant for proteinmembrane-calcium binding.

TUNEL (Terminal Deoxyribonucleotidyl TransferaseMediated Deoxyuridine Nick-End Labeling) Assay

Livers from normal and cycloheximide-treated mice were evaluated for the presence of apoptotic nuclei in formalin-fixed, paraffin- embedded tissue sections with a fluorescent TUNEL assay kit following the manufacturer's directions (Oncogene Research Products Fluorescein-FragEL kit; Calbiochem-Novabiochem Corp.). The number of apoptotic nuclei was determined from 60 fields per slide, containing an average of 36 nuclei per field, examined under 60X magnification.

Blood Clearance and Biodistribution Studies in Normal and Treated Mice

All mice used were adults (6- to 8-wk old) of the BALB/c strain obtained from the breeding facility of the Department of Comparative Medicine, Stanford University. Hepatic apoptosis was induced by intraperitoneal injection of cycloheximide (21) at a dose of 50 mg/ kg dissolved in 0.5 mL of PBS. All animals were anesthetized with a cocktail of ketamine (100 mg/kg intraperitoneally) (Fort Dodge Animal Health) and xylazine (10 mg/kg, intraperitoneally) (Butler) before clearance, biodistribution, and imaging studies. Blood clearance studies were performed by serial retroorbital blood samples obtained after tail vein injection of tracer. Biodistribution assay was performed immediately after sacrifice. Tissue and organ samples were analyzed with a scintillation well counter along with 4 samples of standard activity (1/100 of injected dose) at an energy level of 140 keV with an energy window of 20 keV. Results are expressed as the average of the percentage injected dose (%ID) or %1D per gram (g) of tissue (% ID/g) 1 SD of the mean.

Dynamic Imaging

Dynamic planar images of tracer activity were obtained using the A-SPECT dedicated small animal imaging SPECT γ-camera (Gamma- Medical Instruments) with a 128 128 matrix and a high-resolution, high-sensitivity, parallel-hole collimator. Sedated animals were placed in the anterior supine position and planar whole-body images were collected starting immediately after injection of tracer via tail vein (animals received the same dose of tracer used for static biodistribution studies); serial 1-min images were obtained for 35 min, and serial 3-min images were obtained for another 60 min. Region-of-interest image analyses were performed on planar images using commercially available software (Mirage Software, version 5.3; Segami Corp.). The results of the dynamic image analysis for the activity of each region of interest expressed as counts/pixel are reported as the percentage of wholebody activity also expressed as counts/pixel.

RESULTS

Characterization of ^sup 99m^Tc-Annexin V-128

All mutant proteins were built on the base structure of annexin V- 128 (6). This protein has essentially the same structure as wild- type human annexin V, except for an N-terminal extension of Ala-Gly- Gly-Cys-Gly-His to provide a site for Tc chelation and a Cys316Ser point mutation. Figure 1 shows a calcium titration curve performed with ^sup 99m^Tc-annexin V-128 and RBCs with exposed PS. The curve is identical to that previously obtained with fluorescently labeled annexin V-128. The pK value for binding of ^sup 99m^Tc-annexin V- 128 to RBCs is 30.8 1.0 (mean SD, n = 3), identical to that previously reported for both for wild-type annexin V and for annexin V-128 labeled with fluorescein (6,20). Thus, ^sup 99m^Tc labeling at the N-terminus does not alter binding affinity, consistent with our earlier work with the annexin V-117 protein (12), which has a nearly identical structure.

FIGURE 1. Calcium titration of ^sup 99m^Tc-annexin V-128 binding to RBCs with exposed PS. Line is the fitted function used to determine the median effective concentration (EC50) and Hill coefficient values, from which logarithm of the binding constant (pK value) is calculated (20).

Comparison of HYNIC-Annexin V and Annexin V-128

To verify that 99mTc-annexin V-128 was suitable for detection of cell death, it was compared with ^sup 99m^Tc-HYNICannexin V in normal mice. As seen in Table 1, the biodistribution of the 2 proteins was similar overall, with a few exceptions. The most striking exception was the kidney: ^sup 99m^Tc-annexin V-128 showed 88% less renal uptake than ^sup 99m^Tc-HYNlC-annexin V. Liver and spleen uptake was also somewhat lower for ^sup 99m^Tc-annexin V- 128, possibly reflecting less aggregated material. Small bowel was the only organ showing higher uptake of 99mTc-annexin V-128 (3.9 %ID) compared with ^sup 99m^Tc-HYNIC-annexin V (1.6 %ID). However, this was greatly outweighed by the lower overall abdominal uptake of ^sup 99m^'Tc-annexin V-128 (17 %ID vs. 60 %ID for ^sup 99m^Tc-HYNIC- annexin V).

When mice were treated with cycloheximide to induce apoptosis, the liver and spleen showed a severalfold increase in uptake of ^sup 99m^Tc-annexin V-128 (Fig. 2A). We also verified that uptake of ^sup 99m^Tc-annexin V-128 was consistent with an independent measure of apoptosis. As shown in Figure 2B, in vivo uptake of ^sup 99m^Tc- annexin V-128 in the liver correlated closely with histologic analysis of the degree of apoptosis as judged by the TUNEL assay.

Dynamic Imaging with HYNIC-Annexin V and Annexin V-128

The reason for the striking difference in renal uptake of ^sup 99m^Tc-annexin V-128 compared with that of ^sup 99m^Tc-H YNICannexin V was further explored via dynamic imaging studies (Fig. 3). Renal uptake of each protein is comparable up to about 15 min after injection. After that time, there is a progressive decline in renal radioactivity for the ^sup 99m^Tcannexin V-128 protein, accompanied by a corresponding increase in radioactivity in the bladder. In contrast, for ^sup 99m^Tc-HYNIC-annexin V, radioactivity in the kidney stays roughly constant, and there is minimal bladder excretion during this time. These results were confirmed by static biodistribution measurements performed at 15 min after injection, which confirmed that the renal uptake of ^sup 99m^Tcannexin V-128 and ^sup 99m^Tc-HYNIC-annexin V was comparable at that time (data not shown).

Characterization of Charge and Affinity Mutants

To determine how PS-binding affinity and molecular charge affect biodistribution and target uptake of annexin V, a series of mutants were prepared (Fig. 4; Table 2). The PS-binding affinity of annexin V can be reduced in a stepwise fashion by making point mutations in aspartate or glutamate residues contributing to the AB-helix calciumbinding sites present once in each of the 4 domains (<5). These mutants span a very broad range of membranebinding affinity (as summarized on a logarithmic scale by the pK affinity value) but they all have essentially the same molecular mass as the parent protein, since each mutation changes molecular mass by only 1 Da. Since mutations in the AB-helix calcium-binding sites also alter molecular charge by converting a negatively charged residue to a neutral residue, a second set of mutant proteins was prepared with unaltered affinity but altered molecular charge as control proteins to evaluate the specificity of changes observed with the affinity mutants. Some of these mutant proteins have been previously reported (<5), and others were developed specifically for this study.

TABLE 1

Biodistribution of ^sup 99m^Tc-HYNIC-Annexin V and ^sup 99m^Tc- Annexin V-128

FIGURE 2. (A) Uptake of ^sup 99m^Tc-annexin V-128 in liver and spleen of mice treated with cycloheximide. Groups of 6 mice were either untreated or treated with cycloheximide (50 mg/kg, intraperitoneally). Two hours later, ^sup 99m^Tc-annexin V-128 was injected via tail vein, and organs were harvested 60 min after injection. Significance of the difference between treated and untreated animals was assessed by a 2-tailed t test assuming unequal variances; error bars indicate SD. (B) Correlation between TUNEL positivity and ^sup 99m^Tc-annexin V-128 uptake in liver for treated and untreated mice (6 in each group).

Blood Clearance of Affinity Mutants

The blood clearance of ^sup 99m^Tc-annexin V-128 and selected affinity mutants was measured in mice (Fig. 5). All proteins showed the same rapid clearance kinetics, with only 4% remaining in blood at 60 min. These clearance curves are essentially the same as those previously reported for unlabeled annexin V (16), ^sup 125^I- annexin V and ^sup 123^I-annexin V (UJT), and HYNIC-annexin V (22). Thus, changes in PS-binding affinity do not alter the kinetics of blood clearance of annexin V. Similarly, changes in molecular charge did not alter blood clearance (data not shown).

FIGURE 3. Serial dynamic imaging of kidney and bladder uptake of 99mTc-annexin V-128 and 99mTc-HYNIC-annexin V. Each curve is mean of 4 animals; coefficient of variation at each point is approximately 20%.

Uptake in Apoptotic Organs

To determine the impact of affinity changes on uptake in apoptotic organs, we tested the series of affinity mutants in mice treated with cycloheximide to induce apoptosis of the liver and spleen (Fig. 6A). There was a clear dose-response relationship between the number of mutations a\nd the reduction in organ uptake in the liver and spleen. A single mutation decreased uptake by 50%- 60%, and the presence of 2 or more mutations reduced uptake by 80%- 90%. However, the specific location of the mutation in the molecule (Fig. 4) did not influence the results: Single-site mutants in domains 1 and 4 were equivalent, double mutants in domains 1+4 and domains 2 + 3 were equivalent, and triple mutants in domains 1+2 + 4 and domains 2 + 3 + 4 were equivalent. Thus, the dominant factor determining the uptake in apoptotic tissue is the total number of active domains rather than the specific site of the mutation(s); in other words, membrane-binding sites in each of the 4 domains are equivalent in their influence on target uptake in vivo.

FIGURE 4. Schematic view of 3-dimensional structure of annexin V- 128. View is downward onto membrane-binding face of protein. The 4 domains of protein are numbered, and their approximate locations are indicated by ellipses. Black spheres indicate approximate locations of calcium ions bound to AB-helix calcium-binding sites that have been mutated in this study. Polypeptide backbone is shown as a continuous black line. Location of N-terminal technetium chelation site cannot be seen in this projection because it is on opposite face of molecule, beneath the plane of the page. Structure is based on preliminary coordinates provided by Dr. Barbara Seaton - (personal communication, May 2004).

As a control, we also determined the effect of affinity mutations on the uptake in normal liver and spleen (Fig. 6B). Somewhat unexpectedly, a similar pattern was observed: Organ uptake was progressively reduced by an increasing number of mutations. To verify that this effect on normal organ uptake was not an artifact of concomitant changes in molecular charge that occur in the affinity mutants, a second series of mutant proteins was tested that altered charge without affecting PS-binding affinity (Fig. 7). Charge differences from - 2 to +2 relative to wild-type protein did not affect the uptake in normal liver or spleen, indicating that the effects observed in Figure 8 were due to affinity changes rather than changes in charge per se.

In view of the results obtained with normal liver and spleen, we performed a more extensive evaluation of the effect of PS-binding affinity on biodistribution in normal animals (Fig. 8). Biodistribution was measured with triple and quadruple mutant proteins (i.e., annexin V-137, -143, and -145). Results for individual organs fell into 3 groups. For the spleen, liver, and lung, 75%-85% of uptake was eliminated by eliminating the PS- binding activity of annexin V. For the stomach, small bowel, and heart, about 40%-50% of uptake was lost in the mutants compared with the wild-type protein. For the kidney and large bowel, uptake was unaffected by changes in affinity.

TABLE 2

Summary of Affinity and Charge Mutants

DISCUSSION

Characterization of ^sup 99m^Tc-Annexin V-128 and Comparison with ^sup 99m^Tc-HYNIC-Annexin V

We have recently developed a novel form of annexin V with an endogenous ^sup 99m^Tc chelation site, termed annexin V-128 (6), that has significant advantages over ^sup 99m^TcHYNIC-annexin V. There is a striking difference in the renal handling of ^sup 99m^Tc- labeled annexin V depending on the method of labeling. Initial renal uptake is the same, but subsequent urinary excretion of ^sup 99m^Tc is far more rapid for the ^sup 99m^Tc-annexin V-128 derivative compared with that of the ^sup 99m^Tc-HYNIC-annexin V derivative. The most likely explanation is as follows. Both proteins are filtered equally at the glomerulus, and both are taken up in the proximal tubules by relatively nonspecific uptake systems designed to capture low-molecular-weight proteins (23). Endocytosed proteins are then delivered to the lysosome and degraded by proteases to oligopeptides or amino acids. However, the end products differ greatly in their retention in the kidney. The end product of the degradation of ^sup 99m^Tc-HYNIC-annexin V is probably the lysine adduct of ^sup 99m^Tc-HYNIC complexed to tricine, which has a high degree of retention in lysosomes (24,25), The end product of the metabolism of ^sup 99m^Tc-annexin V-128 is not known but may consist either of a small oligopeptide or possibly pertechnetate alone. In any event, the radioactive end product is much more rapidly excreted from the kidney. Although we did not study metabolism of ^sup 99m^Tc- annexin V-128 in other organs, it seems unlikely that the rapid degradation seen in the kidney would also occur in apoptotic tissues because apoptotic bodies will have lost the multiple systems required for endocytosis, intracellular transport, and degradation of proteins bound to the cell surface. Thus, annexin V is likely to remain intact when bound to the surface of dead cells or their fragments.

FIGURE 5. Blood clearance of affinity mutants. Groups of 4 mice received intravenous injections of ^sup 99m^Tc-labeled protein at time zero. Results are given as mean SD for each mutant.

The improvements in biodistribution achieved with annexin V-128 were obtained without any compromise in its ability to detect apoptosis in vivo, as shown by Figure 2. This protein also has significant advantages for manufacturing compared with HYNIC- annexin V: The end product of recombinant protein production is ready to be labeled directly with ^sup 99m^Tc and does not need to undergo further chemical derivatization, purification, and characterization. The single labeling site at the N-terminus is uniform and well characterized; this region of the protein is on the opposite side of the molecule from the membrane-binding region (5). In contrast, it can be difficult to obtain a reproducible preparation of chemically modified annexin V when amine-directed reagents are used; inevitably, the product is heterogeneous with respect to sites and stoichiometry of labeling. The Tc-labeling procedure is comparable in simplicity to that used with HYNIC- annexin: One simply mixes pertechnetate, tin reagent, and protein and incubates for 20-60 min at 22C or 37C (this study (12)). Overall, ^sup 99m^Tc-annexin V-128 is an attractive candidate to replace ^sup 99m^Tc-HYNIC-annexin V in many applications.

FIGURE 6. (A) Dose-response relationship between number of affinity-reducing mutations and liver and spleen uptake in mice treated with cycloheximide. Each affinity mutant in Table 2 was tested in groups of 4 or 5 mice. Mean values are given for liver and spleen uptake, normalized to uptake observed with wild-type protein. Mutants tested were 128 (wild-type/zero mutations); 131 and 138 (1 mutation); 136 and 139 (2 mutations); 137 and 143 (3 mutations); and 145 (4 mutations). (B) Dose-response relationship between number of affinity-reducing mutations and liver and spleen uptake in normal mice.

FIGURE 7. Effect of molecular charge on liver and spleen uptake of ^sup 99m^Tc-annexin V-128 in normal mice. Each charge mutant in Table 2 was tested in groups of 4 or 5 mice. Mean values are given for liver and spleen uptake.

FIGURE 8. Organ uptake of affinity mutants in normal mice. Triple and quadruple mutants in Table 2 (annexin V-137, -143, and -145) were tested in groups of 4 or 5 mice for each mutant. Results were averaged for all 3 mutant proteins and expressed as percentage of organ uptake observed with wild-type protein (annexin V-128).

Factors Regulating Normal Organ Uptake of Annexin V

This study shows that uptake of annexin V in normal organs is differentially regulated. In some organs, such as liver, spleen, and lung, uptake is an inherent property of the protein and is primarily dependent on the PS-binding affinity of the molecule. In other organs, such as kidney and large bowel, uptake is relatively nonspecific and is not regulated by the PS-binding affinity of the molecule. Some organs, such as stomach, small bowel, and heart, are inbetween, with about half their normal uptake being PS specific. Control experiments showed that these effects were not simply due to charge changes introduced by the point mutations used to reduce binding affinity. Similarly, the observed effects were not due to molecular weight changes, since the point mutations change the protein's molecular mass by only 1-4 Da (6).

There are several possible explanations for the differential effects of PS-binding affinity on organ uptake. It is possible that the results reflect different levels of physiologic apoptosis in different organs. It is also possible that annexin V is binding to apoptotic cells or cell debris that is in the process of being engulfed by phagocytic cells in the liver, spleen, and lung. It is also possible that annexin V is binding to non-PS substances in some normal organs-for example, annexin V is known to bind to anionic polysaccharides such as heparan sulfate under some conditions (26). Finally, annexin V could be taken up by an organspecific endocytic mechanism that is somehow dependent on its membrane-binding affinity. In any event, the results of this study show that the factors governing the biodistribution of annexin V are complex and are not simply a function of nonspecific factors, such as molecular size, charge, or vascular permeability.

Affinity Requirements for Annexin V Uptake in Apoptotic Tissues

Our results indicate clearly that all 4 domains of annexin V are needed for maximum uptake in apoptotic organs in vivo. Although previous work had suggested that most of the in vitro binding affinity of annexin V was concentrated in the first domain (27), our recent work with improved assay methods showed clearly that all 4 domains contribute equally to in vitro binding affinity (6). The results in this study now extend these observations to organ uptake in vivo. It is clear that organ uptake is a function of only the total number of active domains and is not affected by the specific domain(s) that have been inactivated by mu\tation. For example, single-site mutants in domains 1 and 4 behaved equivalently, as did double mutants in domains 1 + 4 versus domains 2 + 3; there was no difference between mutants containing different combinations of individual mutations, provided they contained the same total number of mutations.

This study also confirms the value of our recently developed methods for measuring the affinity of annexin V for membranes in vitro (20). These methods rely on quantitative calcium titration at low ratios of protein to membrane, which are probably most relevant to the in vivo situation. Although we have previously used this method with fluorescently labeled protein, the results of Figure 1 show that the method works equally well for cell-binding assays performed with radioactively labeled protein. We believe that these methods are the best overall means of measuring the membrane- binding affinity of modified annexins and will provide the best overall prediction of in vivo uptake. The logarithm of the binding constant (pK value) quantitatively predicts in vivo uptake: pK values 25% below wild-type are associated with significant loss of in vivo uptake, and pK values below 50% of wild-type are associated with complete loss of in vivo uptake (Table 2; Fig. 6A). It should be noted that the pK value is logarithmic; a decrease of 1 pK unit corresponds to a 10-fold reduction in the equilbrium constant.

CONCLUSION

There are several implications of this work for the further development of annexin V as an imaging agent for cell death in vivo. First, it appears that annexin V derivatives based on a single active domain (18), instead of the full 4 domains of the native protein, will probably lack the binding affinity required to provide effective target localization in vivo. Second, since the binding surface of annexin V encompasses contributions from all 4 domains (6,20), caution should be exercised in amine-directed labeling strategies that affect multiple residues on the same protein molecule, since there are multiple lysine residues present on or near the membrane-binding face of the molecule (5). However, modest changes in molecular charge per se, as can typically occur with modification of single lysine residues or the N-terminus, are unlikely to alter the clearance, biodistribution, or metabolism of the molecule. Finally, the use of the N-terminal endogenous ^sup 99m^Tc chelation sequence appears to have major advantages over the HYNIC chelator with regard to renal retention of ^sup 99m^Tc, with attendant decreased abdominal background and renal radiation dose.

ACKNOWLEDGMENTS

We thank Donald Gibson for preparing experimental materials and Dr. Jean-Luc Vanderheyden of Theseus Imaging Corp. for the North American Scientific 2020 HYNIC-annexin V and tin-tricine kits. This research was supported by U.S. Public Health Service National Institutes of Health grants CA-102348 and EB-000898.

REFERENCES

1. Blankenberg FG, Katsikis PD, Tait JF, et al. In vivo detection and imaging of phosphatidylserine expression during programmed cell death. Proc Natl Acad Sci USA. 1998;95:6349-6354.

2. Blankenberg FG, Katsikis PD, Tait JF, el al. Imaging of apoptosis (programmed cell death) with ^sup 99m^Tc annexin V. J Nucl Med. 1999;40:184-191.

3. Thiagarajan P, Tait JF. Binding of annexin V/placental anticoagulant protein I to platelets: evidence for phosphatidylserine exposure in the procoagulant response of activated platelets. J Biol Chem. 1990;265:17420-17423.

4. Fadok VA, Voelkcr DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM. Exposure of phosphatidylserine on the surface of apoptolic lymphocytes triggers specific recognition and removal by macrophages. J Mol Biol. 1992:148:22072216.

5. Huber R, Berendcs R, Burger A, et al. Crystal and molecular structure of human annexin V after refinement: implications for structure, membrane binding and ion channel formation of the annexin family of proteins. J Mol Biol. 1992;223:683704.

6. Jin M, Smith C, Hsieh HY, Gibson DF, Tait JF. Essential role of B-helix calcium binding sites in annexin V-membrane binding. J Biol Chem. 2004;279:4035140357.

7. Belhocine T, Steinmetz N, Hustinx R, et al. Increased uptake of the apoptosisimaging agent ^sup 99m^Tc recombinant human annexin V in human tumors alter one course of chemotherapy as a predictor of tumor response and patient prognosis. Clin Cancer Res. 2002;8:2766- 2774.

8. Narula J, Acio ER, Narula N, et al. Annexin-V imaging for noninvasive detection of cardiac allograft rejection. Nat Meet. 2001 ;7:1347-1352.

9. Hofstra L, liem IH, Dumont EA, et al. Visualisation of cell death in vivo in patients with acute myocardial infarction. Lancet. 2000;356:209-2I2.

10. Kemcrink GJ, Liu X, Kieffer D, et al. Safety, biodistribution, and dosimetry of WmTc-HYNIC-annexin V, a novel human recombinant annexin V for human application. J Nucl Med. 2003;44:947-952.

11. Stratton JR, Dewhurst TA, Kasina S, et al. Selective uptake of radiolabeled annexin V on acute porcine left atrial thrombi. Circulation. 1995;92:3113-3121.

12. Tail JF, Brown DS, Gibson DF, Blankenberg FG, Strauss HW. Development and characterization of annexin V mutants with endogenous chelation sites for ^sup 99m^Tc. Bioconjug Chem. 2000;11:918-925.

13. Kemerink GJ, Boersma HH, Thimister PW, et al. Biodistribution and dosimetry of ^sup 99m^'Tc-BTAP-annexin-V in humans. Eur J Nucl Med. 2001 ;28:1373-1378.

14. Tait JF, Smith C, Gibson DF. Development of annexin V mutants suitable for labeling with Tc(I)-carbonyl complex. Bioconjug Chem. 2002;13:1119-1123.

15. Zijlstra S, Gunawan J, Burchert W. Synthesis and evaluation of a ^sup 18^F-labelled recombinant annexin-V derivative, for identification and quantification of apoptotic cells with PET. Appl Radiat Isot. 2003;58:201-207.

16. Romisch J, Seiffge D, Reiner G, Paques EP, Heimburger N. In- vivo antilhrombotic potency of placenta protein 4 (annexin V). Thromb Res. 199I;61:93-104.

17. Tait JF, Cerqueira MD, Dewhmst TA, Fujikawa K, Ritchie JL, Slratton JR. Evaluation of annexin V as a platelet-directed thrombus targeting agent. Thromb Res. 1994:75:491-501.

18. Blondel A, Vuillemard C, Tavitian B, et al. A new SPECT tracer for improved apoptosis imaging in tumor bearing mice [abstractj. J Nucl Med. 2003;44(suppl); 98P.

19. Ke S, Wen X, Wu QP, el al. Imaging laxanc-induccd lunior apoplosis using PEGylated, IMIn-labeled annexin V. J Nucl Med. 2004;45:108-115.

20. Tait JF, Gibson DF, Smith C. Measurement of the affinity and coopcralivity of annexin V-mcmbrane binding under conditions of low membrane occupancy. Anal Biochem. 2004;329:112-119.

21. Ledda-Columbano GM, Coni P, Faa G, Manenti G, Columbano A. Rapid induction of apoptosis in rat liver by cyclohexirnide. Am J Pathol. 1992; 140:545-549.

22. Ohtsuki K, Akashi K, Aoka Y, et al. Technelium-99m HYNIC- annexin V: a potential radiopharmaceutical for the in-vivo detection of apoptosis. Eur J Nucl Med. 1999;26:1251-1258.

23. Verroust PJ, Birn H, Nielsen R, Kozyraki R. Chrislcnscn BI. The tandem endocytic receptors mcgalin and cubilin are important proteins in renal pathology. Kidney int. 2002;62:745-756.

24. Ono M, Arano Y, Uehara T, et al. Intraccllular metabolic late of radioactivity after injection of technetium-^sup 99m^-labeled hydra/Jno nicotinamidc dcrivali/.cd proteins. Bioconjug Chem. 1999; 10:386-394.

25. Ono M, Arano Y, Mukai T, et al. Control of radioactivity pharmacokinelics of WmTc-HYNIC-labeled polypeptides derivatized with ternary ligand complexes. Bioconjug Chem. 2002;13:491-501.

26. Capila I, Hernaiz MJ, Mo YD, et al. Annexin V-heparin oligosaccharide complex suggests heparan sulfate-mediated assembly on cell surfaces. Structure (Camb). 2001;9:57-64.

27. Monlaville P, Neumann J-M, Russo-Marie F, Ochsenbein F, Sanson A. A new consensus sequence for phosphatidylserine recognition by annexing. J Biol Chem. 2002;277:24684-24693.

Jonathan F. Tait, MD, PhD1-3; Christina Smith, BS1; and Francis G. Blankenberg, MD4

1 Department of Laboratory Medicine, University of Washington, Seattle, Washington; 2 Medical Genetics, Department of Medicine, University of Washington, Seattle, Washington; 3 Department of Pathology, University of Washington, Seattle, Washington; and 4 Department of Radiology, Stanford University, Palo Alto, California

Received Oct. 18, 2004; revision accepted Jan. 16, 2005.

For correspondence or reprints contact: Jonathan F. Tait, MD, PhD, Department of Laboratory Medicine, University of Washington, Box 357110, Seattle, WA 98195-7110.

E-mail: tait@uwashington.edu

Copyright Society of Nuclear Medicine May 2005

Source: Journal of Nuclear Medicine, The

More News in this Category