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A Novel Approach for 99mTc-Labeling of Red Blood Cells: Evaluation of 99mTc-4SAboroxime as a Blood Pool Imaging Agent Min Liu, Zuoquan Zhao, Wei Fang, and Shuang Liu Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00601 • Publication Date (Web): 17 Nov 2017 Downloaded from http://pubs.acs.org on November 21, 2017

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Bioconjugate Chemistry

A Novel Approach for 99mTc-Labeling of Red Blood Cells: Evaluation of 99mTc-4SAboroxime as a Blood Pool Imaging Agent

Min Liu,1# Zuo-Quan Zhao,2# Wei Fang,2* and Shuang Liu1*

1

School of Health Sciences, Purdue University, IN 47907, USA

2

Department of Nuclear Medicine, Fuwai Hospital, the National Center for Cardiovascular

Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, No.167 North Lishi Road, Xicheng District, Beijing, China, 100037

RUNNING TITLE: 99mTc-4SAboroxime as a Blood Pool Imaging Agent

# These authors contributed equally to this work. *Correspondence should be addressed to: Dr. Shuang Liu, School of Health Sciences, Purdue University, 550 Stadium Mall Drive, West Lafayette, IN 47907, Phone: 765-494-0236, Fax 765-496-1377, Email: [email protected]; or Dr. Wei Fang, Department of Nuclear Medicine, Fuwai Hospital, the National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, No.167 North Lishi Road, Xicheng District, Beijing, China, 100037. Email: [email protected].

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ABSTRACT: Angiography with radiolabeled red blood cells (RBCs) plays an important role in diagnosis and prognosis in vascular diseases. Both in vitro and in vivo methods have been developed for 99m

Tc-labeling of RBCs. However, these methods are complicated and lack reproducibility.

Therefore, it is highly desirable to develop an alternative method for routine RBCs. In this report, we present a novel approach for new

99m

Tc-labeling of

99m

Tc-labeling of RBCs. We prepared a

99m

Tc(III) radiotracer [99mTcCl(CDO)(CDOH)2B-4AS] (99mTc-4ASboroxime: 4AS-B(OH)2

= 4-aminosulfonylphenyl)boronic acid, and CDOH2 = cyclohexanedione dioxime) in >95% radiochemical purity. Imaging and biodistribution studies were performed in Sprague-Dawley (SD) rats. It was found that the blood radioactivity was ~6.0 %ID/g (~90% injected dose for 200 – 225 g SD rats) for

99m

Tc-4ASboroxime with low uptake in the myocardium, kidneys, liver,

lungs and muscle, most likely due to lack of leakage of

99m

Tc-labeled RBCs from intravascular

space. The blood radioactivity was almost unchanged over the 2 h period, suggesting that the binding of

99m

Tc-4ASboroxime to blood components (cells, proteins and plasma) is stable. The

results from γ-counting of the isolated blood components showed that

99m

Tc-4ASboroxime had

>95% of blood radioactivity binding to RBCs, ~1% to albumin, and ~3% remaining free in blood plasma, demonstrating its RBC-specificity. The results from imaging studies in SD rats indicated that 99mTc-4ASboroxime is predominantly distributed in the blood pool. Main blood vessels were well delineated in the head/neck and abdominal regions. This statement was further substantiated by the results from imaging studies in pigs. 99mTc-4ASboroxime is an excellent blood pool agent with potential for diagnosis and prognosis of vascular diseases. Key Words:

99m

Tc(III) complexes, blood pool imaging, SPECT angiography and

red blood cells.

2


99m

Tc-labeled

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INTRODUCTION Angiography plays an important role in the diagnosis and prognosis in vascular diseases.1-8 Computed tomography (CT) and magnetic resonance imaging (MRI) angiography using bloodpool contrast agents provide excellent spatial and temporal resolution of the arteries and veins. However, the low sensitivity of CT and MRI often requires administration of a large quantity of contrast agents, which may increase the risks of allergic reaction or nephrotoxicity. In contrast, radionuclide angiography has been used clinically to determine blood and plasma volumes,9-12 evaluate cardiac function,1-10 diagnose gastrointestinal bleeding,13-18 delineate blood vessels,2-4 and detect neoplasms.19,20 Fundamental to the procurement of high-quality radionuclide bloodpool imaging is the development of an appropriate radiotracer that remains within the intravascular space for a long duration. There are two categories of radiotracers available for blood-pool imaging: radiolabeled red blood cells (RBCs: the most abundant cells accounting for 40 – 45% of total blood volume) and albumin (the most abundant protein comprising 50 – 60% of total blood protein).

99m

Tc is the

preferred radionuclide for SPECT (single photon emission computed tomography) due to its optimal nuclear properties and easy availability at low cost.21-23

99m

Tc-Labeled RBCs play an

important role in blood pool imaging because of their capability to distribute within intravascular pool and to leave this compartment at a slow rate, which allows for acquisition of high quality images with the aid of a gating device, such as electrocardiogram (EKG).24,25 Both the in vitro and in vivo methods have been used for 99mTc-labeling of RBCs.26-29 However, these methods are often

complicated and lack of reproducibility. In the in vitro approach, centrifugation of RBCs is timeconsuming and constitutes the potential for cross-contamination from the blood products. Thus, it is highly desirable to develop an alternative method for 99mTc-labeling of RBCs.

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Figure 1. Chemdraw structures of 99mTc(III) radiotracers [99mTcCl(CDO)(CDOH)2B-R] (99mTcTeboroxime: R = CH3; 99mTc-4Sboroxime: R = 4-PhSO2CH3; and 99mTc-4ASboroxime: R = 4PhSO2NH2). 99mTc-Teboroxime is a FDA-approved radiopharmaceutical for heart imaging. In this study, 99mTc-4ASboroxime was evaluated as a new radiotracer for blood pool imaging. 99m Tc-4Sboroxime was used as a control in the cellular and protein binding experiments. We have been interested in

99m

Tc(III) complexes [99mTcCl(CDO)(CDOH)2B-R] (Figure 1:

CDOH2 = cyclohexanedione dioxime; R = alkyl or aryl groups) as radiotracers for heart imaging.30-35 Recently, we found that the complex [99mTcCl(CDO)(CDOH)2B-4AS] (Figure 1: 99m

Tc-4Sboroxime, 4S-B(OH)2 = 4-(methanesulfonyl)phenylboronic acid) is an excellent heart

imaging agent.35 However, its liver uptake is high.35 In order to minimize the liver radioactivity of

99m

Tc radiotracer, we prepared a new

(Figure 1:

99m

Tc(III) radiotracer [99mTcCl(CDO)(CDOH)2B-4AS]

99m

Tc-4ASboroxime; 4-AS-B(OH)2 = 4-aminosulfonylphenyl)boronic acid). Our

original hypothesis is that replacing the 4-methylsulfonyl functionality in

99m

Tc-4Sboroxime

with the 4-aminosulfonyl group might help to minimize the liver radioactivity of radiotracer because 4-aminosulfonyl is more hydrophilic than 4-methylsulfonyl. We are surprised to find out that more than 90% of the injected dose (%ID) is in the blood pool after intravenous injection of 99m

Tc-4ASboroxime, and >95% of the blood radioactivity is bound to the circulating RBCs. This

surprising discovery has led us to evaluate

99m

Tc-4ASboroxime as a RBC-targeted blood pool

imaging agent. The approach described in this study is novel because it is the first example to use a stable 99mTc radiotracer for routine in vitro and in vivo 99mTc-labeling of RBCs. 4


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RESULTS Radiochemistry.

99m

Tc-4ASboroxime was prepared using a kit formulation (Figure 2).30-35

Heating at 100 oC is required in order to complete radiosynthesis. Its radiochemical purity (RCP) was >95% under the experimental conditions used in this study. Since 4-aminosulfonyl is more water-soluble than 4-methylsulfonyl,

99m

Tc-4ASboroxime (Figure 2: retention time ~7.5 min) is

more hydrophilic than 99mTc-4Sboroxime (~11.5 min). 99mTc-4ASboroxime is stable for > 6 h at room temperature in the diluted solution in the presence of excess NaCl (Figure 2).

Figure 2. Top: The synthetic scheme for radiosynthesis of 99mTc-4ASboroxime. Bottom: Typical radio-HPLC chromatograms of 99mTc-4ASboroxime at 0 and 6 h post-labeling in the solution containing 20 – 30% propylene glycol to demonstrate its solution stability. 5



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A

B

Figure 3. A: Planar images of the SD rat administered with ~45 MBq of 99mTc-4ASboroxime to show its localization in the blood pool and blood vessels. B: Planar image quantification data in the heart region for 99mTc-4ASboroxime and 99mTc-4Sboroxime. The heart radioactivity from quantification of planar images in SD rats (n = 5) included contributions from both the blood pool and myocardium. 6


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Planar Imaging. Figure 3 shows planar images and image quantification data for the SD rats administered with 99mTc-4ASboroxime. Clearly, 99mTc-4ASboroxime has very high uptake in the heart region with a long residual time. It is important to note that the heart radioactivity values obtained from image quantification include the radioactivity in the blood pool and myocardium. Most of the injected

99m

Tc-4ASboroxime is actually in the blood pool (not myocardium) during

the 2 h study period. Main blood vessels in the head/neck and abdominal regions (Figure 3A) are clearly visualized. The radioactivity in the heart region decreases rapidly over the first 5 min after injection (Figure 3B), and then remains relatively unchanged over the next 120 min. These data suggest that the binding of 99mTc-4ASboroxime to blood components (cells and proteins) is highly stable. The bladder radioactivity level is almost undetectable in planar images until 120 min p.i. (Figure 3A), indicating that 99mTc-4ASboroxime has very little renal excretion. We also carried out planar imaging studies by pre-mixing

99m

Tc-4ASboroxime with the fresh SD rat

blood in a syringe right before its injection. Similar results were obtained with the blood-bound 99m

Tc-4ASboroxime (Figure SI1). Therefore, the interaction kinetics of 99mTc-4ASboroxime with

blood components is fast both in vitro and in vivo. Biodistribution Properties. Table 1 summarizes the selected biodistribution data (%ID/g) for

99m

Tc-4ASboroxime in SD rats (200 – 220 g) at 2, 15, 60 and 120 min p.i. Its blood

radioactivity (~6.0 %ID/g) is close to that for 62Cu and 68Ga-labeled albumin in the same animal species.36-40 The total blood radioactivity is 90 – 95% since the total blood volume is 15 – 16 mL for 200-g SD rats.41,42 However, its uptake values in kidneys, liver, lungs and muscle are significantly lower than those reported for

99m

Tc-4Sboroxime,35 and

62

Cu and

albumin at 60 min p.i.36-40 The blood radioactivity and heart uptake of

68

Ga-labeled

99m

Tc-4ASboroxime

remain almost unchanged over the 2 h period (Table 1), suggesting that the interaction between

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99m

Tc-4ASboroxime and blood components is fast and stable. This conclusion is consistent with

the results from planar imaging studies (Figure 3). The uptake of

99m

Tc-4ASboroxime in most

normal organs (Table 1: brain, fat, intestines, kidneys, lungs, muscle, and spleen) also remained relatively unchanged within the experimental errors of biodistribution assay over the 2 h study period. In contrast, the liver uptake of

99m

Tc-4ASboroxime decreases rapidly over the first 15

min (mainly going into spleen), and then stays almost unchanged over the next 2 h, suggesting that

99m

Tc-4ASboroxime has little excretion via the hepatobiliary route. Similar results are

obtained from the in vitro

99m

Tc-labeling approach by pre-mixing

99m

SD rat blood in a syringe before its injection (Table 1). Therefore,

Tc-4ASboroxime with the

99m

Tc-4ASboroxime can be

used for both in vitro and in vivo 99mTc-labeling of RBCs. Table 1. Selected biodistribution data (%ID/g) for 99mTc-4ASboroxime in SD rats (200 – 220 g) at 2, 15, 60 and 120 min p.i. Organ

2 min (n = 5)

15 min (n = 5) 60 min (n = 5)

60 min (n = 3)**

120 min (n = 4)

Blood

6.40±0.84

6.45±0.85

6.37±0.41

5.67±1.05

6.40±1.50

Brain

0.06±0.01

0.06±0.02

0.06±0.01

0.05±0.01

0.09 ±0.04

Fat*

0.12±0.03

0.17±0.07

0.18±0.01

0.15±0.01

0.16±0.06

Heart

0.55±0.12

0.54±0.08

0.51±0.04

0.53±0.04

0.51±0.13

Intestines

0.35±0.13

0.70±0.35

0.90±0.29

0.88±0.30

0.66±0.27

Kidneys

1.66±0.34

1.47±0.35

1.41±0.18

1.55±0.15

1.60±0.60

Liver

2.28±0.70

1.36±0.18

1.21±0.10

1.10±0.20

1.08±0.22

Lungs

1.63±0.21

1.40±0.30

1.13±0.07

1.23±0.05

1.29±0.42

Muscle

0.09±0.02

0.10±0.01

0.09±0.01

0.08±0.02

0.11±0.05

Spleen

1.74±0.49

2.84±0.41

2.72±0.61

2.65±0.33

2.23±1.23

Vessels

0.23±0.02

0.29±0.02

0.29±0.05

0.28±0.04

0.31±0.07

*Fat tissues around blood vessels on the heart; ** Data obtained by mixing the rat whole blood with 99mTc-4ASboroxime in the syringe right before intravenous administration via the tail vein.

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RBC-Specificity. Figure 4 compares the 2-min blood radioactivity and the percentage of RBC-binding for

99m

Tc-4ASboroxime and

99m

Tc-4Sboroxime. The blood radioactivity level is

~6%ID/g for 99mTc-4ASboroxime, ~95% of which is RBC-bound (Figure 4B) with ~1% binding to albumin, and ~3% remaining free in the blood plasma. In contrast, The blood radioactivity is ~0.5%ID/g for 99mTc-4Sboroxime,35 and ~54% of which is associated with RBCs, ~2% binds to albumin and ~44% remains free in blood plasma. 99mTc-4ASboroxime is specific for RBCs.

Figure 4. A: Comparison of 2-min blood radioactivity (%ID/g) between 99mTc-4ASboroxime and 99m Tc-4Sboroxime. The 2-min blood radioactivity data for 99mTc-4Sboroxime was from our previous study.35 B: The RBC-binding % for 99mTc-4ASboroxime and 99mTc-4Sboroxime. These experiments were performed to explore the impact of PhSO2NH2 and PhSO2CH3 groups on the cellular and protein binding capability of 99mTc-4ASboroxime and 99mTc-4Sboroxime.

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Figure 5. The selected 3-dimensional static images of the SD rat administered with ~185 MBq of 99m Tc-4ASboroxime at 0 – 60 min p.i. Images were obtained the using u-SPECT-II scanner. IVC: inferior vena cava; LA: left atrium; RA: right atrium; LV: left ventricle; RV: right ventricle. Major blood vessels were clearly seen throughout the body. Anterior, posterior and side views in the heart region were used to illustrate the presence of blood pool radioactivity. SPECT Imaging in SD rats. Figure 5 displays the 3D images of the SD rat administered with

99m

Tc-4ASboroxime. For comparison purpose, we also obtained SPECT images for the SD

rat administered with

99m

Tc-4Sboroxime (Figure 6). The main blood vessels in the head/neck

regions and lower body parts are well delineated. Even though the aorta is clearly seen, the descending arteries on left and right ventricles are not visualized due to its high blood pool radioactivity accumulation. Microvessels in the liver are also clearly visualized (Figure SI2). If the uptake of

99m

Tc-4ASboroxime were able to perfuse into the tissues of liver, it would have

resulted in more homogeneous radioactivity distribution. This statement is also supported by the low uptake of

99m

Tc-4ASboroxime in the liver (Table 1). These results strongly suggest that the

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interaction between

99m

Tc-4ASboroxime and RBCs is highly stable. It is interesting to note that

the radioactivity distribution pattern is complimentary to each other for 99mTc-4ASboroxime and 99m

Tc-4ASboroxime in the heart.

99m

Tc-4ASboroxime is mainly distributed in the blood pool

with minimal radioactivity in myocardium (Figure 6). In contrast,

99m

Tc-4Sboroxime is a good

radiotracer for heart imaging with the radioactivity being mainly distributed in myocardium with minimal radioactivity in blood pool (Figure 6). These results suggest that

99m

Tc-4ASboroxime

might be useful for determination of blood volumes, measurement of cardiac function, and delineation of blood vessels.

Figure 6. The expanded heart region of the SD rat administered with 99mTc-4ASboroxime (left) and the SPECT image (horizontal long axis) of the SD rat administered with 99mTc-4Sboroxime (right). Images were obtained at 0 – 60 min after injection of 99mTc-4ASboroxime and 0 – 5 min for 99mTc-4Sboroxime using the u-SPECT-II scanner. Anesthesia was induced using an air flow rate of 350 mL/min and ~3.0% isoflurane. IVC: inferior vena cava; LA: left atrium; RA: right atrium; LV: left ventricle; RV: right ventricle. 99mTc-4ASboroxime is an excellent blood pool imaging agent while 99mTc-4Sboroxime is a radiotracer for perfusion imaging.

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Planar Imaging in Pigs. Figure 7 shows the selected whole-body images of a pig (~25 Kg) administered with

99m

Tc-4ASboroxime. Its distribution patterns are similar to those observed in

SD rats (Figure 3), suggesting that there is little difference in its RBC-binding capability in SD rats and pigs. The blood pool radioactivity is almost constant over the 2-h period, indicating that its interaction with RBCs is stable. The main blood vessels in the head/neck and abdominal regions are clearly visualized with good contrast. The liver uptake is relatively low at 5 min p.i., and peaks at ~30 min p.i. There is almost no excretion of

99m

Tc-4ASboroxime during the 2-h

study period through both renal and hepatobiliary routes. These results are consistent with those from planar imaging (Figure 3) and biodistribution studies (Table 1) in SD rats, and further confirm that

99m

Tc-4ASboroxime is an excellent blood pool agent with great potential for

delineation of blood vessels and diagnosis of vascular diseases.

Figure 7. The whole-body images of a pig (~26 Kg) administered with ~370 MBq (10 mCi) of Tc-3SPboroxime at 5 min, 30 min, 60 min and 120 min, respectively, through the ear vein. These images were acquired using a GE Discovery NM630 dual headed detector scanner equipped with a low energy general purpose collimator. Anesthesia was achieved with intravascular injection of 3% sodium pentobarbital (30 mg/kg). The blood pool and the main blood vessels are clearly detected at 5 min after injection, and maintain almost constant over 120 min study period. 99m

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DISCUSSION Traditionally,

99m

Tc-labeling of RBCs can be achieved by two approaches: in vitro and in

vivo. The in vitro approach involves incubation of the whole human blood or pre-separated RBCs with Sn(II) before labeling with 99mTcO4- in the presence of a chelating agent (such as citrate and glucoheptonate).24-29 Kit formulations (e.g. Ultra-Tag® RBC) are commercially available for routine in vitro

99m

Tc-labeling of RBCs.27,28 The in vivo approach involves consecutive short-

interval injections of Sn(II) and 99mTcO4-.29,30 The optimal lag time between two injections is ~30 min.29,30 The RBC-labeling yield is >95% at 30 – 120 min after injection of 99mTcO4-.24,25,29,30 It is believed that the integrity of RBCs is maintained and the radiolabel is stable.24,25 However, the chemistry associated with

99m

Tc-labeling of RBCs is poorly understood for both in vitro and in

vivo methods. It is also not known how 99mTc is attached to RBCs, and why 99mTc-labeled RBCs remain stable in vivo. Both the in vitro and in vivo methods are complicated and lack of reproducibility. In addition, centrifugation of human RBCs in the in vitro approach is timeconsuming and constitutes the potential for cross-contamination from blood products. In this study, we present a new approach for

99m

Tc-labeling of RBCs.

99m

Tc-4ASboroxime

can be prepared in high yield with RCP >98%. The coordination chemistry associated with complexes [99mTcCl(CDO)(CDOH)2B-R] has been well established in the prior literature.43-45 The approach described in this study is convenient and efficient by eliminating the timeconsuming centrifugation step, and avoiding the potential for cross-contamination from blood products. The RBC-binding of

99m

Tc-4ASboroxime is stable and specific (Figure 4B) without

compromising physiologic behavior of RBCs. This statement is further supported by the fact that the blood radioactivity is >90% of the injected dose (Table 1), and remains relatively unchanged over the 2 h study period (Figure 3).

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Injection of

Page 14 of 30

99m

Tc radiotracer into the blood stream exposes it to RBCs, white blood cells

(WBCs), platelets and proteins prior to encountering the tissue. Since the

99m

Tc radiotracer is

free before being taken up by cells, its availability for tissue uptake depends on the extent to which the cellular and protein-bound

99m

Tc radiotracer becomes free during perfusion. Thus,

cellular and protein binding is the main factors that control tissue distribution properties of 99mTc 99m

Tc radiotracer molecules are less available

radiotracer. High blood radioactivity means that

for their uptake in different organs. For 99mTc-4ASboroxime, >90% of the injected dose is in the blood. More than 95% of its blood radioactivity are bound to the cells (RBCs, WBCs and platelets). In the blood circulation, RBCs (4.2 – 6.2 million per microliter) are in large excess over WBCs (4000 – 11000 per microliter) and platelets (150,000 – 350,000 per microliter). Thus, RBCs are most likely the cellular targets for radioactivity for

99m

Tc-4ASboroxime. In contrast, the 2 min blood

99m

Tc-4Sboroxime is only ~5%ID, and 54% of which binds to the RBCs, ~2%

to albumin and ~44% remains free in blood plasma. This may explain why 99mTc-4Sboroxime is a heart imaging agent (Figure 6) and 99mTc-4ASboroxime is useful as a radiotracer for blood pool imaging (Figure 5). Even though the blood radioactivity of 99mTc-4ASboroxime is very closed to that of

62

Cu and

68

Ga-labeled albumin,36-40 its uptake in brain, intestines, kidneys, liver, lungs,

muscle and spleen is lower than those reported for 62Cu and 68Ga-labeled albumin.36-40 The next question is why 99mTc-4ASboroxime and

99m

Tc-4Sboroxime share similar structure

and show so much difference in their biodistribution properties. The answer to this question is most likely related to the binding of 99mTc-4ASboroxime to the enzyme inside or on the surface of RBCs. Since the discovery of sulfonamide antibacterial drugs (such as sulfamethoxazole), a number of sulfonamide-containing drugs have been developed as diuretics (e.g. azosemide), antimigraine agents (e.g. sumatriptan), and cyclooxygenase-II (COX-2)-specific anti-inflammatory

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drugs (e.g. Celecoxib). Celecoxib is also a highly potent inhibitor with binding affinity in nanomolar range for carbonic anhydrases (CA),46 which catalyzes interconversion of carbon dioxide (CO2) and carbonic acid (H2CO3) to maintain the acid-base balance in blood and other tissues. Metal-chelated Celecoxib analogs have been evaluated as CA inhibitors or CA-targeted NMR contrast agents.47,48 Cu(II) complexes of TETA-conjugated sulfonamides have the higher inhibitory potency than sulfonamide ligands alone.49 Crystal structures of CA inhibitors containing triazole–ferrocene or triazole–ruthenocene groups have also been reported.50 The barrel-shaped hydrophobic ferrocene and ruthenocene moieties provide an excellent fit for the hydrophobic binding cavity of CA enzyme.51,52 Alberto and co-workers also found that sulfonamide-containing CA-II inhibitors with the [(Cp-R)Re(CO)3] motif have inhibition constants in low nanomolar range for CA isozymes.53 Since

99m

Tc-4ASboroxime and Celecoxib

share the same benzenesulfonamide group, it is conceivable that it may bind to CA and/or COX2. CA is present in the cytosol of RBCs almost exclusively.54 In contrast, COX-2 is absent in normal tissues and expressed only in response to inflammatory stimuli.55 Thus, the CA enzyme in cytosol of RBCs is likely the target for

99m

Tc-4ASboroxime. This conclusion is supported by

the fact that sulfonamide-containing COX-2 inhibitors exhibit high CA inhibitory potency,46 while non-sulfonamide COX-2 inhibitors shows no CA inhibitory activity.46 This may explain why

99m

Tc-4Sboroxime has much less radioactivity in the blood pool and higher uptake in the

myocardium than 99mTc-4ASboroxime (Figure 6). The number of 99mTc-4ASboroxime molecules binding to each RBC depends on the injected dose. If the injected dose is ~370 MBq (7.2 x 10-11 moles of total

99m/99

Tc from the 99Mo/99mTc

generator with 24 h prior elusion time), the initial blood concentration of 99m/99Tc-4ASboroxime is expected to be ~8.6 x 106 molecules per microliter for a human subject with the blood volume

15



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Page 16 of 30

of ~5 L. The injected number of 99m/99Tc-4ASboroxime molecules is ~4.3 x 1013. Since the total number of RBCs is only ~2.5 x 1013 in the human subject (or ~5 x 106 million per microliter), there is an average of ~1.7 99m/99Tc-4ASboroxime molecules in each RBC. Because of its smaller size, the inclusion of 99mTc-4ASboroxime is not expected to compromise physiologic behavior of RBCs. Since the total number of active sites is larger than the number of

99m/99

Tc-4ASboroxime

molecules in each RBC, the binding of 99mTc-4ASboroxime is not expected to have a significant impact on the overall catalytic activity of RBCs. In addition, 99mTc-labeled RBCs are expected to have much less leakage from the intravascular space because they are significantly larger than the

99m

Tc-labeled human serum albumin. From this view point,

the advantage over

99m

Tc-labeled RBCs will have

99m

Tc-labeled albumin for blood pool imaging and/or SPECT angiography.

Potential clinical applications of

99m

Tc-labeled RBCs include: determination of blood volume,

measurement of cardiac function, delineation of major blood vessels, and detection of hepatic hemangiomas. With the tremendous development of CZT-based high-speed SPECT cameras (such as the Discovery NM 530c from GE Healthcare),56-58 which have much higher sensitivity and spatial resolution, the 99mTc-labeled RBC might expand its applications for diagnosis of deep vein thrombosis, cerebral blood flow and stroke. CONCLUSIONS In this study, we evaluate 99mTc-4ASboroxime as a radiotracer for routine in vitro and in vivo 99m

Tc-labeling of RBCs. The RBC-binding of 99mTc-4ASboroxime is instantaneous, specific and

highly stable. The approach reported in this study is convenient and efficient, eliminates the complicated and time-consuming centrifugation step, and avoids the potential for crosscontamination from blood products.

99m

Tc-4ASboroxime is an excellent blood pool agent, most

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likely targeting carbonic anhydrase in the cytosol of RBCs. Future studies will focus on the CA binding affinity of Re(III) complex [ReCl(CDO)(CDOH)2B-4AS]. EXPERIMENTAL METHODS Materials.

Citric

acid,

γ-cyclodextrin,

cyclohexanedione

dioxime

(CDOH),

diethylenetriaminepentaacetic acid (DTPA), (4-aminosulfonylphenyl)boronic acid (4ASB(OH)2), 4-(methanesulfonyl)phenylboronic acid (4S-B(OH)2), NaCl and SnCl2.2HO2 were purchased from Sigma/Aldrich (St. Louis, MO) or Matrix Scientific (Columbia, SC), and were used without further purification. Na99mTcO4 were obtained from Cardinal HealthCare® (Chicago, IL). Synthesis of 99mTc-4Sboroxime has been described in our previous report.35 Radio-HPLC Method. The radio-HPLC method for analysis of

99m

Tc-4ASboroxime used

an Agilent HP-1100 HPLC system (Agilent Technologies, Santa Clara, CA) equipped with a βram IN/US detector (Tampa, FL) and Zorbax C8 column (4.6 mm x 250 mm, 300 Å pore size; Agilent Technologies, Santa Clara, CA). The flow rate was 1 mL/min. The mobile phase was isocratic with 30% solvent A (10 mM NH4OAc buffer, pH = 6.8) and 70% solvent B (methanol) between 0 and 5 min, followed by a gradient from 70% solvent B at 5 min to and 90% solvent B at 15 min, and isocratic mobile phase with 10% solvent A and 90% solvent B. The RCP was reported as the percentage of area for the expected radiometric peak on each radio-HPLC chromatogram of 99mTc-4ASboroxime. The ITLC method used Gelman Sciences silica-gel strips and a 1:1 (v:v) mixture of acetone and saline as the mobile phase.

99m

Tc-4ASboroxime and

99m

TcO4- migrated to solvent front while [99mTc]colloid stayed at the origin. Radiosynthesis of

99m

Tc-4ASboroxime. To a clean 5 cc vial was added 1 mL solution

containing 2 mg of CDOH2, 4 – 5 mg of 4AS-B(OH)2, 50 – 60 µg of SnCl2·2H2O, 9 mg of citric

17



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Page 18 of 30

acid, 2 mg of DTPA, 50 – 60 mg of NaCl and 20 mg of γ-cyclodextrin, followed by addition of 1.0 mL

TcO4- solution (370 – 1110 MBq). The vial was heated at 100 oC for 10 – 15 min.

99m

After cooling to room temperature, a sample of the resulting solution was diluted with saline containing ~20% propylene glycol to 3.7 MBq/mL. The diluted solution was analyzed by radioHPLC and ITLC. The radiochemical purity (RCP) for

99m

Tc-4ASboroxime was >98% with

minimal amount of [99mTc]colloid (24 h. Animals were anesthetized with intramuscular injection of a mixture of ketamine (80 mg/kg) and xylazine (19 mg/kg) before being used for biodistribution and planar imaging studies.

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Biodistribution Protocol. Twenty SD rats (200 – 220 g) were randomly selected, and divided into 4 groups. Each animal was administered with ~ 110 KBq of 99mTc-4ASboroxime or blood-bound

99m

Tc-4ASboroxime via the tail-vein. Five animals were sacrificed by sodium

pentobarbital overdose (100 – 200 mg/kg) at 2, 15, 60 and 120 min p.i. Blood was withdrawn from heart. Organs of interest (heart, brain, intestines, kidneys, liver, lungs, muscle, and spleen) were harvested, rinsed with saline, dried with absorbent tissues, weighed and counted for radioactivity on a Perkin Elmer Wizard – 1480 γ-counter (Shelton, CT). The organ uptake was reported as the percentage of injected dose per gram of organ tissue (%ID/g). Comparison between two radiotracers was made using one-way ANOVA test (GraphPad Prim 5.0, San Diego, CA). The level of significance was set at p < 0.05. Cellular and Protein-Binding Assay. Blood samples were withdrawn (0.5 – 1.0 mL) from the rat heart at 2 min after injection of

99m

Tc radiotracer, and were then centrifuged at 12,000

rpm (Microfuge 22R Centrifuge, Beckman Coulter, Indianapolis, IN) for 10 – 15 min. The solid (mainly RBCs components) and supernatant were separated, collected and countered on a Perkin Elmer Wizard – 1480 γ-counter (Shelton, CT). The percentage of RBC-binding was calculated as percentage of blood radioactivity. Protein binding of 99mTc-4Sboroxime and 99mTc-4ASboroxime were quantitatively evaluated by ultrafiltration according to the literature method.36,37 Each Amicon Centrifree® (Beverly, MA) ultrafiltration device (30,000 Dalton NMWL) was loaded with 300 – 600 µL of supernatant collected using the procedure above, and centrifuged at 12,000 rpm for 25 – 30 min. The 99mTc activity on the filter and in filtrate was determined by γ-counting. The percentage of free 99mTc radiotracer will be calculated using the literature method.36,37 Dynamic Planar Imaging. Planar imaging was performed in SD rats (n = 5). Each animal was administered with 37 ~ 45 MBq of 99mTc-4ASboroxime or blood-bound 99mTc-4ASboroxime

19



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Page 20 of 30

(prepared by mixing 0.1 mL rat blood with an equal volume of the dose solution containing 99m

Tc-4ASboroxime) via the tail vein injection. The animal was immediately placed prone on a

single head mini γ-camera (Diagnostic Services Inc., NJ). The 1-min images were acquired during first 5 min p.i., followed by 2-min images at 6 – 30, 40, 50 and 60 min p.i. The imaging data were stored digitally in a 128 x 128 matrix. After imaging, animals were returned to a leadshielded cage to recover. Since all new radiotracers had no excretion via hepatobiliary and renal routes, planar images were analyzed by drawing regions of the heart (the heart radioactivity) and whole body (the injected radioactivity into each animal). The background was corrected by drawing the region right above heart. The results were expressed as the percentage of injected radioactivity (%ID). The curve fit of myocardial washout kinetics was determined using GraphPad Prim 5.0 (GraphPad Software, Inc., San Diego, CA). The data were reported as an average ± standard deviation based on the results from 4 – 6 animals at each time point. Protocol for SPECT in SD Rats. Static imaging was performed using the u-SPECT-II/CT scanner (Milabs, Utrecht, The Netherlands) equipped with a 1.0 mm multi-pinhole collimator. The SD rat was placed into a shielded chamber connected to an isoflurane anesthesia unit (Univentor, Zejtun, Malta). Anesthesia was induced using an air flow rate of 350 mL/min and ~3.0% isoflurane, and maintained using an air flow of ~250 mL/min with ~2.5% isoflurane during image data acquisition (75 projections over 30 min per frame, 2 frames). The animal was administered with

99m

Tc-4ASboroxime (~185 MBq) in 0.5 mL saline containing 20 ~ 30%

propylene vial the tail-vein injection. Rectangular scan in regions of interest (ROIs) were selected one the basis of orthogonal X-ray images provided by the CT. After image data acquisition, the animal was translated into the attached CT scanner and imaged using the ‘normal’ acquisition settings (2 degree intervals) at 45 kV and 500 µA. After CT acquisition, the

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animal was allowed to recover in a lead-shielded cage. Image reconstruction was performed using a POSEM (pixelated ordered subsets by expectation maximization) algorithm with 6 iterations and 16 subsets. After reconstruction, SPECT data were re-sampled to equivalent voxel sizes. The images were further rendered and visualized using the PMOD software (PMOD Technologies, Zurich, Switzerland). A 3D-Guassian filter (1.0 mm FWHM) was applied to smooth noise, and the LUTs (look up tables) were adjusted for good visual contrast. The images were visualized as orthogonal slices and maximum intensity projections. Static Imaging Protocol in Pigs. The whole body static scan was performed in normal pigs (23 – 26 Kg) using a GE Discovery NM630 dual headed detector scanner with a low energy general purpose collimator. The imaging protocol was approved by the Fu Wai Hospital Animal Care and Use Committee (Beijing, China). The pig was supine on the scanning table after anesthesia with intravascular injection of 3% sodium pentobarbital (30 mg/kg). Additional sodium pentobarbital were used to maintain anesthesia.

99m

Tc-4ASboroxime (370 MBq/5mL)

was injected into the ear vein. The image data acquisitions began at 5 min, 30 min, 60 min and 120 min, respectively, after administration at a rate of 15cm/min in the whole body scan mode. Anterior and posterior position whole-body images were obtained at four time points. Supporting Information Available: Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxxxxxxx. Figure SI1. Planar images of the SD rat administered with 99mTc-4ASboroxime. (PDF) Figure SI2. Image of the SD rat administered with 99mTc-4ASboroxime. (PDF) AUTHOR INFORMATION Corresponding Authors: S.L. e-mail: [email protected] W.F. e-mail: [email protected]. Conflict of interest: The authors declare no competing financial interest. 21



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Acknowledgement. This work was supported, in part, by Purdue University, R21 EB017237-01 (S.L.) from the National Institute of Biomedical Imaging and Bioengineering (NIBIB), and grants 81771872/81320108014 from the National Nature Science Foundation of China (YZ). References (1)

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