Radioiodinated Pentixather for SPECT Imaging of the Chemokine

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Radioiodinated Pentixather for SPECT Imaging of the Chemokine Receptor CXCR4 Expression in Rat Myocardial Infarction/Reperfusion Models Jindian Li, Chenyu Peng, Zhide Guo, Changrong Shi, Rongqiang Zhuang, Xingfang Hong, Xiangyu Wang, Duo Xu, Pu Zhang, Deliang Zhang, Ting Liu, Xinhui Su, and Xianzhong Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02553 • Publication Date (Web): 12 Jul 2018 Downloaded from http://pubs.acs.org on July 14, 2018

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Analytical Chemistry

Radioiodinated Pentixather for SPECT Imaging of the Chemokine Receptor CXCR4 Expression in Rat Myocardial Infarction/Reperfusion Models

Jindian Li1, Chenyu Peng1, Zhide Guo1, Changrong Shi1, Rongqiang Zhuang1, Xingfang Hong2, Xiangyu Wang1, Duo Xu1, Pu Zhang1, Deliang Zhang1, Ting Liu1, Xinhui Su3 and Xianzhong Zhang1*

1

State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics & Center

for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen 361102, China. 2

Laboratory of Pathogen Biology, School of Basic Medical Sciences, Dali University,

Dali 671000, China. 3

Zhongshan Hospital Affiliated to Xiamen University, Xiamen 361004, China.

Running title: 125I-pentixather for CXCR4 imaging Corresponding

author:

Xianzhong

Zhang,

PhD,

Professor,

E-mail:

[email protected]; Phone/Fax: +86-592-2880645. Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiang’an South Rd., Xiang’an district, Xiamen 361102, China.

1

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 25

Abstract: The purpose of this study is to develop a specific CXCR4-targeting radioiodinated agent (125I/131I-pentixather) for single-photon emission computed tomography

(SPECT)

infarction/reperfusion 125

imaging

(MI/R)

rat

of

CXCR4

models.

After

expression SPECT/CT

in

myocardial

imaging

with

I-pentixather at 4, 12, 36 h, 3 and 7 d after MI/R, the models were validated by ex

vivo autoradiography, TTC staining and immunohistochemistry, and in vivo echocardiography and classical

99m

Tc-MIBI perfusion imaging, respectively. The

SPECT/CT images showed that the infarcted myocardium (IM) could be visualized with high quality as early as 4 h and reached the maximum at 3 d after MI/R, and the CXCR4 upregulation is still visible at 7 d after MI/R. In the biodistribution study, high uptakes in the IM (0.99 ± 0.13, 1.52 ± 0.29, 1.75 ± 0.22, 1.94 ± 0.27 and 0.61 ± 0.14 %ID/g at 4, 12, 36 h, 3 and 7 d after MI/R respectively) were observed and which much higher than that of normal myocardium. The highest uptake was reached at 3 d after MI/R, which agreed well with the SPECT results. In addition, both the radioactivity uptakes of IM in the biodistribution and SPECT imaging could be blocked effectively by excess amount AMD3465, indicating the high specificity of radioiodinated pentixather to CXCR4. Based on the promising properties of 125

I-pentixather, it may serve as a powerful CXCR4 expression diagnostic probe for

evaluating lesion and monitoring therapy response in patients with cardiovascular diseases.

Key words: CXCR4; 131/125I-pentixather; SPECT Imaging; Myocardial Infarction

2


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Introduction Precision medicine demands a precise understanding of the biological process of human diseases1. Molecular imaging enables to provide noninvasive visualization, characterization, and quantification of the biologic processes of human diseases to guide the development of precision medicine and to tailor individualized treatment for better therapeutic outcomes2. Myocardial infarction (MI) is the leading cause of morbidity and mortality worldwide3. Up to 1 billion myocardial cells die post myocardial infarction4. Cardiomyocyte necrosis triggers an intense inflammatory cascade and progenitor cell recruitment5. Current clinical cardiac imaging modalities include anatomy, perfusion, function, and myocardium viability assessment, but less involved insight into the specific molecular pathways in the repair process. The chemokine receptor CXCR4 plays a very pivotal role in the homing and recruitment of progenitor and stem cells to the ischemic zone, which has emerged as a therapeutic target to support tissue repair6-9. Stromal cell derived factor 1 (SDF-1) binds to its receptor CXCR4 regulating adhesion and migration of inflammatory cells and increases homing of CXCR4-positive stem/progenitor cells to ischemic tissue. Several SPECT and positron emitting tomography (PET) imaging probes for CXCR4 expression have been applied in several diseases models10-18. However, only 99m

Tc-MAS3-SDF-1α and 68Ga-pentixafor have been applied to myocardial infarction

imaging. Frangioni et.al reported

99m

Tc-MAS3-SDF-1α could be used as a sensitive

and specific probe for CXCR4 expression in vivo and be able to quantify changes in CXCR4 expression after myocardium infarction. However, the infarct myocardium uptake of

99m

Tc-MAS3-SDF-1α was only 0.57 ± 0.04 %ID/g and they did not exhibit

myocardial imaging results18.

68

Ga-pentixafor, a radiolabeled CXCR4 ligand, has

been developed successfully and is the first noninvasive detection probe of CXCR4 expression in the human myocardium after acute myocardial infarction19,20. However, it still has some disadvantages. Firstly, Static PET images were acquired at 60 min after injection of

68

Ga-pentixafor in rats and human, which may delay the optimal

treatment time for patients with myocardial infarction. Secondly, uptake in bone 3



Page 4 of 25

marrow was up to 3.0 %ID/g may result in radiation damage. Thirdly, blood level and infarct uptake were approximately equal (2.0 vs. 2.2) and the infarcted-remote myocardium ratio was 1.7, therefore, the high blood pool effect and low infarcted-remote myocardium ratio could reduce the quality of infarcted myocardium (IM) imaging. The structure of drug determines its pharmacological activity. Lipophilic chelate ligand, 4-(aminomethyl) benzoic acid-DOTA, may result in the slow blood clearance and the high uptake in bone marrow of the cyclic peptide pentixather. Therefore, we suppose that

131 125

I/

I directed labeled tyrosine of pentixather may have faster plasma

clearance, lower bone marrow uptake and higher imaging quality such as higher IM-to-normal myocardium and IM-to-blood ratios. In addition, SPECT imaging is cheaper than PET imaging and suitable for a larger range. Moreover, quantitative SPECT such as Siemens symbia intevo xSPECT system has been received FDA clearance and becoming more and more popular in the world. Therefore, the development of new CXCR4-targeting SPECT imaging probes has great value in clinical applications. In this study, we aimed to develop a

131 125

I/ I-pentixather tracer for noninvasive

visualization and quantification expression of CXCR4 at early and late time points after IM/R. The tracer of radioiodinated pentixather was evaluated in vitro stability, pharmacokinetics,

ex

vivo

biodistribution,

autoradiography,

and

immunohistochemistry, and in vivo SPECT/CT imaging.

EXPERIMENTAL SECTION Materials and Methods. Cyclo(D-Tyr1-D-[NMe]Orn2-Arg3-Nal4-Gly5) (pentixather) was purchased from Nanjing Peptide Biotech Ltd (Nanjing, China) with a purity > 95%. Iodogen (1,3,4,6-tetrachloro-3α,

6α-diphenylglycouril)

was

obtained

from

Pierce

Biotechnology (ZI Camp Jouven, France). Na131I was supplied by Zhongshan Hospital Affiliated to Xiamen University. Na125I was obtained from China Isotope & Radiation Corporation (Beijing, China). Male adult Wistar rats (250 ~ 280 g) were

4


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purchased from Shanghai Slakey Laboratory Animal Co. Ltd. (Shanghai, China). The radioactivity counts were measured with an automatic γ-counter (WIZARD 2480, Perkin-Elmer, USA). SPECT imaging study was performed using a microSPECT/CT scanner (Mediso, HUNGARY). Radiosynthesis of

131 125

I/ I-pentixather. The iodogen-coating method was employed

for radioiodination of pentixather with a Na131I or Na125I solution to form 131

I-pentixather or

125

I-pentixather21. The radiosynthesis route of

131

125

I-pentixather was shown in Scheme 1. Briefly, the pentixather (50 µg) was

I-pentixather and

dissolved in 50 µL of normal saline and transferred to an Eppendorf tube coated with 80 µg of iodogen (the oxidant iodogen was dissolved in dichloromethane solvent and then coated on the wall of tube). Upon addition of Na131I (55.5 MBq) or Na125I (74 MBq), the reaction vessel was briefly vortexed and then allowed to proceed for 10 min at room temperature. The reaction was terminated by transferring the reaction solution to a clean tube. Radiochemical purity and separation of 125

131

I-pentixather or

I-pentixather from unlabeled precursor was achieved using HPLC (Thermo Fisher

Dionex UltiMate 3000) with the Nucleosil C18 column (250 × 4 mm, 10 µm, 100 Å) and chromatograms were collected at 254 nm. Gradient: 25%–50% methanol (0.1% trifluoroacetic acid) in water (0.1% trifluoroacetic acid) within 25 min with flow rate at 1 mL/min. The HPLC product fraction was identified by high-resolution mass spectrum and then dried with nitrogen. Afterward, it was dissolved and diluted in normal saline to the desired concentration. Stability Studies in vitro. To observe the stability of the radiolabeled compound in normal saline and rat plasma, 50 µL

131

I-pentixather (37 MBq) solution was added to

450 µL normal saline or rat plasma and incubated at 37 °C for 30 and 60 min. Plasma proteins were precipitated by adding equal acetonitrile and removed by centrifugation (13500 rpm, 5 min)22. The radiochemical purity of 131I-pentixather was determined by HPLC as described above. Determination of Partition Coefficient. The partition coefficient (log P) was determined by the “shake-flask” method23. 185 ~ 222 kBq (5 ~ 6 µCi) 131I-pentixather was added to Eppendorf tube containing 1 mL phosphate-buffered saline (PBS, 0.1 M, 5



pH = 7.4) and 1 mL n-octanol (n = 3), respectively. The mixture was vortexed for 10 min followed by centrifugation (12,000 rpm for 10 min). The counts in 100 µL aliquots of both octanol and PBS phase were measured using an automatic γ-counter. The octanol/buffer partition coefficient was calculated as P = (cpm in the organic phase − background cpm)/(cpm in the aqueous phase - background cpm) and was expressed as log P. An average log P value was obtained from those ratios. Pharmacokinetics. The rats (n = 3) were intravenously injected with 25 MBq/kg of 131

I-pentixather. After 5, 10, 30 min, 1, 2, and 4 h injection, 10 µL blood was collected

through the tail vein, respectively24. The radioactive counts of blood samples were measured using an automatic γ-counter corrected for background radiation and physical decay. Measured activity was expressed as megabecquerel per liter of blood (MBq/L). The pharmacokinetic parameters were calculated by the statistical moment method of the non-compartment model using Drug and Statistics for Windows 2.0 software (SAS Inc., Cary, NC). MI/R model. All the animal procedures were in accordance with the Guide for the Care and Use of Laboratory Animals and were reviewed and approved by Xiamen University’s animal care and use committee. Rat MI/R models were conducted as previously described21. Briefly, the rat was intraperitoneally anesthetized with chloral hydrate (10%, 3 mL/kg), then intubated and artificially ventilated with room air using a small animal ventilator (RWD407, Shenzhen, China). Afterward, open chest surgery was performed along the third and fourth intercostal space, cut the pericardium to expose the left ventricle. Left anterior descending (LAD) coronary artery was ligated using a 4-0 silk suture by a detachable knot at 2 mm below the junction of the left atrial appendage and the pulmonary conus. The end of suture was left outside of the thorax after the chest wall was closed. The suture end was pulled to coronary reperfusion 60 minutes after LAD occlusion. For preventing infection, the rats were intramuscularly injected with 80000 U Penicillin. Echocardiography. Transthoracic echocardiography was performed at baseline and 3 h after coronary ligation, as described previously25,26. Rats were anesthetized using 3% isoflurane. After the hairs of the anterior chest were removed using chemical hair 6


Page 6 of 25

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remover, the rats were placed on the scanning table maintaining normothermia (37 °C) and monitored by a rectal thermometer. Ultrasound gel was applied to the chest. Echocardiograms were performed with dedicated small animal high-resolution echocardiography system (Vevo 2100 system, VisualSonics, Canada) equipped with a transducer (MS250) with a frequency of 25 MHz. A two-dimensional guided M-mode of the parasternal short axis at the papillary muscle level was obtained to measure standard parameters. Biodistribution. Biodistribution study was performed to quantify the absolute tracer uptake in the interested tissues. In this study, acute infarct models with 60 min of ischemia followed by 4, 12, 36 h, 3 and 7 d of reperfusion were used. Eighteen MI/R rats were randomly divided into six groups (n = 3). Each rat was injected with 131

I-pentixather (3.7 MBq) via the tail vein. The rats were sacrificed at 30 min

post-injection (p.i.). Then the tissues of interest were sampled and weighed, and the radioactivity was determined separately by using γ-counter. Corrections were made for background radiation and physical decay during counting. The results were presented as a percentage of the injected dose per gram of tissues (%ID/g). To further confirm CXCR4 receptor binding specificity of

131

I-pentixather, blocking

study was performed by injecting of 2 mg/kg of AMD3465 (a CXCR4 specific antagonist as blocking agent) at 5 min before

131

I-pentixather (3.7 MBq)

administration in the group of 3 d after reperfusion. SPECT/CT Imaging. To evaluate CXCR4 receptor expression after MI/R, longitudinal SPECT/CT imaging was conducted at 4, 12, 36 h, 3 and 7 d after myocardial infarction using 125I-pentixather as the imaging probe. To validate the MI model, microSPECT/CT imaging was acquired at 60 min after intravenous injection a dose of 111~148 MBq of

99m

Tc-MIBI to each rat of 36 h and 3 d after myocardial

infarction27, which were performed at 24 h before

125

I-pentixather injected. The static

pinhole SPECT imaging was performed at 30 min after intravenous injection of 125

I-pentixather (66.6 MBq/300 µL) through the tail vein. CT data were acquired using

an X-ray voltage biased to 50 kVp with a 670 µA anode current, and the projections were 720°. The SPECT imaging was performed with a preclinical SPECT/CT scanner 7



Page 8 of 25

(Mediso, HUNGARY) equipped with low energy high-resolution collimators. The acquiring parameters were as follows: energy peak of 28 keV for

125

I, window width

of 20%, matrix of 256 × 256, medium zoom, and frame: 30 s. The CXCR4 specificity was verified in a separate competition study through intravenous injection of CXCR4-specific AMD3465 (2 mg/kg) 5 min before 125

I-pentixather injection in rats at 3 d after MI/R (n = 3). In order to further quantify

the IM-to-viable myocardium ratio of

125

I-pentixather by autoradiography, the rats

were sacrificed immediately after SPECT/CT imaging. Autoradiography and TTC Staining. After SPECT/CT imaging, the infarcted and viable myocardium were obtained and cut into 2 mm thick short-axis blocks. One-half of sections were stained in a 2 % buffered 2, 3, 5-triphenyl-2H-tetrazolium chloride (TTC) solution for 15 min at 37 °C and digitally photographed28,29. Subsequently, the sections were exposed to a high-performance storage phosphor screen (super resolution screen; Canberra-Packard, Ontario, Canada) for 4 h. Phosphor Imager scanner (Cyclone® Plus; PerkinElmer, USA) was utilized to read the screen. Immunohistochemistry. Slices adjacent to those used for TTC staining were selected to 10% formalin-fixed, paraffin-embedded, and then were cut into 5-µm sections for H&E staining and immunohistochemistry12. Myocardium sections were cleared with xylene, rehydrated and antigens retrieved by heating sections in citrate buffer solution (pH 6.0). Peroxidase activity was quenched with 3% H2O2 for 15 min, afterward, the sections were incubated in 5% bovine serum albumin for 20 min, washed with PBS, and incubated with recombinant rabbit monoclonal antibodies against CXCR4 (Abcam, clone UMB2; Cambridge, UK). A negative control was incubated with secondary antibody only to check the specificity of the antibody. After further washing with PBS, the sections were incubated with secondary antibody goat anti-rabbit IgG conjugated with horseradish peroxidase. Diaminobenzidine (DAB) was used as a substrate chromogen to visualize the relevant antigen, and hematoxylin was used for counterstaining. Then sections were covered with neutral balsam and were examined under a Leica Microscope (Leica, Wetzlar, Germany).

8


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RESULTS AND DISCUSSION Radiosynthesis and Partition Coefficient. The radiochemical purity was greater than 98% measured by gradient reversed-phase HPLC (Figure 1A). The specific activities of 131I-pentixather and 125I-pentixather were calculated as approximately 800 GBq/mmol (21.6 Ci/mmol) and 1100 GBq/mmol (29.7 Ci/mmol), respectively. The log P value of

131

I-pentixather was measured as -1.12 ± 0.03 (n = 3) indicating the

hydrophilicity of this radioiodinated tracer. Stability Studies in Rat Serum and Pharmacokinetics. For in vitro stability analysis, 131I-pentixather was intact after incubated for 30 and 60 min at 37 °C in normal saline (Figure 1B and C) or rat plasma (Figure 1D and E), suggesting that 131

I-pentixather had very good stability in vitro. Time-activity curve of blood after

intravenous administration of 131I-pentixather to normal rats is shown in Figure 2, indicating that the fast blood clearance of half-life of

131

131

I-pentixather. The blood elimination

I-pentixather was 0.83 ± 0.22 h, which was propitious to the early

SPECT imaging of myocardial infarction after intravenous injection. Major pharmacokinetics parameters are summarized in Table 1. Echocardiography and

99m

Tc-MIBI Perfusion Imaging. The MI/R rat model was

confirmed by ST-T segment elevation using electrocardiography of a special small animal echocardiography system (Figure 3A and B). M-mode ultrasound (mid-ventricle level) showed that the akinesis of the left ventricular wall significantly decreased in fractional shortening compared with before ischemic. Cardiograph showed ST segment elevations at MI/R rat. Left ventricular ejection fraction (LVEF) and fractional shortening (FS) were decreased from 85.4 ± 3.5% to 47.5 ± 2.2% and 54.9 ± 2.8% to 24.3 ± 1.2% in rats after ischemia-reperfusion respectively. The SPECT

myocardial

perfusion

imaging

of

MI/R

rat

with

99m

Tc-MIBI

displayedradioactivity defect area clearly in the anterolateral wall of the heart (Figure 3D, yellow arrow) when compared with that of normal rat (Figure 3C, white arrow). The results of echocardiography and

99m

Tc-MIBI perfusion imaging showed that the

MI/R rat model was successful by left anterior descending coronary artery occlusion. Biodistribution. The radioactivity uptake in the infarcted area versus normal 9



Page 10 of 25

myocardium of 131I-pentixather at 30 min p.i. in IM/R rats was summarized in Table 2. The radioactivity uptakes in the infarcted area were 0.99 ± 0.13, 1.52 ± 0.29, 1.75 ± 0.22, 1.94 ± 0.27 and 0.61 ± 0.14 %ID/g, and reached the maximum at 3 d after MI/R, displaying the tendency rising up at the beginning and declining in late time points. While the uptakes in the normal myocardium were 0.34 ± 0.04, 0.41 ± 0.09, 0.35 ± 0.11, 0.32 ± 0.05 and 0.28 ± 0.07 %ID/g, maintaining a low radioactivity level (Table 2 and Figure 5A). High IM-to-normal myocardium, IM-to-blood, and IM-to-lung ratios were observed at 4, 12, 36 h, 3 and 7 d after MI/R, and peaked at 3 d after MI/R (Figure 5B). 131

However, the IM-to-liver ratio was relatively lower. The uptake of

I-pentixather in the IM (1.94 ± 0.27 %ID/g at 30 min p.i. of 3 d after MI/R rats)

could be blocked effectively by excess amount CXCR4 antagonist AMD3465 (0.52 ± 0.17 %ID/g at 30 min p.i. of 3 d after MI/R rats) which indicated the good specificity of radioiodinated pentixather for CXCR4-targeting. When compared to the previously reported 68Ga-pentixafor, which has been used as a CXCR4-targeted imaging agent with high target specificity in cardiovascular disease imaging19,

131

I-pentixather had much higher ratios of the IM-to-normal myocardium

(6.0) and IM-to-lung (7.7) at 30 min p.i. in rats at 3 d after MI/R (Figure 5B). At the same time, the fast blood clearance (0.36 %ID/g at 30 min p.i.) lead to high IM-to-blood ratio as well. While for 68Ga-pentixafor, its uptakes in the blood and IM were approximately equal (2.0 vs. 2.2 %ID/g) and the infarcted-remote myocardium ratio was 1.7 at 60 min after injection in rats at 3 d after MI/R. Although the absolute 131

I-pentixather uptake in the IM was slightly lower than that of 68Ga-pentixafor (1.9

vs 2.2 %ID/g), the faster blood clearance and higher IM-normal myocardium ratio of 131

I-pentixather might be a great benefit to early imaging of IM with SPECT.

Additionally, less lung uptake of

131

I-pentixather may reduce radiation-induced lung

injury. The low MI-to-liver ratio has not been marked effect on myocardial imaging because the liver is located in the abdominal cavity. SPECT/CT Imaging. The representative trans-axial SPECT/CT images of MI/R rats with

99m

Tc-MIBI and

125

I-pentixather were shown in Figure 3D and E. The

hyper-enhanced region at the anterior LV wall on 10


125

I-pentixather-SPECT/CT

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matched well to the perfusion deficit area on

99m

Tc-MIBI-SPECT/CT in rats at 36 h

and 3 d after MI/R. Compared with the normal myocardium, the increased radioactivity in the infarcted area was observed as early as 4 h after MI/R and reached the maximum at 3 d after MI/R. These results were agreed well with that of biodistribution study. Although the radioactivity signal was decreased in the IM after 3 d, it is still visible at 7 d after MI/R, which indicating the high sensitive of 125

I-pentixather to noninvasively visualize CXCR4 upregulation. While for the

imaging with

68

Ga-pentixafor, the IM at 7 d after MI/R could not be detected. The

better sensitive of 68

125

I-pentixather might due to the higher affinity for CXCR4 than

Ga-pentixafor. Therefore,

125

I-pentixather was able to image CXCR4 expression

from 4 h to 7 d after MI/R with significantly improved imaging quality when compared to

68

Ga-pentixafor. Blocking study demonstrated that

125

I-pentixather

uptake in the IM could be blocked by excess amount CXCR4 antagonist AMD3465 (Figure 3 E6). It further substantiated the specificity of CXCR4-targeting imaging. Autoradiography and TTC Staining. Typical autoradiographic images and TTC staining photographs at different time points were shown in Figure 4A. At 4, 12, 36 h, 3 and 7 d after MI/R, increased radioactive intensity was clearly shown in the infarcted area, evidenced by TTC stains. Quantification data (Figure 5C) demonstrated that the trend of the uptake ratio of 125I-pentixather reached a maximum at 3 d during 4 h to 7 d periods after MI/R, which is consistent with SPECT imaging results. Once blocked with excess amount AMD3465, the decreased uptake of 125

I-pentixather was also reflected in the auto-radiographic images. Ex vivo

autoradiography confirmed specific

125

I-pentixather binding in the infarct territory as

indicated by concomitant TTC Staining. Immunohistochemistry. To further investigate the mechanism of increased 125

I-pentixather uptake after MI/R, we performed CXCR4 immunostaining at 3 d after

the surgery. As shown in Figure 4B, at 3 d after MI/R, the radioactivity signal corresponds to increased CXCR4 immunostaining and immunofluorescence in the damaged region as indicated by concomitant H&E staining, indicating signal specificity. 11



This study evaluated

125

Page 12 of 25

I-pentixather as a molecular imaging tracer to visualize

and quantify the temporal changes of CXCR4 expression in rat MI/R models. Increased focal tracer retention was observed as early as 4 h after MI/R and the 125

I-pentixather accumulation peaked at 3 d in the infarcted zones. In addition,

blocking studies with AMD3465 confirmed specificity, which suggests that the in vivo signal primarily includes CXCR4-positive cells. Therefore, the imaging signal of 125

I-pentixather specifically indicates elevated CXCR4 expression in the infarcted

myocardial region. In the present study,

125

I (half-life: 60 d) was selected for MI imaging due to its

low γ-energy suitable for small animal SPECT imaging. However,

125

I or

131

I are not

suitable for clinical use due to too long half-life with 125I and too high energy with 131I. Therefore, 123I (half-life: 13.02 h, 159 keV) or

124

I (half-life: 4.18 d, 511 keV) with a

shorter half-life and appropriate energy should be considered for clinical SPECT or PET imaging in the future. On the other hand, mice or rats are not ideal animals for modeling myocardial infarction due to limited spatial resolution with nuclear imaging (SPECT/CT or PET/CT). Instead, rabbit myocardial infarction/reperfusion models perform much better in this aspect30-32. It is known that CXCR4 plays an important homeostatic function by mediating the homing of leukocytes, bone-marrow-derived progenitor cells, hematopoietic stem cells (SCs) and endothelial progenitor cells and regulating their mobilization into peripheral tissues upon injury or stress. Additionally, up-regulation of CXCR4 by cardiac myocytes has been demonstrated to occur after 36 to 48 h after acute infarction33. Because the image is a composite signal of all cells and cell types present in the respective tissue region, hence, the precise cell population contributing to the in vivo 125I-pentixather signal cannot be identified in the present study. In summary, longitudinal SPECT imaging using

125

I-pentixather provided

temporal information on CXCR4 changes after MI/R in a noninvasive mode. Moreover, the results of SPECT imaging was further validated by ex vivo autoradiography,

in

vivo

99m

Tc-MIBI

myocardial

perfusion

imaging,

and

corresponding TTC and H&E staining. However, one inherent limitation is that the 12


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precise cell population cannot be identified in the present study.

CONCLUSIONS 125

I-pentixather preserves the chemical structure and biologic activity of

pentixather to the most extent compared to other radioisotopes labeled pentixather cyclic peptide. Noninvasive SPECT imaging with

125

I-pentixather identifies regional

CXCR4 upregulation as early as 4 h after MI/R. What’s more, it is able to noninvasively visualize CXCR4 upregulation at 7 d after MI/R. In addition, 125

I-pentixather showed better imaging quality of MI/R due to the high

target-to-nontarget ratios. The present work lays a foundation for further studies testing the usefulness of targeted CXCR4 imaging to determine outcome after MI and to guide individual therapeutic interventions aimed at improved myocardial repair and regeneration through modulation of CXCR4.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected]. ORCID Xianzhong Zhang: 0000-0001-8591-8301 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (81471707), the National Key Basic Research Program of China (2014CB744503), and the Scientific Research Foundation of State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics (2016ZY002).

REFERENCES 13



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Scheme 1. Radiosynthesis route of 131I-pentixather and 125I-pentixather.

17



Figure 1. HPLC analysis of

131

I-pentixather after the preparation (A: radiochemistry

purity) and incubated in physiological saline for 30 min (B) and 60 min (C), and rat plasma for 30 min (D) and 60 min (E), respectively for in vitro stability test.

18


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Figure 2. Time-activity curve of 131I-pentixather in the blood of healthy rats. Data are expressed as mean ± SD (n = 3).

19



Page 20 of 25

Table 1. Major pharmacokinetics parameters derived by non-compartmental modeling after iv. administration of 131I-pentixather at 25 MBq/kg in healthy rats within 4 h p.i. (n = 3). Parameter

Unit

Value

AUC(0−t)

MBq/L·h

198.458 ± 17.785

AUC(0−∞)

MBq/L·h

243.378 ± 34.405

t1/2z

h

0.830 ± 0.219

Tmax

h

0.083 ± 0.000

CLz

L/h/kg

0.104 ± 0.016

Cmax

MBq/L

225.988 ± 17.986

AUC(0−t) and AUC(0−∞): area under the curve. t1/2z: elimination half-life. Tmax: peak time. CLz: clearance. Cmax: peak concentration. The values are expressed as mean ± SD.

20


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Table 2. Biodistribution of 131I-pentixather in IM/R Wistar rat at 30 min p.i. Data are expressed as average percentage injected dose per gram of tissue plus or minus standard deviation (%ID/g ± SD, n = 3). Tissues

Times after MI/R 4h

12 h

36 h

3d

3 d + block

7d

Blood

0.32 ± 0.06

0.47 ± 0.05

0.33 ± 0.07

0.36 ± 0.07

0.38 ± 0.06

0.46 ± 0.10

brain

0.02 ± 0.00

0.03 ± 0.00

0.02 ± 0.00

0.01 ± 0.00

0.01 ± 0.00

0.03 ± 0.00

thyroid

1.16 ± 0.21

0.96 ± 0.23

1.55 ± 0.35

1.07 ± 0.09

1.35 ± 0.22

1.39 ± 0.28

Liver

4.79 ± 0.53

5.46 ± 0.68

4.5 ± 0.41

5.57 ± 0.72

4.79 ± 0.64

3.77 ± 0.66

Spleen

4.50 ± 0.70

5.54 ± 0.66

4.72 ± 0.20

5.27 ± 1.21

5.52 ± 1.12

3.20 ± 0.79

Lung

0.25 ± 0.03

0.38 ± 0.9

0.29 ± 0.05

0.25 ± 0.06

0.27 ± 0.04

0.23 ± 0.05

Kidney

4.33 ± 0.90

4.51 ± 0.65

3.89 ± 0.57

3.53 ± 0.88

3. 35 ± 0.71

2.39 ± 0.59

Stomach

0.51 ± 0.04

0.45 ± 0.11

0.46 ± 0.09

0.38 ± 0.13

0.40 ± 0.12

0.32 ± 0.09

Intestinal

0.49 ± 0.07

0.94 ± 0.12

0.49 ± 0.06

0.66 ± 0.16

0.53 ± 0.12

0.47 ± 0.16

marrow

0.64 ± 0.19

1.03 ± 0.21

0.88 ± 0.12

0.86 ± 0.22

0.65 ± 0.17

0.72 ± 0.28

Muscle

0.09 ± 0.02

0.27 ± 0.06

0.13 ± 0.03

0.09 ± 0.02

0.11 ± 0.03

0.12 ± 0.02

NM

0.34 ± 0.04

0.41 ± 0.09

0.35 ± 0.11

0.32 ± 0.05

0.33 ± 0.07

0.28 ± 0.07

IM

0.99 ± 0.13

1.52 ± 0.29

1.75 ± 0.22

1.94 ± 0.27

0.52 ± 0.17

0.61 ± 0.14

NM (Normal myocardium), IM (Infarcted myocardium).

21



Figure 3. Characterization of the MI/R models and representative transaxial SPECT/CT images using

125

I-pentixather and

99m

Tc-MIBI at different times after

MI/R. A/B: M-mode ultrasound showed that the akinesis of the left ventricular wall after ischemia (B, white arrow) significantly decreased in fractional shortening compared with that of before ischemia (A, yellow arrow) and cardiograph showed ST segment significant elevations after ischemia. C/D: 99mTc-MIBI myocardial perfusion imaging displayed that

99m

Tc-MIBI accumulation in the anterolateral wall (D, yellow

arrow) decreased in the heart with MI/R compared with the heart of normal rat (C, white arrow). E: Representative trans-axial SPECT/CT images of the different reperfusion times after IM/R rat models with uptake of radioactivity with arrows (E1-E5). The

125

125

125

I-pentixather at 30 min p.i. The high

I-pentixather in the IM areas was indicated by yellow

I-pentixather uptake peaked at 3 d after IM/R and could be

blocked by excess amount AMD3465 (E6).

22


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Figure 4. A: Representative autoradiographs of

125

I-pentixather radioactivity uptake

in the myocardium (first row) and TTC staining (second row) at different time points after MI/R. B: Immunohistochemistry, immunofluorescence and HE staining of CXCR4 in the connected area between infarcted and normal myocardium of model rats at 3 d after IM/R.

23



Page 24 of 25

Figure 5. A: The quantification of 131I-pentixather (30 min p.i) uptake after MI/R over time, expressed in %ID/g of infarcted myocardium (IM) and normal myocardium (NM). B: The %ID/g ratio of infarcted myocardium/normal myocardium (IM/NM), infarcted

myocardium/blood,

infarcted

myocardium/lung,

and

infarcted

myocardium/liver. C: The quantitative analysis of autoradiography in IM and NM at different times after SPECT/CT imaging with 125I-pentixather.

24


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TOC

25


Radioiodinated Pentixather for SPECT Imaging of the Chemokine

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