Research Article

Imaging the Cytokine Receptor CXCR4 in Atherosclerotic Plaques with [⁶⁸Ga]-APD, A Novel Radiotracer on Computer Simulation Approach

https://doi.org/10.21203/rs.3.rs-1331010/v1

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Background

C-X-C motif chemokine receptor 4 (CXCR4) plays a prominent role in inflammation, atherosclerosis, and cancer biology. Therefore, CXCR4 represents a promising target for molecular imaging in cardiovascular diseases such as atherosclerosis and arterial wall injury. CXCR4 and its cognate ligand, stromal cell–derived factor 1α (SDF-1α), induced monocyte recruitment to the injured endothelium and subsequent plaque formation is the crucial progression of atherosclerosis. CXCR4 was been proved to be intensively expressed on monocytes/macrophage. [⁶⁸Ga]-APD, based on the structure of CXCR4 antagonists TIQ-15, had designed by computer simulation as a PET tracer for imaging activated macrophages within the atherosclerotic lesions. The aim of this study was to evaluate the biological characteristics of [⁶⁸Ga]-APD, and compared with the PET tracers targeting atherosclerotic lesion such as [¹⁸F]-FDG, [¹⁸F]-NaF, and [⁶⁸Ga]-Pentixafor in apolipoprotein-E-deficient (ApoE^−/−) mice.

Results

The specification and quality of APD was identified by Mass, NMR and HPLC. After being labeled with Ga-68 under acetate buffer (pH≒5.5), radiochemical purity was over 90% and stable for more than 4 hours in 37℃human serum. After being injected from tail vein on ApoE^−/− atherosclerotic mice model, hydrophilic [⁶⁸Ga]-APD was quickly eliminate from the kidney and bladder. [⁶⁸Ga]-APD could accumulate in the atherosclerotic aorta site and CXCR4 expression organs. The highest target/background ratio (TBR) on atherosclerotic sites were 17.68 ± 0.71 (n = 3) on high-fat diet ApoE^−/− mice for 12 weeks after [⁶⁸Ga]-APD injection within 1 hour. However, [⁶⁸Ga]-Pentixafor was only 2.06 ± 0.67 (n = 3) on the same mice model. Competitive study represented that CXCR4 antagonist AMD3465 could effectively block the uptake of [⁶⁸Ga]-APD on the atherosclerotic site and CXCR4 expression organs. Comparing with [¹⁸F]-FDG and [¹⁸F]-NaF, [⁶⁸Ga]-APD represented relatively better TBR and specificity on the imaging of atherosclerotic lesions.

Conclusion

In vivo evaluation of CXCR4 expression in ApoE^−/− mice revealed the uptake of [⁶⁸Ga]-APD mainly accumulated in the atherosclerotic aorta site. Moreover, the TBR of [⁶⁸Ga]-APD was 8 times higher than [⁶⁸Ga]-Pentixafor, a radiotracer targeting CXCR4 and under phase II clinical trial, indicating this novel tracer [⁶⁸Ga]-APD is more feasible as a surrogate marker for inflammatory atherosclerosis.

CXCR4

Apo E-/-

Atherosclerosis

APD

Pentixafor

Although the diet management and medical improvement in treating cardiovascular diseases (CVD), atherosclerotic plaque-induced stroke and coronary heart disease (CHD), heart attack, or angina are one of the leading causes of mortality and morbidity worldwide. (1, 2). The pathogenesis of atherosclerosis is characterized by the chronic accumulation of lipids and pro-inflammatory immune cells to the focal arterial wall and progressively narrows the lumen of the artery (3–5). Owing to the unstable plaques by the pathologic mechanisms of inflammation, atherosclerotic plaque formation can be diagnosis from the mechanism of inflammation (6, 7). Within the inflammation and different aspects of the atherosclerotic plaque, activated macrophages play the most interesting target in the atherosclerotic lesion (8).

Imaging atherosclerosis with PET/CT characterizes atherosclerotic plaques on a molecular level and enables the quantification of arterial calcification (9). Many Functional PET imaging tracers have been tried to targeting molecular elements of atherosclerosis of increasing the glucose metabolism by the inflammatory cells (10–13) and detect micro-calcifications around the arterial wall (14–16), such as [¹⁸F]-FDG and [¹⁸F]-NaF, respectively.

The 7-transmembrane helix G-protein–coupled receptor, Chemokine C-X-C motif receptor 4 (CXCR4), and its cognate ligand, stromal cell–derived factor 1a (SDF-1a/CXCL12), induced monocyte recruitment to the injured endothelium and subsequent plaque formation is the crucial progression of atherosclerosis. CXCR4 is also been proven to be intensively expressed on monocytes/macrophage (17–21). Recently, CXCR4-directed of macrophages PET imaging with [⁶⁸Ga]-radiolabeled cyclic peptide, Pentixafor, has been provided to imaging neoplasms, hematologic, stroke, atherosclerosis, and myocardial infarction in humans by the mechanism of overexpression CXCR4 (22–25). In addition, Tetra-azamacrocycles based small-molecule CXCR4 antagonists, such as AMD3100 and AMD3465, are also developed and proven to bind on human and mouse variants CXCR4 receptor. Orit Jacobson et al. had developed a one step radiosynthesis of [⁶⁴Cu]- AMD3100 and exhibited a promising agent for visualization of CXCR4-positive tumors and metastases (26). However, Copper(II) cyclam complexes are unlikely to have sufficient electrostatic/H-bonding between cyclam amine groups with aspartate residue side chains (171 and 262) and lead to high nonspecific hepatic accumulation to retain the metal ion in vivo (27).

The atherosclerotic-prone apolipoprotein E-deficient (ApoE^−/−) mice were first developed in 1992 by homologous recombination of embryonic stem cells and currently use in the pre-clinical atherosclerotic model by downregulation of cholesterol transporter ATP-binding cassette subfamily A member 1 levels (28). Poor lipoprotein clearance of atherosclerosis-prone apolipoprotein E-deficient (Apoe^−/−) mice display the accumulation of cholesterol ester-enriched particles in the blood and promote the development of atherosclerotic plaques. ApoE^−/− mice is well established for studying atherosclerotic lesions by high-fat chow diet or even a regular diet for several weeks. The atherosclerotic lesions in ApoE^−/− mice resemble human lesions in their predilection sites and progression to the fibro-proliferative stage (29).

In this study, we investigate the biological characteristics of novel CXCR4 agent [⁶⁸Ga]-APD, and compared with other PET tracers targeting atherosclerotic lesion such as [¹⁸F]-FDG, [¹⁸F]-NaF, and [⁶⁸Ga]-Pentixafor in the ApoE^−/−mice model with high fat diet.

General

Computer simulation BIOVIA Discovery Studio designed APD from Tetrahydroisoquinoline-Based CXCR4 Antagonist TIQ-15 (Fig.1) by screening million’s chemical fragments with chemical binding energy and CDOCKER on CXCR4 specific amino acids residue by H-bond, pi-pi interaction (30). APD synthesized at RDD LAB. INC, Taiwan and determined its specifications by NMR (JNM-ECZ500R/S1, JOEL USA, Inc), Mass (AB Sciex (Concord, ON, Canada) 4000QTRAP® mass spectrometry) and high-performance liquid chromatography (HPLC, Waters-2695, with UV and radio detector). Radio-thin-layer chromatography was performed on instant TLC chromatogram sheets (Sigma Chemical Company, USA), eluting with citrate buffer (pH=5.5) and Bioscan AR-2000 (Eckert & Ziegler, Germany). Pentixafor purchased from UNION CHEMICAL IND. CO., LTD. Taiwan. [¹⁸F]-FDG and [¹⁸F]-NaF provided from Institute of Nuclear Energy Research, Taoyuan, Taiwan.

Preparation and radiochemical purity (RCP) analysis

APD (0.01~0.1mg) dissolved in acetate buffer (pH 5.0~6.0) and then labeled with [⁶⁸Ga]-GaCl₃ (1~10mCi) for at least 20 minutes under 60~70℃.To determine the amount of impurity free Ga-68, the sample was chromatographed on ITLC-SG (Sigma Chemical Company, USA) using 0.1 M citrate buffer (pH 5.5) as the mobile phase. Free Ga-68 migrated with the solvent front, whereas labeled [⁶⁸Ga]-APD remained at the origin. A Waters system pump (Germering, D) and radiometric detection (Bioscan, Washington DC), were used for RP-HPLC analysis. C-18 column (Waters 5 μm, 80 Å, 250 × 4.6 mm). Flow rates of 0.8 mL/min were employed as the following: a linear gradient eluent starting from 30% A (0.1% TFA in water) and 70% B (0.1% TFA in acetonitrile), increasing to 90% B for 10 min, remaining constant at 90% B for another 15–30 min. The RCP was calculated expressing the peak corresponding to [⁶⁸Ga]-APD as percentage of the total activity in the radio-chromatogram. The injected activity on the HPLC-column was in the order of 5 MBq. RCP of [⁶⁸Ga]‐Pentixafor was also determined by HPLC followed as previously described (31).

Partition Coefficient (n-Octanol/PBS) Determination

To determine the hydrophilicity of [⁶⁸Ga]‐APD, the octanol/water partition coefficients (log P) were measured. Briefly, [⁶⁸Ga]-APD (7.4 MBq) with phosphate-buffered saline (PBS, 5.0 mL, 0.15 M, pH 7.4) and n-octanol (5 mL) within a test tube. After vortexing for 1 min, tube was placed on bench for 3 min in room temperature. Samples (from each phase, 100 μL) were counted its radioactivity by using gamma-counter. The tests were reported as mean ± SD (n≧3), independently.

Mice and PET/CT Acquisition

ApoE^-/- mice experimental protocol had been approved by the Institutional Animal Care and Use Committee (IACUC) in the Institute of Nuclear Energy Research (Taoyuan, Taiwan) and followed the ARRIVE guidelines and the USPHS Policy on Human Care and Use of Laboratory Animals. Female ApoE^-/- mice (provided from Taiwan University Hospital) (aged >8 weeks) housed in a controlled environment (22 ± 2°C and 50 ± 5% relative humidity) with a 12-h light-and-dark cycle and received a high-fat diet for accelerating atherosclerotic sites. PET/CT imaging was performed on a Bioscan scanner (matrix size, 128 × 128 × 159; CT attenuation-corrected; non-scatter corrected) (Washington DC, USA) following a intravenous tail vein injection of approximately 14.8 MBq (0.4 mCi) of [⁶⁸Ga]‐APD into ApoE^-/- mice (3–5 mice per group). Dynamic scans were acquired in list mode format for at least 120 min, and sorted into 22 frames, 0.5-mm sinogram bins for image reconstruction (4 × 15 s, 4 × 60 s, 11 × 300 s, 3 × 600 s). Mice were anesthetized with isoflurane (3% for induction and 2% for maintenance) throughout the experiment.

ApoE^-/- mice was imaged with [⁶⁸Ga]‐APD (11.1 MBq) on 2, 8 and 12 weeks of high-fat diet to characterize atherosclerotic lesions. For competitive inhibition assay on [⁶⁸Ga]‐APD, a CXCR4 inhibitor AMD3465 (25 mg/kg) was injected before administration of 11.1 MBq of [⁶⁸Ga]‐APD on high-fat diet ApoE^-/- mice for 12 weeks. [⁶⁸Ga]‐Pentixafor (11.1 MBq) was also administrated on the same mice model for comparing the efficacy on atherosclerotic lesions with [⁶⁸Ga]‐APD. [¹⁸F]-FDG PET/CT imaging was been performed after 6 h fasting period to ensure serum glucose levels below 130 mg/dL. To estimate the radioactivity concentration, volumes of interests had defined on coregistered PET/CT images using PMOD software. Target/Background Ratio (TBR) had calculated by placing a circular 2-mm volume of all interest organs/tissues around a site. TBRs were been calculated as focal uptake divided by blood pool.

Autoradiographic studies on atherosclerosis

To visualize the distribution of [⁶⁸Ga]‐APD in the heart and aorta, autoradiography was performed using 20-μm-thick fresh-frozen heart and aorta sections derived from ApoE^-/- mice. The specimens were exposed to phosphor imaging plates (MS-2040, Fuji Photo Film Co. Ltd.) for 2 h in the [⁶⁸Ga]‐APD examination. Subsequently, the radioactivity distributions were imaged by using imaging analyzer BAS 4000 (Fuji Photo Film Co. Ltd.).

Statistical analysis

The statistical significance of comparisons of radioactivity in organs in PET studies was based on 2-sided, 2-sample Student t-tests. For all tests, P < 0.05 was considered statistically significance and represented on figures. Results were reported as mean ± SD (n≧3).

Synthesis and Characterization

The novel CXCR4 antagonist structure of APD was represented. (Fig.1) The binding energy and CDOCKER energy of APD were -300.33 kcal/mol and -57.01kJ/mol, respectively. However, the binding energy of TIQ-15 was only -88 kcal/mol. After the synthesis, chemical characterization had verified by NMR, mass spectrum and HPLC. ¹H NMR (D₂O) d 8.46-8.45 (m, 1 H), 7.84-7.80 (m, 2.H), 7.49-7.47 (d, 1H), 7.41-7.34(m, 3H), 3.70-3.90 (m,6H), 2.78-2.47 (m, 20H), 1.73-1.67 (m, 2H). ¹³C NMR (D₂O) d160.30, 157.53, 148.16, 138.61, 137.96, 124.63, 123.08, 122.84, 120.45, 59.98, 53.29, 50.30, 45.93, 45.10, 43.92, 43.29, 23.31. The molecular weight and purity of APD was measured as [M+H]⁺=441.51 and over 98%, respectively.

Radiochemical purity (RCP) and Partition Coefficient (n-Octanol/PBS) Determination

APD (0.01~0.1mg) dissolved in acetate buffer (pH 5.0~6.0) and then labeled with [⁶⁸Ga]-GaCl₃ (1~10mCi) for at least 20 mins under 60~70℃.Radiochemical purity of [⁶⁸Ga]-APD was calculated by paper chromatography on SG-ITLC strip with citrate buffer (pH5.5) (Fig. 2A). Free Ga-68 would migrate to solvent front, whereas [⁶⁸Ga]-APD remained nearly at the origin. The chromatogram represented that the radiochemical purity of [⁶⁸Ga]-APD was over 90%. In addition, according to the Radio-HPLC chromatogram (Fig. 2B), the retention time of [⁶⁸Ga]-APD and impurity-free Ga-68 was represented at about 13.2 min and 3.5 min, respectively. The radiochemical purity of [⁶⁸Ga]-APD was also verified over 90% by HPLC analysis. Furthermore, [⁶⁸Ga]-APD was stable under room temperature and even in 37℃human serum for over 4 hours. Its primary decomposition of radio-product appeared to be free Ga-68. The partition coefficient of [⁶⁸Ga]-APD was determined as 0.040±0.0029 (n=4) which was shown its good hydrophilicity.

PET/CT Studies on ApoE^-/- atherosclerotic model mice

In the competitive inhibition study, the PET/CT images represented that [⁶⁸Ga]-APD could be apparently blocked the uptake on atherosclerotic lesion by pre-injection of a dose (5 mg/kg) of CXCR4 inhibitor AMD3465 on high fat diet ApoE^-/-mice for 12weeks. Furthermore, the pre-dosing with AMD-3465 could also significantly reduce the TBR of [⁶⁸Ga]-APD at CXCR4-overexpression organs, such as liver, lung, and kidney (27). These evidences provided that [⁶⁸Ga]-APD was specific binding on CXCR4 (P<0.05). (Fig.3)

In comparing the PET images of [⁶⁸Ga]-APD (11.1 MBq) on the different duration ApoE^-/-mice, the TBR was 7.52 ± 0.83, 9.65 ± 0.69 and 17.68 ± 0.71 on 2, 8 and 12 weeks of high fat diet, respectively. The TBR of [⁶⁸Ga]-APD was decreased with time (n=3) (Fig.4). Kidney and bladder had higher tracer uptake, providing that [⁶⁸Ga]-APD might primarily excrete from the kidney to bladder.

For comparing the imaging efficacy on atherosclerotic lesions between [⁶⁸Ga]-APD and [⁶⁸Ga]-Pentixafor (RCP>95%), tracers (11.1 MBq) were injected on high-fat diet ApoE^-/- mice for 12 weeks. The TBR result represented that [⁶⁸Ga]-APD had about 8 times higher than [⁶⁸Ga]-Pentixafor during the imaging time (Fig.5). The results of TBR on [⁶⁸Ga]-Pentixafor was almost matched as previously described (22-25).

[¹⁸F]-FDG injection was been performed after fasting for at least 6 h to ensure serum glucose levels below 130 mg/dL. The PET image represents that [¹⁸F]-FDG accumulated mainly on the myocardium. However, the highest TBR of [¹⁸F]-FDG was only 1.53±0.75 (n=3) on the atherosclerotic lesions on high-fat diet ApoE^-/- mice for 10 weeks. Furthermore, [¹⁸F]-NaF could not effectively target atherosclerotic site by the reason of lacking calcified lesions on only 10 weeks of high-fat diet. (Fig.6)

Autoradiographic studies on atherosclerosis

From the PET/CT image of [⁶⁸Ga]-APD (Fig 6), there was tracer specific targeting in the chest region. For verified the distribution of [⁶⁸Ga]-APD, the slide autoradiogram of [⁶⁸Ga]-APD (Fig.7) had shown obvious high radioactivity accumulating in the aorta region but not on the heart.

This study reports a novel CXCR4 antagonist structure APD was been designed by replacing over million’s chemical fragments on the structure of TIQ-15 and evaluating its docking efficacy by computer simulation approach. After being labeled with Ga-68, [⁶⁸Ga]-APD represent good sensitivity and specificity to CXCR4 expression atherosclerotic lesions.

Atherosclerotic plaque formation, development, and progression are complex and involve many factors, including endothelial dysfunction, elevated cholesterol and immune activity. As plaques in the arterial walls build and progress, the rupture on the arteries may be occluded distally and block blood flow into heart muscle, brain, and other parts of the body (1). Inflammation is an important component of atherosclerotic plaque vulnerability. Owing to the glucose uptake in active and lipid-rich macrophages on inflammatory plaques, it is likely to use [¹⁸F]-FDG as a marker to detect small lesions of atherosclerotic plaques. However, [¹⁸F]-FDG plaque imaging is limited by non-specificity on atherosclerotic lesions. In addition, the constant motion of cardiac cycle and the significant uptake of [¹⁸F]-FDG in the myocardium has decreased the enthusiasm to use this tracer in atherosclerotic imaging (25). [¹⁸F]-NaF have significant advantages in the assessment of atherosclerotic plaques imaging with regards to its more accurately and precisely in detecting and characterizing plaques than [¹⁸F]-FDG. Therefore, the future of PET imaging to assess atherosclerosis will predict to heavily rely on [¹⁸F]-NaF (32).

Oxidized low-density lipoprotein (Ox-LDL) and vascular hypoxic process might significantly induce macrophage to overexpression CXCR4 and enhanced the atherosclerotic progression. In comparison within early or advanced atherosclerotic lesions, Ilze Bot et al. had found that CXCR4 expression was significantly pronounced in advanced unstable lesions (33). Remarkably, the expression of CXCR4 protein was co-localized with macrophage expression in inflamed vulnerable plaque lesions. This finding might support the concept that CXCR4 played a recruitment role for leukocytes into the vessel wall and could serve as a good biomarker for atherosclerotic imaging (27).

There are many PET tracers have tried to image atherosclerotic changes based on the macrophage to expression CXCR4. [⁶⁸Ga]-Pentixafor is one of the successful CXCR4 clinical trial tracers to image atherosclerosis with a clear view of vascular uptake and a relatively clear background. Xiang Li et al. had shown that patients with higher uptake of [⁶⁸Ga]-Pentixafor represented a higher incidence of metabolic syndrome and might suggest its clinical potential in the evaluation of diseased vessels (34). Derlin T. et al. had also shown that accumulation of [⁶⁸Ga]-Pentixafor and [¹⁸F]-FDG represented only a weak correlation by observed in non-calcified lesions (35).

Tetrahydroisoquinoline-Based CXCR4 antagonists TIQ-15 had been found to have good oral bioavailability and its active structure was confirmed to interaction with ASP 97, Tyr116 and Glu288 residue on CXCR4 (36). In this study, a novel CXCR4 tracer APD was been designed from the structure of TIQ-15 by computer simulation approach. The selected structure of APD was been screened by replacing over million’s chemical fragments on the structure of TIQ-15 and evaluating its docking efficacy. The simulating binding energy of APD and TIQ-15 was − 300.33 and − 88kcal/mol, respectively. In our preliminary study, [⁶⁸Ga]-APD had showed specific binding on CXCR4-expression MDA-MB231 breast cancer cells and could be blocked the cellular uptake by using CXCR4 antagonist AMD3465. The PET image of [⁶⁸Ga]-APD on ApoE^−/− atherosclerotic model had shown high selective uptake on atherosclerotic lesions. Owing to its high hydrophilicity (Partition coefficient = 0.040 ± 0.0029), [⁶⁸Ga]-APD PET image showed a quick excretion from kidney and with a relatively low background. CXCR4 antagonist AMD465 (5 mg/kg) could effectively reduce the uptake on atherosclerotic plaque, liver, lung and kidney at 60 min before tracer administration which suggested its specific binding on CXCR4 (Fig. 3). Further experiments demonstrated that more than 90% of urine radioactivity was intact [⁶⁸Ga]-APD. Human serum stability assays also showed that [⁶⁸Ga]-APD could be stable for over 4 h under 37℃ human serum. For confirming the distribution of isotope labeled APD on the atherosclerotic sites, tissue frozen section had also been done after the bio-distributed of [⁶⁸Ga]-APD (RCP > 90%) for 30 mins on ApoE^−/− mice. The autoradiogram represented that [⁶⁸Ga]-APD was major distributed on aorta, but not in the heart (Fig. 7).

Atherosclerotic plaques may stabilize or reduce in size, lipid content, foam cell content, and macrophage inflammation through Macrophage Reverse Cholesterol Transport (RCT) mechanism by eliminating oxidized low-density lipoprotein (Ox-LDL) from atherosclerotic plaques. (37, 38). Early detection and therapy is the key to improving the atherosclerotic prognosis at present. In this study, [⁶⁸Ga]-APD PET image provides the possibility in the early diagnosis of atherosclerosis with high sensitivity and specificity than [⁶⁸Ga]-Pentixafor by targeting CXCR4-overexpression macrophage.

In vivo evaluation of CXCR4 expression in ApoE^−/− mice revealed the uptake of [⁶⁸Ga]-APD mainly accumulated in the atherosclerotic aorta site. Moreover, the TBR of [⁶⁸Ga]-APD was 8 times higher than [⁶⁸Ga]-Pentixafor, a radiotracer targeting CXCR4 and under phase II clinical trial, indicating this novel tracer [⁶⁸Ga]-APD is more feasible as a surrogate marker for inflammatory atherosclerosis.

CXCR4: C-X-C motif chemokine receptor 4; SDF-1a: stromal cell–derived factor 1a; APD: 6-Aminomethyl- pyridin-2-ylmethyl)-pyridin-2-ylmethyl-[3-(1,4,7,10tetraaza-cyclododec-1-yl)-propyl]-amine; ApoE^-/-:apolipoprotein-E-deficient; TBR: target/background ratio; RCP: radiochemical purity.

Ethics approval and consent to participate

ApoE^-/- mice experimental protocol had approved by the Institutional Animal Care and Use Committee (IACUC) in the Institute of Nuclear Energy Research (Taoyuan, Taiwan) and followed the ARRIVE guidelines and the USPHS Policy on Human Care and Use of Laboratory Animals. ApoE^-/- mice had gotten the consent to provide from Taiwan University Hospital.

Consent for publication

Not applicable

Availability of data and material

All the raw data of this study, including PET images, can be obtained through the corresponding authors on reasonable request.

Competing interests

The authors declare that no other potential conflict of interest relevant to this article exist.

Funding

Funding was all provided by Institute of Nuclear Energy Research, Taiwan.

Authors' contributions

CH conducted experiments and wrote the manuscript. CY, CC and CP reviewed the study design and helped to cultured cells, raised mice and conducted mice’s experiments. All authors read and approved the final manuscript.

Acknowledgements

I would like to express my deep and sincere gratitude to research colleagues, Dr. Mei-Hui Wang, Dr. Hung-Wen Yu, Dr. Ying-Hsia Shih and all of the teams’ member for his genuine support throughout this research work. I thank the management of Institute of Nuclear Energy Research for their financial support to do this work. I also thank the directors Dr. Shiou-Shiow Farn, Dr. Jeng-Hung Lee and Dr. Jeng-Jong Wang and Dr. Chih-Hsien Chang for their genuine support to complete this thesis successfully. Finally, my thanks go to all the people who have supported me to complete the research work directly or indirectly.

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Editor invited by journal
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