Scavenging strategy for specific activity improvement: application to a new CXCR4-specific cyclopentapeptide positron emission tomography tracer (original) (raw)
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Bioorganic & Medicinal Chemistry, 2012
The expression of the chemokine receptor CXCR4 in tumors is associated with tumor aggressiveness and poor prognosis for the patient and contributes to metastatic seeding. Therefore it is of high interest to find a specific PET tracer for the imaging of CXCR4 expression in tumors. The aim of this study was the synthesis, 68 Ga labeling and first evaluation of DOTA-4-FBn-TN14003 as a potential PET tracer for this purpose. DOTA-4-FBn-TN14003 was synthesized using solid phase peptide synthesis and radiolabeling of this versatile precursor was performed with 68 Ga, which was obtained from a 68 Ge/ 68 Ga generator. 68 Ga-DOTA-4-FBn-TN14003 was reproducibly obtained in isolated radiochemical yields of 72.5 ± 4.9% with an excellent radiochemical purity of >99.5%. Specific activities of up to 29.8 ± 3.1 GBq/lmol were achieved. In competition binding assays with SDF-1a, human T cell lymphoma Jurkat cells expressed high levels of CXCR4 whereas human breast cancer MDA-MB-231 cells expressed significantly lower levels of this chemokine receptor. The inhibition constants (IC 50) of Ga-DOTA-4-FBn-TN14003 and 4-FBn-TN14003 to CXCR4 were determined in a competition assay against 125 I-SDF-1a using Jurkat as well as MDA-MB-231 cells. The IC 50 values of Ga-DOTA-4-FBn-TN14003 (1.99 ± 0.31 nM) and 4-FBn-TN14003 (4.07 ± 1.00 nM) proved to be comparable, indicating negligible influence of the metal complex. These results suggest 68 Ga-DOTA-4-FBn-TN14003 as a promising agent for the imaging of CXCR4 expression in tumors and metastases.
Development and Evaluation of an 18F-Radiolabeled Monocyclam Derivative for Imaging CXCR4 Expression
Molecular Pharmaceutics, 2019
C-X-C chemokine receptor type 4 (CXCR4) is a protein that in humans is encoded by the CXCR4 gene and binds the ligand CXCL12 (also known as SDF-1). The CXCR4-CXCL12 interaction in cancer elicits biological activities that result in tumor progression and has accordingly been the subject of significant investigation for detection and treatment of disease. Peptidic antagonists have been labeled with a variety of radioisotopes for detection of CXCR4, but methodology utilizing small molecules have predominantly used radiometals. We report here the development of a 18 Fradiolabeled cyclam-based small molecule radioprobe, [ 18 F]MCFB, for imaging CXCR4 expression. The IC50 of [ 19 F]MCFB for CXCR4 was similar to that of AMD3465 (111.3 and 89.9 nM, respectively). In vitro binding assays show that the tracer depicted differential CXCR4 expression, which was blocked in the presence of AMD3465, demonstrating specificity of [ 18 F]MCFB. Positron emission tomography (PET) imaging studies showed distinct uptake of radioprobe in lymphoma and breast cancer xenografts. High liver and kidney uptake were seen with [ 18 F]MCFB leading us to further examine the basis of its pharmacokinetics in relation to the tracer's cationic nature, thus, the role of organic cation transporters (OCTs). Substrate competition following the intravenous injection of metformin led to a marked decrease in urinary excretion of [ 18 F]MCFB, with moderate changes observed in other organs, including the liver. Our results suggest involvement of OCTs in renal elimination of the tracer. In conclusion, the 18 F radiolabeled monocyclam, [ 18 F]MCFB, has potential to detect tumor CXCR4 in non-hepatic tissue.
Improvement of CXCR4 tracer specificity for PET imaging
Journal of Controlled Release, 2012
Tumors expressing the chemokine receptor CXCR4 have been reported to be more aggressive and to produce more metastatic seeding in specific organs, such as the bone marrow. However, evaluation of tumors for CXCR4 expression requires testing of ex vivo biopsy samples, and is not routinely done in cancer management. In prior work to address this issue, we and others have developed tracers for positron emission tomography (PET) that targeted CXCR4, but in addition to binding to CXCR4 these tracers also bound to red blood cells (and to other unrelated targets) in vivo. Here we report two new tracers based on the CXCR4 peptide antagonist 4F-benzoyl-TN14003 (T140) that bind to CXCR4, but not to undesired targets. These tracers, NOTA-NFB and DOTA-NFB, show slight reductions in both 1) binding affinities for CXCR4 and 2) inhibition of CXCL12 induced migration, compared to T140, in vitro. Both NOTA-NFB and DOTA-NFB specifically accumulate in CXCR4-positive, but not CXCR4-negative, tumor xenografts in mice and allow clear visualization of CXCR4 expression by PET. Evaluation of NOTA-NFB and DOTA-NFB for their potential to mobilize immune cells and progenitor cells from the bone marrow to the peripheral blood revealed slightly reduced, but still comparable, results to the parent molecule T140. The tracers reported here may allow the evaluation of CXCR4 expression in primary tumors and metastatic nodules, and enable better informed, more personalized treatment for patients with cancer.
PET Imaging of CXCR4 Receptors in Cancer by a New Optimized Ligand
ChemMedChem, 2011
Nowadays, personalized medicine is considered to be of utmost importance to target the different causes of identical phenotypes. For example, cancer of the same type can significantly differ in its biochemical phenotypes and thus its molecular profile between patients. The disease-specific characterization of malignant cells at the molecular level is a prerequisite for targeted therapy and personalized treatment. Positron emission tomography (PET) and its combination with computer tomography (PET/CT) and magnetic resonance tomography (PET/MRT) in modern hybrid systems offer the possibility to localize and quantify biochemical function by means of PET with anatomical (CT) and morphological (MRT) information. For this purpose, radiolabeled probes are used that target, for example, enzyme activities, transport systems, and surface receptors with high affinity and specificity. We describe the development of the first gallium-68 (t 1/2 = 68 min) ligand for the G-protein-coupled receptor CXCR4 and preliminary demonstrate its potential for in vivo imaging of CXCR4 expression using a mouse model with a human small-cell lung cancer xenograft. This ligand offers the possibility to be used as an initial tool for diagnosis in an approach of personalized medicine for treating CXCR4-related cancer.
High-Contrast CXCR4-Targeted 18FPET Imaging Using a Potent and Selective Antagonist
C-X-C chemokine receptor 4 (CXCR4) is highly expressed in cancers, contributing to proliferation, metastasis, and a poor prognosis. The noninvasive imaging of CXCR4 can enable the detection and characterization of aggressive cancers with poor outcomes. Currently, no 18 F-labeled CXCR4 positron emission tomography (PET) radiotracer has demonstrated imaging contrast comparable to [ 68 Ga]Ga-Pentixafor, a CXCR4-targeting radioligand. We, therefore, aimed to develop a high-contrast CXCR4targeting radiotracer by incorporating a hydrophilic linker and trifluoroborate radioprosthesis to LY2510924, a known CXCR4 antagonist. A carboxy-ammoniomethyl-trifluoroborate (PepBF 3) moiety was conjugated to the LY2510924-derived peptide possessing a triglutamate linker via amide bond formation to obtain BL08, whereas an alkyne ammoniomethyl-trifluoroborate (AMBF 3) moiety was conjugated using the copper-catalyzed [3+2] cycloaddition click reaction to obtain BL09. BL08 and BL09 were radiolabeled with [ 18 F]fluoride ion using 18 F− 19 F isotope exchange. Pentixafor was radiolabeled with [ 68 Ga]GaCl 3. Side-by-side PET imaging and biodistribution studies were performed on immunocompromised mice bearing Daudi Burkitt lymphoma xenografts. The biodistribution of [ 18 F]BL08 and [ 18 F]BL09 showed tumor uptake at 2 h postinjection (p.i.) (5.67 ± 1.25%ID/g and 5.83 ± 0.92%ID/g, respectively), which were concordant with the results of PET imaging. [ 18 F]BL08 had low background activity, providing tumor-to-blood,-muscle, and-liver ratios of 72 ± 20, 339 ± 81, and 14 ± 3 (2 h p.i.), respectively. [ 18 F]BL09 behaved similarly, with ratios of 64 ± 20, 239 ± 72, and 17 ± 3 (2 h p.i.), respectively. This resulted in high-contrast visualization of tumors on PET imaging for both radiotracers. [ 18 F]BL08 exhibited lower kidney uptake (2.2 ± 0.5%ID/g) compared to [ 18 F]BL09 (7.6 ± 1.0%ID/g) at 2 h p.i. [ 18 F]BL08 and [ 18 F]BL09 demonstrated higher tumor-to-blood,-muscle, and-liver ratios compared to [ 68 Ga]Ga-Pentixafor (18.9 ± 2.7, 95.4 ± 36.7, and 5.9 ± 0.7 at 2 h p.i., respectively). In conclusion, [ 18 F]BL08 and [ 18 F]BL09 enable high-contrast visualization of CXCR4 expression in Daudi xenografts. Based on high tumor-to-organ ratios, [ 18 F]BL08 may prove a valuable new tool for CXCR4-targeted PET imaging with potential for translation. The use of a PepBF 3 moiety is a new approach for the orthogonal conjugation of organotrifluoroborates for 18 F-labeling of peptides.
Journal of Nuclear Medicine, 2019
Expression of the chemokine receptor chemokine C-X-C motif receptor 4 (CXCR4) plays an important role in cancer metastasis, autoimmune diseases and during stem cell based repair processes after stroke and myocardial infarction. Previously reported positron emission tomography (PET) imaging agents targeting CXCR4 suffer from either high non-specific uptake or only bind to the human form of the receptor. The objective of this study is to develop a high stability copper-64 labelled small molecule PET agent for imaging both human and murine CXCR4 chemokine receptors. Methods: Synthesis, radiochemistry, stability and radioligand binding assays were performed for the novel tracer 64 Cu-CuCB-Bicyclam. In vivo dynamic PET studies were carried out on mice bearing U87 (CXCR4 low) and U87.CXCR4 (CXCR4 high) tumors. Biodistribution and receptor blocking studies were carried out on CD1-IGS immunocompetent mice. CXCR4 expression on tumor and liver disaggregates was confirmed using a combination of immunohistochemistry, quantitative polymerase chain reaction (qPCR) and western blot. Results: 64 Cu-CuCB-Bicyclam has a high affinity for both the human and murine variants of the CXCR4 receptor (IC50 = 8 nM (human)/ 2 nM (murine)) and can be obtained from the parent chelator that has low affinity. In vitro and in vivo studies demonstrate specific uptake in CXCR4 expressing cells that can be blocked by >90% using a higher affinity antagonist, with limited uptake in non-CXCR4 expressing organs and high in vivo stability. The tracer was also able to selectively displace the CXCR4 antagonists AMD3100 and AMD3465 from the liver. Conclusions: The application of the tetraazamacrocyclic small molecule 64 Cu-CuCB-Bicyclam is demonstrated as an imaging agent 2 for the CXCR4 receptor that is likely to be applicable across a range of species. It has high affinity and stability and is suitable for preclinical research in immunocompetent murine models.
Preclinical evaluation of a CXCR4-specific 68Ga-labelled TN14003 derivative for cancer PET imaging
Bioorganic & Medicinal Chemistry, 2014
Molecular imaging is an ideal platform for non-invasive detection and assessment of cancer. In recent years, the targeted imaging of CXCR4, a chemokine receptor that has been associated with tumour metastasis, has become an area of intensive research. In our pursuit of a CXCR4-specific radiotracer, we designed and synthesised a novel derivative of the CXCR4 peptidic antagonist TN14003, CCIC16, which is amenable to radiolabelling by chelation with a range of PET and SPECT radiometals, such as 68 Ga, 64 Cu and 111 In as well as 18 F (Al 18 F). Potent in vitro binding affinity and inhibition of signalling-dependent cell migration by unlabelled CCIC16 were confirmed by a threefold uptake in CXCR4-over-expressing cells compared to their isogenic counterparts. Furthermore, in vivo experiments demonstrated the favourable pharmacokinetic properties of the 68 Ga-labelled tracer 68 Ga-CCIC16, along with its CXCR4specific accumulation in tissues with desirable contrast (tumour-to-muscle ratio: 9.5). The specificity of our tracer was confirmed by blocking experiments. Taking into account the attractive intrinsic PET imaging properties of 68 Ga, the comprehensive preclinical evaluation presented here suggests that 68 Ga-CCIC16 is a promising PET tracer for the specific imaging of CXCR4-expressing tumours.
[99mTc]O2-AMD3100 as a SPECT tracer for CXCR4 receptor imaging
Nuclear Medicine and Biology, 2013
Purpose: CXCR4 plays an important role in HIV infection, tumor progression, neurogenesis, and inflammation. In-vivo imaging of CXCR4 could provide more insight in the role of this receptor in health and disease. The aim of this study was to investigate [ 99m Tc]O 2-AMD3100 as a potential SPECT tracer for imaging of CXCR4. Method: AMD3100 was labelled with [ 99m Tc]pertechnetate. A cysteine challenge assay was performed to test the tracer stability. Heterologous and homologous receptor binding assay and internalization assay were performed in CXCR4 expressing Jurkat-T cells. Ex vivo biodistribution was studied in healthy mice at 30, 60, and 120 min after tracer injection. Tumor uptake of the tracer was determined by microSPECT imaging in nude mice xenografted with human PC-3 prostate tumor. Specificity of tracer uptake was determined by blocking studies using an excess of unlabelled AMD3100. Results: AMD3100 was labelled with technetium-99 m with a radiochemical yield of N98%. The tracer was stable in PBS and mouse plasma for at least 6 h at 37°C. Heterologous and homologous binding assays with AMD3100 showed IC 50 values of 240 ± 10 μM, and 92 ± 5 μM for [ 125 I]SDF-1α and [ 99m Tc]O 2-AMD3100 respectively, with negligible receptor internalisation. The tracer showed high uptake in liver, lungs, spleen, thymus, intestine and bone. Blocking dose of AMD3100.8HCl (20 mg/kg) decreased the uptake in these organs (p b 0.05). [ 99m Tc]O 2-AMD3100 showed specific tumor accumulation in mice bearing PC-3 xenografts model. Time activity curves (TAC) in AMD3100 pre-treated animals tracer showed 1.7 times less tumor uptake as compared to control animals (p b 0.05). Conclusion: [ 99m Tc]O 2-AMD3100 is readily labelled, is stable in plasma and displays a favourable binding affinity for the CXCR4 receptors. [ 99m Tc O 2-AMD3100 shows specific binding in organs with high CXCR4 expression and in CXCR4 positive tumors. These results justify further evaluation of this radiopharmaceutical as a potential biomarker for the non-invasive imaging of CXCR4 receptors.
Translational …, 2011
Pathology is fundamental in grading, staging, and treatment planning of malignancies. One relatively novel biomarker that may become more important in therapy and diagnostics is the chemokine receptor 4 (CXCR4). Ac-TZ14011 peptide derivatives, functionalized with a radiolabel, can be used for molecular imaging of tumors. Direct fluorescent labeling of the small peptide Ac-TZ14011 with the fluorescent dye fluorescein isothiocyanate (FITC), however, provides an alternative for the detection of CXCR4 expression levels in cells and tumor tissue. In this study, Ac-TZ14011-FITC was validated for CXCR4 staining in human breast cancer cell lines MDAMB231 and MDAMB231CXCR4+ during flow cytometric analysis. Its efficacy was compared to commercially available antibodies. Competition experiments validated the staining specificity. Confocal imaging revealed that CXCR4 staining was predominantly found on the cell membrane and/or in vesicles formed after endocytosis. Next to being able to differentiate “high” and “low” CXCR4-expressing tumor cells, the fluorescent peptide demonstrates potential in fluorescent immunohistochemistry of tumor tissue. Ac-TZ14011-FITC was able to differentiate MDAMB231 from MDAMB231CXCR4+ tumor cells and tissue, proving its applicability in the detection of differences in CXCR4 expression levels.