Development and Evaluation of an 18F-Radiolabeled Monocyclam Derivative for Imaging CXCR4 Expression.

In humans, C-X-C chemokine receptor type 4 (CXCR4) is a protein that 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 the disease. Peptidic antagonists have been labeled with a variety of radioisotopes for the detection of CXCR4, but the methodology utilizing small molecules has predominantly used radiometals. We report here the development of a 18F-radiolabeled cyclam-based small molecule radioprobe, [18F]MCFB, for imaging CXCR4 expression. The IC50 value of [19F]MCFB for CXCR4 was similar to that of AMD3465 (111.3 and 89.8 nM, respectively). In vitro binding assays show that the tracer depicted a differential CXCR4 expression, which was blocked in the presence of AMD3465, demonstrating the specificity of [18F]MCFB. Positron emission tomography (PET) imaging studies showed a distinct uptake of the radioprobe in lymphoma and breast cancer xenografts. High liver and kidney uptakes were seen with [18F]MCFB, leading us to further examine the basis of its pharmacokinetics in relation to the tracer's cationic nature and thus the role of organic cation transporters (OCTs). Substrate competition following the intravenous injection of metformin led to a marked decrease in the urinary excretion of [18F]MCFB, with moderate changes observed in other organs, including the liver. Our results suggest involvement of OCTs in the renal elimination of the tracer. In conclusion, the 18F-radiolabeled monocyclam, [18F]MCFB, has potential to detect tumor CXCR4 in nonhepatic tissues.

Development of a PET Tracer for OGA with Improved Kinetics in the Living Brain.

O-GlcNAcylation is thought to play a role in the development of tau pathology in Alzheimer's disease because of its ability to modulate tau's aggregation propensity. O-GlcNAcylation is regulated by 2 enzymes: O-GlcNAc transferase and O-GlcNAcase (OGA). Development of a PET tracer would therefore be an essential tool for developing therapeutic small-molecule inhibitors of OGA, enabling clinical testing of target engagement and dose selection. Methods: A collection of small-molecule compounds was screened for inhibitory activity and high-affinity binding to OGA, as well as favorable PET tracer attributes (multidrug resistance protein 1 efflux, central nervous system PET multiparameter optimization, etc.). Two lead compounds with high affinity and selectivity for OGA were selected for further profiling, including OGA binding to tissue homogenate using a radioligand competition binding assay. In vivo pharmacokinetics were established using a microdosing approach with unlabeled compounds in rats. In vivo imaging studies were performed in rodents and nonhuman primates (NHPs) with 11C-labeled compounds. Results: Two selected candidates, BIO-735 and BIO-578, displayed promising attributes in vitro. After radiolabeling with tritium, [3H]BIO-735 and [3H]BIO-578 binding in rodent brain homogenates demonstrated dissociation constants of 0.6 and 2.3 nM, respectively. Binding was inhibited, concentration-dependently, by homologous compounds and thiamet G, a well-characterized and structurally diverse OGA inhibitor. Imaging studies in rats and NHPs showed both tracers had high uptake in the brain and inhibition of binding to OGA in the presence of a nonradioactive compound. However, only BIO-578 demonstrated reversible binding kinetics within the time frame of a PET study with a 11C-labeled molecule to enable quantification using kinetic modeling. Specificity of tracer uptake was confirmed with a 10 mg/kg blocking dose of thiamet G. Conclusion: We describe the development and testing of 2 11C PET tracers targeting the protein OGA. The lead compound BIO-578 demonstrated high affinity and selectivity for OGA in rodent and human postmortem brain tissue, leading to its further testing in NHPs. NHP PET imaging studies showed that the tracer had excellent brain kinetics, with full inhibition of specific binding by thiamet G. These results suggest that the tracer [11C]BIO-578 is well suited for further characterization in humans.

Identification of ABC Transporter Interaction of a Novel Cyanoquinoline Radiotracer and Implications for Tumour Imaging by Positron Emission Tomography.

BACKGROUND: The epidermal growth factor receptor (EGFR) is overexpressed in many cancers including lung, ovarian, breast, head and neck and brain. Mutation of this receptor has been shown to play a crucial role in the response of non-small cell lung carcinoma (NSCLC) to EGFR-targeted therapies. It is envisaged that imaging of EGFR using positron emission tomography (PET) could aid in selection of patients for treatment with novel inhibitors. We recognised multi-drug resistant phenotype as a threat to development of successful imaging agents. In this report, we describe discovery of a novel cyanoquinoline radiotracer that lacks ABC transporter activity. METHODS: Cellular retention of the prototype cyanoquinoline [18F](2E)-N-{4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxyquinolin-6-yl}-4-({[1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl]methyl}amino)-but-2-enamide ([18F]FED6) and [18F](2E)-N-{4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxyquinolin-6-yl}-4-[({1-[(2R,5S)-3-fluoro-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]-1H-1,2,3-triazol-4-yl}methyl)amino]but-2-enamide ([18F]FED20) were evaluated to establish potential for imaging specificity. The substrate specificity of a number of cyanoquinolines towards ABC transporters was investigated in cell lines proficient or deficient in ABCB1 or ABCG2. RESULTS: FED6 demonstrated substrate specificity for both ABCG2 and ABCB1, a property that was not observed for all cyanoquinolines tested, suggesting scope for designing novel probes. ABC transporter activity was confirmed by attenuating the activity of transporters with drug inhibitors or siRNA. We synthesized a more hydrophilic compound [18F]FED20 to overcome ABC transporter activity. FED20 lacked substrate specificity for both ABCB1 and ABCG2, and maintained a strong affinity for EGFR. Furthermore, FED20 showed higher inhibitory affinity for active mutant EGFR versus wild-type or resistant mutant EGFR; this property resulted in higher [18F]FED20 cellular retention in active mutant EGFR expressing NSCLC. CONCLUSION: [18F]FED20 binds EGFR but is devoid of ABC transporter activity, thus, has potential for EGFR imaging.