Tobias L. Ross

Radiolabeling of Nanoparticles and Polymers for PET Imaging

Nanomedicine has become an emerging field in imaging and therapy of malignancies. Nanodimensional drug delivery systems have already been used in the clinic, as carriers for sensitive chemotherapeutics or highly toxic substances. In addition, those nanodimensional structures are further able to carry and deliver radionuclides. In the development process, non-invasive imaging by means of positron emission tomography (PET) represents an ideal tool for investigations of pharmacological profiles and to find the optimal nanodimensional architecture of the aimed-at drug delivery system. Furthermore, in a personalized therapy approach, molecular imaging modalities are essential for patient screening/selection and monitoring. Hence, labeling methods for potential drug delivery systems are an indispensable need to provide the radiolabeled analog. In this review, we describe and discuss various approaches and methods for the labeling of potential drug delivery systems using positron emitters.

Fluorine-18 click radiosynthesis and preclinical evaluation of a new 18F-labeled folic acid derivative

The folate receptor (FR) is highly expressed on most epithelial cancer cells, while normal cells show only restricted expression of FR. As a result, the FR is an ideal target for receptor-based molecular imaging and therapy of cancer and has become a promising target in oncology. To date, several folate-based chemotherapeutics and imaging probes such as radiopharmaceuticals for single photon emission computed tomography (SPECT) have been developed. However, an (18)F-labeled folic acid derivative suitable for positron emission tomography (PET) imaging that can be routinely applied is still lacking. In this study, a new fluorinated and radiofluorinated folic acid derivative, (18/19)F-click folate, was synthesized using click chemistry. In a convenient and very efficient two-step radiosynthesis, the isolated (18)F-click folate was obtained in good radiochemical yields of 25-35% with a specific activity of 160+/-70 GBq/micromol after <or=90 min synthesis time. The new compound was pharmacologically evaluated in vitro and in vivo. The affinity of the non-radioactive (19)F-click folate to the FR was determined in displacement studies with FR expressing KB tumor cells using (3)H-folic acid. In these in vitro binding studies, a nanomolar affinity with a K(i) of 9.76+/-3.13 nM was found for (19)F-click folate. The (18)F-labeled click folate derivative was then applied for in vivo PET studies and ex vivo biodistribution experiments using nude mice bearing KB tumor xenografts. The post mortem dissection experiments showed a high specific uptake of (18)F-click folate derivative in FR-expressing tissues. Uptake in KB tumor xenografts and kidneys (FR-positive tissue) amounted to 3.13%ID/g (94% specific blockade) and 16.53%ID/g (75% specific blockade), respectively. PET imaging using (18)F-click folate permitted a visualization of KB tumors, and blockade studies confirmed the specific accumulation of the radiotracer in vivo. However, strong hepatobiliary excretion of the new tracer led to elevated accumulation of radioactivity in the abdominal region. In conclusion, the click chemistry approach is convenient to accomplish and provided high radiochemical yields of (18)F-click folate. The new tracer showed good in vitro but limited in vivo properties. Ultimately, the (18)F-click folate emphasizes the potential of (18)F-labeled folates for receptor-based tumor PET imaging.

Molecular Imaging of the Chemokine Receptor CXCR4 After Acute Myocardial Infarction.

OBJECTIVES An assay for molecular imaging of myocardial CXCR4 expression was evaluated, in order to obtain mechanistic insights noninvasively based on quantitative positron emission tomography (PET). BACKGROUND The chemokine receptor CXCR4 has emerged as a therapeutic target after acute myocardial infarction (AMI), because of its role in inflammatory and progenitor cell recruitment. METHODS PET with the specific CXCR4 ligand, gallium-68 ((68)Ga)-pentixafor, was performed in mice (n = 53) and compared with ex vivo autoradiography, immunohistochemistry, and left ventricular flow cytometry. In addition, 12 patients were imaged at 2 to 8 days after AMI. RESULTS In mice, (68)Ga-pentixafor identified regional CXCR4 upregulation in the infarct region, peaking at 3 days (infarct/remote [I/R] ratio 1.5 ± 0.2 at 3 days vs. 1.2 ± 0.3 at 7 days; p = 0.03), corresponding to a flow cytometry-based peak of CD45+ leukocytes and immunohistochemical detection of CD68+ macrophages and Ly6G+ granulocytes. Blockade with the CXCR4 antagonist AMD3100 abolished the signal. No specific uptake was found in sham-operated or control animals. Long-term treatment with oral enalapril attenuated the CXCR4 signal (I/R 1.2 ± 0.2 at 3 days and 1.0 ± 0.0.1 at 7 days; p = 0.01 vs. untreated). Patients showed variable degrees of CXCR4 upregulation in the infarct region. No single clinical parameter allowed for prediction of CXCR4 signal strength. At multivariate analysis, a combination of infarct size and time after reperfusion predicted the CXCR4 infarct signal (rmultiple = 0.73; p = 0.03). Infarct signal in the myocardium was paralleled by elevated pentixafor uptake in bone marrow (r = 0.61; p = 0.04), which highlighted systemic interactions. CONCLUSIONS Targeted PET imaging with (68)Ga-pentixafor identifies the global and regional CXCR4 expression pattern in myocardium and systemic organs. CXCR4 upregulation after AMI coincides with inflammatory cell infiltration, but shows interindividual variability in patients. This may have implications for the response to CXCR4- or other inflammation-targeted therapy, and for subsequent ventricular remodeling.

5-(2-18F-Fluoroethoxy)-l-Tryptophan as a Substrate of System L Transport for Tumor Imaging by PET

Large neutral l-amino acids are substrates of system L amino acid transporters. The level of one of these, LAT1, is increased in many tumors. Aromatic l-amino acids may also be substrates of aromatic l-amino acid decarboxylase (AADC), the level of which is enhanced in endocrine tumors. Increased amino acid uptake and subsequent decarboxylation result in the intracellular accumulation of the amino acid and its decarboxylation product. 18F- and 11C-labeled neutral aromatic amino acids, such as l-3,4-dihydroxy-6-18F-fluorophenylalanine (18F-FDOPA) and 5-hydroxy-l-[β-11C]tryptophan, are thus successfully used in PET to image endocrine tumors. However, 5-hydroxy-l-[β-11C]tryptophan has a relatively short physical half-life (20 min). In this work, we evaluated the in vitro and in vivo characteristics of the 18F-labeled tryptophan analog 5-(2-18F-fluoroethoxy)-l-tryptophan (18F-l-FEHTP) as a PET probe for tumor imaging. Methods: 18F-l-FEHTP was synthesized by no-carrier-added 18F fluorination of 5-hydroxy-l-tryptophan. In vitro cell uptake and efflux of 18F-l-FEHTP and 18F-FDOPA were studied with NCI-H69 endocrine small cell lung cancer cells, PC-3 pseudoendocrine prostate cancer cells, and MDA-MB-231 exocrine breast cancer cells. Small-animal PET was performed with the respective xenograft-bearing mice. Tissues were analyzed for potential metabolites. Results: 18F-l-FEHTP specific activity and radiochemical purity were 50–150 GBq/μmol and greater than 95%, respectively. In vitro cell uptake of 18F-l-FEHTP was between 48% and 113% of added radioactivity per milligram of protein within 60 min at 37°C and was blocked by greater than 95% in all tested cell lines by the LAT1/2 inhibitor 2-amino-2-norboranecarboxylic acid. 18F-FDOPA uptake ranged from 26% to 53%/mg. PET studies revealed similar xenograft-to-reference tissue ratios for 18F-l-FEHTP and 18F-FDOPA at 30–45 min after injection. In contrast to the 18F-FDOPA PET results, pretreatment with the AADC inhibitor S-carbidopa did not affect the 18F-l-FEHTP PET results. No decarboxylation products of 18F-l-FEHTP were detected in the xenograft homogenates. Conclusion: 18F-l-FEHTP accumulates in endocrine and nonendocrine tumor models via LAT1 transport but is not decarboxylated by AADC. 18F-l-FEHTP may thus serve as a PET probe for tumor imaging and quantification of tumor LAT1 activity. These findings are of interest in view of the ongoing evaluation of LAT1 substrates and inhibitors for cancer therapy.

18F-Labeling Using Click Cycloadditions

Due to expanding applications of positron emission tomography (PET) there is a demand for developing new techniques to introduce fluorine-18 (t 1/2 = 109.8 min). Considering that most novel PET tracers are sensitive biomolecules and that direct introduction of fluorine-18 often needs harsh conditions, the insertion of 18F in those molecules poses an exceeding challenge. Two major challenges during 18F-labeling are a regioselective introduction and a fast and high yielding way under mild conditions. Furthermore, attention has to be paid to functionalities, which are usually present in complex structures of the target molecule. The Cu-catalyzed azide-alkyne cycloaddition (CuAAC) and several copper-free click reactions represent such methods for radiolabeling of sensitive molecules under the above-mentioned criteria. This minireview will provide a quick overview about the development of novel 18F-labeled prosthetic groups for click cycloadditions and will summarize recent trends in copper-catalyzed and copper-free click 18F-cycloadditions.

Serial Quantitative TSPO-Targeted PET Reveals Peak Microglial Activation up to 2 Weeks After an Epileptogenic Brain Insult

Experimental and clinical evidence suggests that neuroinflammation, triggered by epileptogenic insults, contributes to seizure development. We used translocator protein–targeted molecular imaging to obtain further insights into the role of microglial activation during epileptogenesis. Methods: As epileptogenic insult, a status epilepticus (SE) was induced in rats by lithium pilocarpine. Rats were subjected to 11C-PK11195 PET scans before SE; at 4 h after SE; at 1, 2, 5, 7, 14, and 22 d after SE; and at 14–16 wk after SE. For data evaluation, brain regions were outlined by coregistration with a standard rat brain atlas, and percentage injected dose/cm3 and binding potential (simplified reference tissue model with cerebellar gray matter as a reference region) were calculated. For autoradiography and immunohistochemical evaluation, additional rats were decapitated without prior SE or 2, 5, or 14 d after SE. Results: After SE, increases in 11C-PK11195 uptake and binding potential were evident in epileptogenesis-associated brain regions, such as the hippocampus, thalamus, or piriform cortex, but not in the cerebellum beginning at 2–5 d and persisting at least 3 wk after SE. Maximal regional signal was observed at 1–2 wk after SE. Autoradiography confirmed the spatiotemporal profile. Immunohistochemical evaluation revealed microglial and astroglial activation as well as neuronal cell loss in epileptogenesis-associated brain regions at all investigated time points. The time course of microglial activation was consistent with that demonstrated by tracer techniques. Conclusion: Translocator protein–targeted PET is a reliable tool for identifying brain inflammation during epileptogenesis. Neuroinflammation mainly affects brain regions commonly associated with seizure generation and spread. Definition of the time profile of neuroinflammation may facilitate the development of inflammation-targeted, antiepileptogenic therapy.

Comparison of standard and delayed imaging to improve the detection rate of [(68)Ga]PSMA I&T PET/CT in patients with biochemical recurrence or prostate-specific antigen persistence after primary therapy for prostate cancer.

PurposeThe aim of this study was to assess the value of dual-time point imaging in PET/CT for detection of biochemically recurrent or persistent prostate cancer, using the prostate-specific membrane antigen (PSMA) ligand [68Ga]PSMA I&T.Methods240 patients who underwent a [68Ga]PSMA I&T PET/CT in the context of biochemical relapse of prostate cancer were included in this retrospective analysis. Imaging consisted of a standard whole-body PET/CT (1 h p.i.), followed by delayed (3 h p.i.) imaging of the abdomen. PSA-stratified proportions of positive PET/CT results, standardized uptake values and target-to-background ratios were analyzed, and compared between standard and delayed imaging.ResultsThe overall detection rates of [68Ga]PSMA I&T PET/CT were 94.2, 71.8, 58.6, 55.9 and 38.9% for PSA levels of ≥2, 1 to <2, 0.5 to <1, >0.2 to <0.5, and 0.01 to 0.2 ng/mL, respectively. Although the target-to-background ratio improved significantly over time (P < 0.0001), the majority (96.6%) of all lesions suggestive of recurrent disease could already be detected in standard imaging. Delayed imaging at 3 h p.i. exclusively identified pathologic findings in 5.4% (10/184) of abnormal [68Ga]PSMA I&T PET/CT scans, and exclusively detected 3.4% (38/1134) of all lesions suggestive of recurrent disease.Conclusions[68Ga]PSMA I&T PET/CT shows high detection rates in patients with prostate-specific antigen persistence or biochemical recurrence of prostate cancer. Delayed imaging can detect lesions with improved contrast compared to standard imaging. However, the impact on detection rates was limited in this study.

18F-labeled folic acid derivatives for imaging of the folate receptor via positron emission tomography†

The folate receptor (FR) is already known as a proven target in diagnostics and therapy of cancer. Furthermore, the FR is involved in inflammatory and autoimmune diseases. The major advantage as a valuable target is its strongly limited expression in healthy tissues. Over the past two decades, several folic acid-based radiopharmaceuticals addressing the FR have been developed, and some of them show great potential for applications in clinical routine. However, most of these radiofolates were developed for single photon emission computed tomography imaging, and only a few can be used for positron emission tomography (PET) imaging. The development of suitable (18) F-labeled derivatives for PET imaging of the FR has aroused great interest and recent studies revealed very promising candidates for further development and translation into human applications. In this review, we focus on the development of (18) F-labeled folic acid derivatives for PET imaging of the FR and discuss various radiochemical strategies and approaches towards (18) F-folates. Besides radiochemistry and (18) F-labeling, we briefly look into the crucial pharmacological parameters and the preclinical in vivo performance of those (18) F-folates.

Clinical Molecular Imaging of Chemokine Receptor CXCR4 Expression in Atherosclerotic Plaque using 68Ga-Pentixafor PET: Correlation with Cardiovascular Risk Factors and Calcified Plaque Burden

The CXC-motif chemokine receptor 4 (CXCR4) represents a promising target for molecular imaging of different CXCR4-positive cell types in cardiovascular diseases such as atherosclerosis and arterial wall injury. The aim of this study was to assess the prevalence, pattern, and clinical correlates of arterial wall accumulation of 68Ga-pentixafor, a specific CXCR4 ligand for PET. Methods: The data for 51 patients who underwent 68Ga-pentixafor PET/CT for noncardiovascular indications were retrospectively analyzed. Tracer accumulation in the vessel wall of major arteries was analyzed qualitatively and semiquantitatively by blood-pool–corrected target-to-background ratios. Tracer uptake was compared with calcified plaque burden and cardiovascular risk factors. Results: Focal arterial uptake of 68Ga-pentixafor was seen at 1,411 sites in 51 (100%) of patients. 68Ga-pentixafor uptake was significantly associated with calcified plaque burden (P < 0.0001) and cardiovascular risk factors including age (P < 0.0001), arterial hypertension (P < 0.0001), hypercholesterolemia (P = 0.0005), history of smoking (P = 0.01), and prior cardiovascular events (P = 0.0004). Both the prevalence (P < 0.0001) and the signal intensity (P = 0.009) of 68Ga-pentixafor uptake increased as the number of risk factors increased. Conclusion: 68Ga-pentixafor PET/CT is suitable for noninvasive, highly specific PET imaging of CXCR4 expression in the atherosclerotic arterial wall. Arterial wall 68Ga-pentixafor uptake is significantly associated with surrogate markers of atherosclerosis and is linked to the presence of cardiovascular risk factors. 68Ga-pentixafor signal is higher in patients with a high-risk profile and may hold promise for identification of vulnerable plaque.

Initial Experience with Volumetric 68Ga-PSMA I&T PET/CT for Assessment of Whole-body Tumor Burden as a Quantitative Imaging Biomarker in Patients with Prostate Cancer

A quantitative imaging biomarker is desirable to provide a comprehensive measure of whole-body tumor burden in patients with metastatic prostate cancer, and to standardize the evaluation of treatment-related changes. Therefore, we evaluated whether prostate-specific membrane antigen (PSMA) ligand PET/CT may be applied to provide PSMA-derived volumetric parameters for quantification of whole-body tumor burden. Methods: One hundred one patients who underwent 68Ga-PSMA I&T PET/CT because of increasing prostate-specific antigen (PSA) levels after radical prostatectomy were included in this retrospective analysis. Tracer uptake was quantified using SUVs. Volumetric parameters, that is, PSMA-derived tumor volume (PSMA-TV) and total lesion PSMA (TL-PSMA), were calculated for each patient using a 3-dimensional segmentation and computerized volumetry technique and compared with serum PSA levels. In a group of 10 patients, volumetric parameters were applied for treatment monitoring. Results: Volumetric parameters, that is, whole-body PSMA-TV and whole-body TL-PSMA, demonstrated a statistically significant correlation with PSA levels (P < 0.0001) as a surrogate marker of tumor burden, whereas SUVmax (P = 0.22) or SUVmean (P = 0.45) did not. Treatment response and treatment failure were paralleled by concordant changes in both whole-body PSMA-TV and whole-body TL-PSMA (P = 0.02), whereas neither the change in SUVmax (P = 1.0) nor the change in SUVmean (P = 1.0) concordantly paralleled changes in PSA levels. Conclusion: PSMA-derived volumetric parameters provide a quantitative imaging biomarker for whole-body tumor burden, capable of standardizing quantitative changes in PET imaging of patients with metastatic prostate cancer and of facilitating therapy monitoring.