Automated sulfur-[18F]fluoride exchange radiolabelling of a prostate specific membrane antigen (PSMA) targeted ligand using the GE FASTlab™ cassette-based platform.

Sulfur-[18F]fluoride exchange radiochemistry is a rapid and convenient method for incorporating fluorine-18 into biologically active molecules. We report a fully automated radiolabelling procedure for the synthesis of a [18F]SO3F-bearing prostate specific membrane antigen (PSMA) targeted ligand ([18F]5) using the GE FASTLab™ cassette-based platform in a 25.0 ± 2.6% radiochemical yield (decay corrected). Uptake in vitro and in vivo correlated with PSMA expression, and the radioligand exhibited favourable biodistribution and pharmacokinetic profiles.

Design, synthesis, and evaluation of a novel PET imaging agent targeting lipofuscin in senescent cells.

Promoting a senescent phenotype to suppress tumour progression may present an alternative strategy for treating cancer and encourages the development of positron emission tomography (PET) imaging biomarkers for assessing response to treatment. The accumulation of lipofuscin deposits in senescent cells is visualised using the pathology stain Sudan Black B (SBB) which is an emerging biomarker of senescence. We describe the design, synthesis and evaluation of [18F]fluoroethyltriazole-SBB ([18F]FET-SBB), a fluorine-18 radiolabelled derivative of SBB. The in vitro uptake of [18F]FET-SBB in a senescent cell line corelated with lipofuscin deposits; in vivo PET imaging and metabolite analysis confirm a favourable pharmacokinetic and metabolic profile for further studies of in vivo models of senescence.

A kit-based aluminium-[18F]fluoride approach to radiolabelled microbubbles.

The production of 18F-labelled microbubbles (MBs) via the aluminium-[18F]fluoride ([18F]AlF) radiolabelling method and facile inverse-electron-demand Diels-Alder (IEDDA) 'click' chemistry is reported. An [18F]AlF-NODA-labelled tetrazine was synthesised in excellent radiochemical yield (>95% RCY) and efficiently conjugated to a trans-cyclooctene (TCO) functionalised phospholipid (40-50% RCY), which was incorporated into MBs (40-50% RCY). To demonstrate the potential of producing 18F-labelled MBs for clinical studies, we also describe a kit-based approach which is amenable for use in a hospital radiopharmacy setting.

Consideration of Metabolite Efflux in Radiolabelled Choline Kinetics.

Hypoxia is a complex microenvironmental condition known to regulate choline kinase α (CHKA) activity and choline transport through transcription factor hypoxia-inducible factor-1α (HIF-1α) and, therefore, may confound the uptake of choline radiotracer [18F]fluoromethyl-[1,2-2H4]-choline ([18F]-D4-FCH). The aim of this study was to investigate how hypoxia affects the choline radiotracer dynamics. Three underlying mechanisms by which hypoxia could potentially alter the uptake of the choline radiotracer, [18F]-D4-FCH, were investigated: 18F-D4-FCH import, CHKA phosphorylation activity, and the efflux of [18F]-D4-FCH and its phosphorylated product [18F]-D4-FCHP. The effects of hypoxia on [18F]-D4-FCH uptake were studied in CHKA-overexpressing cell lines of prostate cancer, PC-3, and breast cancer MDA-MB-231 cells. The mechanisms of radiotracer efflux were assessed by the cell uptake and immunofluorescence in vitro and examined in vivo (n = 24). The mathematical modelling methodology was further developed to verify the efflux hypothesis using [18F]-D4-FCH dynamic PET scans from non-small cell lung cancer (NSCLC) patients (n = 17). We report a novel finding involving the export of phosphorylated [18F]-D4-FCH and [18F]-D4-FCHP via HIF-1α-responsive efflux transporters, including ABCB4, when the HIF-1α level is augmented. This is supported by a graphical analysis of human data with a compartmental model (M2T6k + k5) that accounts for the efflux. Hypoxia/HIF-1α increases the efflux of phosphorylated radiolabelled choline species, thus supporting the consideration of efflux in the modelling of radiotracer dynamics.

The aluminium-[18F]fluoride revolution: simple radiochemistry with a big impact for radiolabelled biomolecules.

The aluminium-[18F]fluoride ([18F]AlF) radiolabelling method combines the favourable decay characteristics of fluorine-18 with the convenience and familiarity of metal-based radiochemistry and has been used to parallel gallium-68 radiopharmaceutical developments. As such, the [18F]AlF method is popular and widely implemented in the development of radiopharmaceuticals for the clinic. In this review, we capture the current status of [18F]AlF-based technology and reflect upon its impact on nuclear medicine, as well as offering our perspective on what the future holds for this unique radiolabelling method.

Effective Detection and Monitoring of Glioma Using [18F]FPIA PET Imaging.

BACKGROUND: Reprogrammed cellular metabolism is a cancer hallmark. In addition to increased glycolysis, the oxidation of acetate in the citric acid cycle is another common metabolic phenotype. We have recently developed a novel fluorine-18-labelled trimethylacetate-based radiotracer, [18F]fluoro-pivalic acid ([18F]FPIA), for imaging the transcellular flux of short-chain fatty acids, and investigated whether this radiotracer can be used for the detection of glioma growth. METHODS: We evaluated the potential of [18F]FPIA PET to monitor tumor growth in orthotopic patient-derived (HSJD-GBM-001) and cell line-derived (U87, LN229) glioma xenografts, and also included [18F]FDG PET for comparison. We assessed proliferation (Ki-67) and the expression of lipid metabolism and transport proteins (CPT1, SLC22A2, SLC22A5, SLC25A20) by immunohistochemistry, along with etomoxir treatment to provide insights into [18F]FPIA uptake. RESULTS: Longitudinal PET imaging showed gradual increase in [18F]FPIA uptake in orthotopic glioma models with disease progression (p < 0.0001), and high tumor-to-brain contrast compared to [18F]FDG (p < 0.0001). [18F]FPIA uptake correlated positively with Ki-67 (p < 0.01), SLC22A5 (p < 0.001) and SLC25A20 (p = 0.001), and negatively with CPT1 (p < 0.01) and SLC22A2 (p < 0.01). Etomoxir reduced [18F]FPIA uptake, which correlated with decreased Ki-67 (p < 0.05). CONCLUSIONS: Our findings support the use of [18F]FPIA PET for the detection and longitudinal monitoring of glioma, showing a positive correlation with tumor proliferation, and suggest transcellular flux-mediated radiotracer uptake.

Novel Non-Congeneric Derivatives of the Choline Kinase Alpha Inhibitor ICL-CCIC-0019.

Choline kinase alpha (CHKA) is a promising target for the development of cancer therapeutics. We have previously reported ICL-CCIC-0019, a potent CHKA inhibitor with high cellular activity but with some unfavorable pharmacological properties. In this work, we present an active analogue of ICL-CCIC-0019 bearing a piperazine handle (CK146) to facilitate further structural elaboration of the pharmacophore and thus improve the biological profile. Two different strategies were evaluated in this study: (1) a prodrug approach whereby selective CHKA inhibition could be achieved through modulating the activity of CK146, via the incorporation of an ε-(Ac) Lys motif, cleavable by elevated levels of histone deacetylase (HDAC) and cathepsin L (CTSL) in tumour cells; (2) a prostate-specific membrane antigen (PSMA) receptor targeted delivery strategy. Prodrug (CK145) and PSMA-targeted (CK147) derivatives were successfully synthesized and evaluated in vitro. While the exploitation of CK146 in those two strategies did not deliver the expected results, important and informative structure-activity relationships were observed and have been reported.

Detecting hypoxia in vitro using 18F-pretargeted IEDDA "click" chemistry in live cells.

We have exemplified a pretargeted approach to interrogate hypoxia in live cells using radioactive bioorthogonal inverse electron demand Diels-Alder (IEDDA) "click" chemistry. Our novel 18F-tetrazine probe ([18F]FB-Tz) and 2-nitroimidazole-based TCO targeting molecule (8) showed statistically significant (P < 0.0001) uptake in hypoxic cells (ca. 90 %ID per mg) vs. normoxic cells (<10 %ID per mg) in a 60 min incubation of [18F]FB-Tz. This is the first time that an intracellularly targeted small-molecule for IEDDA "click" has been used in conjunction with a radioactive reporter molecule in live cells and may be a useful tool with far-reaching applicability for a variety of applications.

Radiolabelling an 18F biologic via facile IEDDA "click" chemistry on the GE FASTLab™ platform.

The use of biologics in positron emission tomography (PET) imaging is an important area of radiopharmaceutical development and new automated methods are required to facilitate their production. We report an automated radiosynthesis method to produce a radiolabelled biologic via facile inverse electron demand Diels-Alder (IEDDA) "click" chemistry on a single GE FASTLab™ cassette. We exemplified the method by producing a fluorine-18 radiolabelled interleukin-2 (IL2) radioconjugate from a trans-cyclooctene (TCO) modified IL2 precursor. The radioconjugate was produced using a fully automated radiosynthesis on a single FASTLab™ cassette in a decay-corrected radiochemical yield (RCY, d.c.) of 19.8 ± 2.6% in 110 min (from start of synthesis); the molar activity was 132.3 ± 14.6 GBq µmol-1. The in vitro uptake of [18F]TTCO-IL2 correlated with the differential receptor expression (CD25, CD122, CD132) in PC3, NK-92 and activated human PBMCs. The automated method may be adapted for the radiosynthesis of any TCO-modified protein via IEDDA chemistry.

Development of a fluorine-18 radiolabelled fluorescent chalcone: evaluated for detecting glycogen.

BACKGROUND: Glycogen is a multibranched polysaccharide of glucose produced by cells to store energy and plays a key role in cancer. A previously reported fluorescent probe (CDg4) was shown to selectively bind glycogen in mouse embryonic stem cells, however the molecule was not evaluated in cancer cells. We report the synthesis and biological evaluation of a dual-modality imaging probe based on CDg4, for positron emission tomography (PET) and fluorescence microscopy. RESULTS: A fluorine-18 radiolabelled derivative of CDg4, ([18F]5) for in vivo quantification of total glycogen levels in cancer cells was developed and synthesised in 170 min with a non-decay corrected radiochemical yield (RCY n.d.c) of 5.1 ± 0.9% (n = 4) in > 98% radiochemical purity. Compound 5 and [18F]5 were evaluated in vitro for their potential to bind glycogen, but only 5 showed accumulation by fluorescence microscopy. The accumulation of 5 was determined to be specific as fluorescent signal diminished upon the digestion of carbohydrate polymers with α-amylase. PET imaging in non-tumour bearing mice highlighted rapid hepato-biliary-intestinal elimination of [18F]5 and almost complete metabolic degradation after 60 min in the liver, plasma and urine, confirmed by radioactive metabolite analysis. CONCLUSIONS: Fluorescent compound 5 selectively accumulated in glycogen containing cancer cells, identified by fluorescence microscopy; however, rapid in vivo metabolic degradation precludes further investigation of [18F]5 as a PET radiopharmaceutical.

Chemistry Considerations for the Clinical Translation of Oncology PET Radiopharmaceuticals.

Positron emission tomography (PET) has proven to be an invaluable tool in the staging and management of disease in oncology; however, [18F]fluorodeoxyglucose ([18F]FDG) remains the most widely used PET radiopharmaceutical despite the large financial investment in novel radiotracer development. We report our perspective and experience of translating radiopharmaceuticals into clinical studies, discussing the PET development pipeline from a chemistry perspective. We hope that, by identifying potential points of attrition along the pipeline and suggesting solutions to these problems, we may help others take their preclinical radiotracers into human studies. This review focuses primarily on the development of fluorine-18 radiopharmaceuticals, although the broader field of radiometal chemistry is considered where the translation journey is similar.

[18F]FET-ßAG-TOCA: The Design, Evaluation and Clinical Translation of a Fluorinated Octreotide.

The success of Lutathera™ ([177Lu]Lu-DOTA-TATE) in the NETTER-1 clinical trial as a peptide receptor radionuclide therapy (PRRT) for somatostatin receptor expressing (SSTR) neuroendocrine tumours (NET) is likely to increase the demand for patient stratification by positron emission tomography (PET). The current gold standard of gallium-68 radiolabelled somatostatin analogues (e.g., [68Ga]Ga-DOTA-TATE) works effectively, but access is constrained by the limited availability and scalability of gallium-68 radiopharmaceutical production. The aim of this review is three-fold: firstly, we discuss the peptide library design, biological evaluation and clinical translation of [18F]fluoroethyltriazole-ßAG-TOCA ([18F]FET-ßAG-TOCA), our fluorine-18 radiolabelled octreotide; secondly, to exemplify the potential of the 2-[18F]fluoroethylazide prosthetic group and copper-catalysed azide-alkyne cycloaddition (CuAAC) chemistry in accessing good manufacturing practice (GMP) compatible radiopharmaceuticals; thirdly, we aim to illustrate a framework for the translation of similarly radiolabelled peptides, in which in vivo pharmacokinetics drives candidate selection, supported by robust radiochemistry methodology and a route to GMP production. It is hoped that this review will continue to inspire the development and translation of fluorine-18 radiolabelled peptides into clinical studies for the benefit of patients.

Clinical translation of 18F-fluoropivalate - a PET tracer for imaging short-chain fatty acid metabolism: safety, biodistribution, and dosimetry in fed and fasted healthy volunteers.

BACKGROUND: Fatty acids derived de novo or taken up from the extracellular space are an essential source of nutrient for cell growth and proliferation. Radiopharmaceuticals including 11C-acetate, and 18F-FAC (2-18F-fluoroacetate), have previously been used to study short-chain fatty acid (SCFA) metabolism. We developed 18F-fluoropivalate (18F-FPIA; 3-18F-fluoro-2,2-dimethylpropionic acid) bearing a gem-dimethyl substituent to assert metabolic stability for studying SCFA metabolism. We report the safety, biodistribution, and internal radiation dosimetry profile of 18F-FPIA in 24 healthy volunteers and the effect of dietary conditions. MATERIALS AND METHODS: Healthy volunteer male and female subjects were enrolled (n = 24), and grouped into 12 fed and 12 fasted. Non-esterified fatty acids (NEFA) and carnitine blood measurements were assessed. Subjects received 159.48 MBq (range, 47.31-164.66 MBq) of 18F-FPIA. Radiochemical purity was > 99%. Safety data were obtained during and 24 h after radiotracer administration. Subjects underwent detailed multiple whole-body PET/CT scanning with sampling of venous bloods for radioactivity and radioactive metabolite quantification. Regions of interest were defined to derive individual and mean organ residence times; effective dose was calculated using OLINDA 1.1. RESULTS: All subjects tolerated 18F-FPIA with no adverse events. Over 90% of radiotracer was present in plasma at 60 min post-injection. The organs receiving highest absorbed dose (in mGy/MBq) were the liver (0.070 ± 0.023), kidneys (0.043 ± 0.013), gallbladder wall (0.026 ± 0.003), and urinary bladder (0.021 ± 0.004); otherwise there was low tissue uptake. The calculated effective dose using mean organ residence times over all 24 subjects was 0.0154 mSv/MBq (SD ± 0.0010). No differences in biodistribution or dosimetry were seen in fed and fasted subjects, though systemic NEFA and carnitine levels reflected fasted and fed states. CONCLUSION: The favourable safety, imaging, and dosimetric profile makes 18F-FPIA a promising candidate radiotracer for tracing SCFA metabolism.

Synthesis of a benzoxazinthione derivative of tanaproget and pharmacological evaluation for PET imaging of PR expression.

BACKGROUND: The histological evaluation of estrogen receptor (ER) and progesterone receptor (PR) expression in breast cancer lesions from biopsy tissue can stratify patients to receive endocrine therapy. Furthermore, PR expression can predict response to selective estrogen receptor modulators (SERMs). Current immunohistochemical approaches to PR detection are limited by sampling error associated with biopsy and lack of standardised protocols; positron emission tomography (PET) using receptor targeted radiopharmaceuticals to provide quantitative, whole-body imaging may overcome these limitations. PR expression has been successfully imaged with PET in the clinical setting, however investigation into new radioligands with improved pharmacokinetics and metabolic stability is desirable. RESULTS: We report the synthesis of a focused library of non-steroidal PR ligands evaluated for use as PET radioligands. A lead candidate ([18F]2) with low nanomolar activity was selected and radiolabelled with a radiochemical yield of 2.29 ± 2.31% (decay-corrected), radiochemical purity (RCP) > 95% and a molar activity of 2.5 ± 1.6 GBq/µmol. Cell uptake studies showed a significant and specific accumulation of [18F]2 in T47D (PR++) breast cancer cell compared to MDA-MB-231 (PR-) control; however, in vivo evaluation was confounded by rapid defluorination of the radioligand. In vitro metabolite analysis of 2 in MLM confirmed defluorination and oxidative metabolism of the thiocarbamate to oxocarbamate moiety by mass spectrometry. CONCLUSIONS: A route to access [18F]2 was developed to allow in vitro and in vivo evaluation, albeit with low radiochemical yield and modest molar activity. [18F]2 demonstrated selective uptake in PR++ T47D cells which could be blocked in a dose dependent manner with progesterone. However, [18F]2 showed poor in vivo metabolic stability with rapid defluorination within the time frame of the imaging protocol.

Affibody-Based PET Imaging to Guide EGFR-Targeted Cancer Therapy in Head and Neck Squamous Cell Cancer Models.

In head and neck squamous cell cancer, the human epidermal growth factor receptor 1 (EGFR) is the dominant signaling molecule among all members of the family. So far, cetuximab is the only approved anti-EGFR monoclonal antibody used for the treatment of head and neck squamous cell cancer, but despite the benefits of adding it to standard treatment regimens, attempts to define a predictive biomarker to stratify patients for cetuximab treatment have been unsuccessful. We hypothesized that imaging with EGFR-specific radioligands may facilitate noninvasive measurement of EGFR expression across the entire tumor burden and allow for dynamic monitoring of cetuximab-mediated changes in receptor expression. Methods: EGFR-specific Affibody molecule (ZEGFR:03115) was radiolabeled with 89Zr and 18F. The radioligands were characterized in vitro and in mice bearing subcutaneous tumors with varying levels of EGFR expression. The protein dose for imaging studies was assessed by injecting 89Zr-deferoxamine-ZEGFR:03115 (2.4-3.6 MBq, 2 µg) either together with or 30 min after increasing amounts of unlabeled ZEGFR:03115 (1, 5, 10, 15, and 20 µg). PET images were acquired at 3, 24, and 48 h after injection, and the image quantification data were correlated with the biodistribution results. The EGFR expression and biodistribution of the tracer were assessed ex vivo by immunohistochemistry, Western blot, and autoradiography. To downregulate the EGFR level, treatment with cetuximab was performed, and 18F-aluminium fluoride-NOTA-ZEGFR:03115 (12 µg, 1.5-2 MBq/mouse) was used to monitor receptor changes. Results: In vivo studies demonstrated that coinjecting 10 µg of nonlabeled molecules with 89Zr-deferoxamine-ZEGFR:03115 allows for clear tumor visualization 3 h after injection. The radioconjugate tumor accumulation was EGFR-specific, and PET imaging data showed a clear differentiation between xenografts with varying EGFR expression levels. A strong correlation was observed between PET analysis, ex vivo estimates of tracer concentration, and receptor expression in tumor tissues. Additionally, 18F-aluminium fluoride-NOTA-ZEGFR:03115 could measure receptor downregulation in response to EGFR inhibition. Conclusion: ZEGFR:03115-based radioconjugates can assess different levels of EGFR level in vivo and measure receptor expression changes in response to cetuximab, indicating a potential for assessment of adequate treatment dosing with anti-EGFR antibodies.

HER3-Mediated Resistance to Hsp90 Inhibition Detected in Breast Cancer Xenografts by Affibody-Based PET Imaging.

Purpose: Recent studies have highlighted a role of HER3 in HER2-driven cancers (e.g., breast cancer), implicating the upregulation of the receptor in resistance to HER-targeted therapies and Hsp90 inhibitors (e.g., AUY922). Therefore, we have developed an affibody-based PET radioconjugate that quantitatively assesses HER3 changes induced by Hsp90 inhibition in vivoExperimental Design: ZHER3:8698 affibody molecules were conjugated via the C-terminus cysteine to DFO-maleimide for 89Zr radiolabeling. The probe was characterized in vitro and in vivo in a panel of human breast cell lines and xenograft models with varying HER3 receptor levels. In addition, the radioconjugate was investigated as a tool to monitor the outcome of AUY922, an Hsp90 inhibitor, in an MCF-7 xenograft model.Results: We demonstrated that 89Zr-DFO-ZHER3:8698 can track changes in receptor expression in HER3-positive xenograft models and monitor the outcome of AUY922 treatment. Our in vitro findings showed that MCF-7 cells, which are phenotypically different from BT474, develop resistance to treatment with AUY922 through HER3/IGF-1Rß-mediated signaling. Of note, the lack of response in vitro due to HER3 recovery was confirmed in vivo using 89Zr-DFO-ZHER3:8698-based imaging. Upon AUY922 treatment, higher radioconjugate uptake was detected in treated MCF-7 xenografts, correlating with an AUY922-induced HER3 upregulation concomitant with an increase in IGF-1Rß expression.Conclusions: These data underline the potential of HER3-based PET imaging to noninvasively provide information about HER3 expression and to identify patients not responding to targeted therapies due to HER3 recovery. Clin Cancer Res; 24(8); 1853-65. ©2018 AACR.

Efficient [(18)F]AlF Radiolabeling of ZHER3:8698 Affibody Molecule for Imaging of HER3 Positive Tumors.

The human epidermal growth factor receptor 3 (HER3) is overexpressed in several cancers, being linked to a more resistant phenotype and hence leading to poor patient prognosis. Imaging HER3 is challenging owing to the modest receptor number (<50000 receptors/cell) in overexpressing cancer cells. Therefore, to image HER3 in vivo, high target affinity PET probes need to be developed. This work describes two different [(18)F]AlF radiolabeling strategies of the ZHER3:8698 affibody molecule specifically targeting HER3. The one-pot radiolabeling of ZHER3:8698 performed at 100 °C and using 1,4,7-triazanonane-1,4,7-triacetate (NOTA) as chelator resulted in radiolabeled products with variable purity attributed to radioconjugate thermolysis. An alternative approach based on the inverse electron demand Diels-Alder (IEDDA) reaction between a novel tetrazine functionalized 1,4,7-triazacyclononane-1,4-diacetate (NODA) chelator and the trans-cyclooctene (TCO) functionalized affibody molecule was also investigated. This method enabled the radiolabeling of the protein at room temperature. The [(18)F]AlF-NOTA-ZHER3:8698 and [(18)F]AlF-NODA-ZHER3:8698 conjugates showed a specific uptake at 1 h after injection in high HER3-expressing MCF-7 tumors of 4.36 ± 0.92% ID/g and 4.96 ± 0.65% ID/g, respectively. The current results are encouraging for further investigation of [(18)F]AlF-NOTA-ZHER3:8698 as a HER3 imaging agent.

Development of PDT/PET Theranostics: Synthesis and Biological Evaluation of an (18)F-Radiolabeled Water-Soluble Porphyrin.

Synthesis of the first water-soluble porphyrin radiolabeled with fluorine-18 is described: a new molecular theranostic agent which integrates the therapeutic selectivity of photodynamic therapy (PDT) with the imaging efficacy of positron emission tomography (PET). Generation of the theranostic was carried out through the conjugation of a cationic water-soluble porphyrin bearing an azide functionality to a fluorine-18 radiolabeled prosthetic bearing an alkyne functionality through click conjugation, with excellent yields obtained in both cold and hot synthesis. Biological evaluation of the synthesized structures shows the first example of an (18)F-radiolabeled porphyrin retaining photocytotoxicity following radiolabeling and demonstrable conjugate uptake and potential application as a radiotracer in vivo. The promising results gained from biological evaluation demonstrate the potential of this structure as a clinically relevant theranostic agent, offering exciting possibilities for the simultaneous imaging and photodynamic treatment of tumors.

PET Imaging of Steroid Hormone Receptor Expression.

Steroid hormone receptor (SHR) expression and changes in SHR expression compared to basal levels, whether upregulated, downregulated, or mutated, form a distinguishing feature of some breast, ovarian, and prostate cancers. These receptors act to induce tumor proliferation. In the imaging context, total expression together with modulation of expression can yield predictive and prognostic information. Currently, biopsy for histologic assessment of SHR expression is routine for breast and prostate cancer; however, the technique is not well suited to the heterogeneous tumor environment and can lead to incorrect receptor expression assignment, which precludes effective treatment. The development of positron emission tomography (PET) radioligands to image receptor expression may overcome the difficulties associated with tumor heterogeneity and facilitate the assessment of metastatic disease.