Radiofluorination of non-activated aromatic prosthetic groups for synthesis and evaluation of fluorine-18 labelled ghrelin(1-8) analogues.

The growth hormone secretagogue receptor 1a (GHSR) is differentially expressed in various disease states compared to healthy tissues and thus is a target for molecular imaging. The endogenous ligand for the GHSR is ghrelin, a 28 amino acid peptide with a unique octanoyl group on the serine-3 residue. A recently reported ghrelin analogue revealed the successful use of fluorine-containing, polycyclic aromatic groups in place of the octanoyl side chain, thereby providing potential access to new 18F-PET imaging probes. The peptide [Inp1,Dpr3(6-FN),1Nal4,Thr8]ghrelin(1-8) amide (1) showed sub-nanomolar receptor affinity (IC50 = 0.11 nM) toward the GHSR making it the strongest affinity ghrelin analogue reported to date. However, attempts to label such non-activated aromatic groups with fluoride-18 through conventional substitution methods resulted in low radiochemical yields, impractical for use in vivo. Since larger, non-activated aromatic groups appear to be of value for incorporating fluorine into ghrelin(1-8) analogues, an additional peptide bearing a 4'-fluorobiphenyl-4-carboxyl (4'-FBC) group in place of the octanoyl side chain was also of interest. Herein, we describe the radiosynthesis of [Inp1,Dpr3([18F]6-FN),1Nal4,Thr8]ghrelin(1-8) amide ([18F]1) and [Inp1,Dpr3([18F]4'-FBC),1Nal4,Thr8]ghrelin(1-8) amide ([18F]2) using a prosthetic group approach from iodonium ylide precursors as well as initial in vitro and in vivo evaluation of [18F]1 as a potential PET tracer for targeted imaging of the GHSR.

A dual modality 99mTc/Re(i)-labelled T140 analogue for imaging of CXCR4 expression.

The C-X-C chemokine receptor 4 (CXCR4) has been shown to be overexpressed in at least 23 types of cancer, including prostate cancer which has been shown to have a significant distinction of expression rates between cancerous compared to healthy or benign tissue. In an attempt to exploit the difference in expression, we have synthesized a derivative of T140, a peptide antagonist for CXCR4, containing a fluorescent 4-amino-1,8-naphthalimide appended with a di-(2-picolyl)amine binding unit to chelate rhenium or technetium-99m for fluorescence or SPECT imaging. The rhenium-coordinated variant was shown to have similar binding affinity for the receptor as T140 and showed specific uptake by fluorescence microscopy in CXCR4 expressing cells. The peptide was radiolabelled with technetium-99m in decay corrected radiochemical yields ranging from 60-85%, radiochemical purities >95%, and molar activities of 36-44 GBq µmol-1. The technetium-99m labelled peptide showed two-fold higher uptake in U87 cells expressing CXCR4 compared to non-transfected cells. Ex vivo biodistribution studies were performed using the technetium-99m labelled peptide in NOD/SCID mice bearing tumors derived from U87 cells with CXCR4. Tumor uptake of 0.51 ± 0.09% ID g-1 was observed two-hours post-injection. Our novel T140 derivative is suitable for imaging of CXCR4 expression by confocal microscopy. Further structural modifications to the peptide or metal complex may result in improved biodistribution for use in SPECT imaging of CXCR4 expressing tumors.

Development and Characterization of an 18F-labeled Ghrelin Peptidomimetic for Imaging the Cardiac Growth Hormone Secretagogue Receptor.

One-third of patients with heart disease develop heart failure, which is diagnosed through imaging and detection of circulating biomarkers. Imaging strategies reveal morphologic and functional changes but fall short of detecting molecular abnormalities that can lead to heart failure, and circulating biomarkers are not cardiac specific. Thus, there is critical need for biomarkers that are endogenous to myocardial tissues. The cardiac growth hormone secretagogue receptor 1a (GHSR1a), which binds the hormone ghrelin, is a potential biomarker for heart failure. We have synthesized and characterized a novel ghrelin peptidomimetic tracer, an 18F-labeled analogue of G-7039, for positron emission tomography (PET) imaging of cardiac GHSR1a. In vitro analysis showed enhanced serum stability compared to natural ghrelin and significantly increased cellular uptake in GHSR1a-expressing OVCAR cells. Biodistribution studies in mice showed that tissue uptake of the tracer was independent of circulating ghrelin levels, and there was negligible cardiac uptake and high uptake in the liver, intestines, and kidneys. Specificity of tracer uptake was assessed using ghsr -/- mice; both static and dynamic PET imaging revealed no difference in cardiac uptake, and there was no significant correlation between cardiac standardized uptake values and GHSR1a expression. Our study lays the groundwork for further refinement of peptidomimetic PET tracers targeting cardiac GHSR1a.

Synthesis and evaluation of optical and PET GLP-1 peptide analogues for GLP-1R imaging.

A fluorescein-GLP-1 (7-37) analog was generated to determine GLP-1R distribution in various cell types of the pancreas in both strains of mice and receptor-specific uptake was confirmed by blocking with exendin-4. Biodistribution studies were carried out using 68Ga-labeled GLP-1(7-37) peptides in CD1 and C57BL/6 mice. In addition, immunocompromised mice bearing GLP-1R-expressing insulinomas were evaluated by positron emission tomography (PET) imaging and ex vivo biodistribution studies. The optical GLP-1 probe strongly colocalized with immunofluorescence for insulin and glucagon, and more weakly with amylase (exocrine pancreas) and cytokeratin 19 (ductal cells), confirming its application for in situ GLP-1R imaging in various pancreatic cell types. Insulinomas were clearly visualized by in vivo PET. Reducing the peptide positive charge decreased renal retention as well as tumor uptake. Results demonstrate the application of the developed GLP-1 peptide analogues for in situ (optical) and in vivo (PET) imaging of GLP-1R expression.

Synthesis and biological characterization of a promising F-18 PET tracer for vesicular acetylcholine transporter.

Nine fluorine-containing vesicular acetylcholine transporter (VAChT) inhibitors were synthesized and screened as potential PET tracers for imaging the VAChT. Compound 18a was one of the most promising carbonyl-containing benzovesamicol analogs; the minus enantiomer, (-)-18a displayed high potency (VAChT Ki=0.59 ± 0.06 nM) and high selectivity for VAChT versus σ receptors (>10,000-fold). The radiosynthesis of (-)-[(18)F]18a was accomplished by a two-step procedure with 30-40% radiochemical yield. Preliminary biodistribution studies of (-)-[(18)F]18a in adult male Sprague-Dawley rats at 5, 30, 60 and 120 min post-injection (p.i.) were promising. The total brain uptake of (-)-[(18)F]18a was 0.684%ID/g at 5 min p.i. and by 120 min p.i. slowly washed out to 0.409 %ID/g; evaluation of regional brain uptake showed stable levels of ∼0.800 %ID/g from 5 to 120 min p.i in the VAChT-enriched striatal tissue of rats, indicating the tracer had crossed the blood brain barrier and was retained in the striatum. Subsequent microPET brain imaging studies of (-)-[(18)F]18a in nonhuman primates (NHPs) showed high striatal accumulation in the NHP brain; the standardized uptake value (SUV) for striatum reached a maximum value of 5.1 at 15 min p.i. The time-activity curve for the target striatal region displayed a slow and gradual decreasing trend 15 min after injection, while clearance of the radioactivity from the cerebellar reference region was much more rapid. Pretreatment of NHPs with 0.25mg/kg of the VAChT inhibitor (-)-vesamicol resulted in a ∼90% decrease of striatal uptake compared to baseline studies. HPLC metabolite analysis of NHP plasma revealed that (-)-[(18)F]18a had a good in vivo stability. Together, these preliminary results suggest (-)-[(18)F]18a is a promising PET tracer candidate for imaging VAChT in the brain of living subjects.

Synthesis and biological evaluation of 18F-labled 2-phenylindole derivatives as PET imaging probes for ß-amyloid plaques.

A novel series of fluorinated 2-phenylindole derivatives were synthesized and evaluated as ß-amyloid imaging probes for PET. The in vitro inhibition assay demonstrated that their binding affinities for Aß(1-42) aggregates ranged from 28.4 to 1097.8 nM. One ligand was labeled with (18)F ([(18)F]1a) for its high affinity (K(i)=28.4 nM), which was also confirmed by in vitro autoradiography experiments on brain sections of transgenic mouse (C57BL6, APPswe/PSEN1, 11 months old, male). In vivo biodistribution experiments in normal mice showed that this radiotracer displayed high initial uptake (5.82±0.51% ID/g at 2 min) into and moderate washout (2.77±0.31% ID/g at 60 min) from the brain. [(18)F]1a could be developed as a promising new PET imaging probe for Aß plaques although necessary modifications are still needed.

Protease-activated receptor 2 (PAR2)-targeting peptide derivatives for positron emission tomography (PET) imaging.

The proteolytically-activated G protein-coupled receptor (GPCR) protease-activated receptor 2 (PAR2), is implicated in various cancers and inflammatory diseases. Synthetic ligands and in vitro imaging probes targeting this receptor have been developed with low nanomolar affinity, however, no in vivo imaging probes exist for PAR2. Here, we report the strategic design, synthesis, and biological evaluation of a series of novel 4-fluorobenzoylated PAR2-targeting peptides derived from 2f-LIGRLO-NH2 (2f-LI-) and Isox-Cha-Chg-Xaa-NH2 (Isox-) peptide families, where the 4-fluorobenzoyl moiety acts as the 19F-standard of an 18F-labeled probe for potential use in in vivo imaging. We found that several of the 4-fluorobenzoylated peptides from the 2f-LI-family exhibited PAR2 selectivity with moderate potency (EC50 = 151-252 nM), whereas several from the Isox-family exhibited PAR2 selectivity with high potency (EC50 = 13-42 nM). Our lead candidate, Isox-Cha-Chg-Ala-Arg-Dpr(4FB)-NH2 (EC50 = 13 nM), was successfully synthesized with fluorine-18 with a radiochemical yield of 37%, radiochemical purity of >98%, molar activity of 20 GBq/µmol, and an end of synthesis time of 125 min. Biodistribution studies and preliminary PET imaging of the tracer in mice showed predominantly renal clearance. This 18F-labeled tracer is the first reported PAR2 imaging agent with potential for use in vivo. Future work will explore the use of this tracer in cancer xenografts and inflammation models involving upregulation of PAR2 expression.

Synthesis and in vitro evaluation of α-synuclein ligands.

Accumulation of misfolded α-synuclein in Lewy bodies and Lewy neurites is the pathological hallmark of Parkinson's disease (PD). To identify ligands having high binding potency toward aggregated α-synuclein, we synthesized a series of phenothiazine derivatives and assessed their binding affinity to recombinant α-synuclein fibrils using a fluorescent thioflavin T competition assay. Among 16 new analogues, the in vitro data suggest that compound 11b has high affinity to α-synuclein fibrils (K(i)=32.10 ± 1.25 nM) and compounds 11d, 16a and16b have moderate affinity to α-synuclein fibrils (K(i)≈50-100 nM). Further optimization of the structure of these analogues may yield compounds with high affinity and selectivity for aggregated α-synuclein.

Plasma radio-metabolite analysis of PET tracers for dynamic PET imaging: TLC and autoradiography.

BACKGROUND: In molecular imaging with dynamic PET, the binding and dissociation of a targeted tracer is characterized by kinetics modeling which requires the arterial concentration of the tracer to be measured accurately. Once in the body the radiolabeled parent tracer may be subjected to hydrolysis, demethylation/dealkylation and other biochemical processes, resulting in the production and accumulation of different metabolites in blood which can be labeled with the same PET radionuclide as the parent. Since these radio-metabolites cannot be distinguished by PET scanning from the parent tracer, their contribution to the arterial concentration curve has to be removed for the accurate estimation of kinetic parameters from kinetic analysis of dynamic PET. High-performance liquid chromatography has been used to separate and measure radio-metabolites in blood plasma; however, the method is labor intensive and remains a challenge to implement for each individual patient. The purpose of this study is to develop an alternate technique based on thin layer chromatography (TLC) and a sensitive commercial autoradiography system (Beaver, Ai4R, Nantes, France) to measure radio-metabolites in blood plasma of two targeted tracers-[18F]FAZA and [18F]FEPPA, for imaging hypoxia and inflammation, respectively. RESULTS: Radioactivity as low as 17 Bq in 2 µL of pig's plasma can be detected on the TLC plate using autoradiography. Peaks corresponding to the parent tracer and radio-metabolites could be distinguished in the line profile through each sample (n = 8) in the autoradiographic image. Significant intersubject and intra-subject variability in radio-metabolites production could be observed with both tracers. For [18F]FEPPA, 50% of plasma activity was from radio-metabolites as early as 5-min post injection, while for [18F]FAZA, significant metabolites did not appear until 50-min post. Simulation study investigating the effect of radio-metabolite in the estimation of kinetic parameters indicated that 32-400% parameter error can result without radio-metabolites correction. CONCLUSION: TLC coupled with autoradiography is a good alternative to high-performance liquid chromatography for radio-metabolite correction. The advantages of requiring only small blood samples (~ 100 µL) and of analyzing multiple samples simultaneously, make the method suitable for individual dynamic PET studies.

18F-Labeled PET Probe Targeting Enhancer of Zeste Homologue 2 (EZH2) for Cancer Imaging.

The enzyme enhancer of zeste homologue 2 (EZH2) plays a catalytic role in histone methylation (H3K27me3), one of the epigenetic modifications that is dysregulated in cancer. The development of a positron emission tomography (PET) imaging agent targeting EZH2 has the potential to provide a method of stratifying patients for epigenetic therapies. In this study, we designed and synthesized a series of fluoroethyl analogs based upon the structure of EZH2 inhibitors UNC1999 and EPZ6438. Among the candidate compounds, 20b exhibited a high binding affinity to EZH2 (IC50 = 6 nM) with selectivity versus EZH1 (IC50 = 200 nM) by SAM competition assay, and furthermore, EZH2 inhibition was demonstrated in the pancreatic cancer cell line PANC-1 (IC50 = 9.8 nM). [18F]20b was synthesized successfully and showed 5-fold higher uptake in PANC-1 cells than in MCF-7 cells. MicroPET imaging in a PANC-1 cell xenograft mouse model indicates that [18F]20b has specific binding to EZH2, which was identified by ex vivo Western blot analysis of the tumor tissue.

Nitrone-Modified Gold Nanoparticles: Synthesis, Characterization, and Their Potential as 18F-Labeled Positron Emission Tomography Probes via I-SPANC.

A novel bioorthogonal gold nanoparticle (AuNP) template displaying interfacial nitrone functional groups for bioorthogonal interfacial strain-promoted alkyne-nitrone cycloaddition reactions has been synthesized. These nitrone-AuNPs were characterized in detail using 1H nuclear magnetic resonance spectroscopy, transmission electron microscopy, thermogravimetric analysis, and X-ray photoelectron spectroscopy, and a nanoparticle raw formula was calculated. The ability to control the conjugation of molecules of interest at the molecular level onto the nitrone-AuNP template allowed us to create a novel methodology for the synthesis of AuNP-based radiolabeled probes.

Peptidomimetic growth hormone secretagogue derivatives for positron emission tomography imaging of the ghrelin receptor.

The ghrelin receptor is a seven-transmembrane (7-TM) receptor known to have an increased level of expression in human carcinoma and heart failure. Recent work has focused on the synthesis of positron emission tomography (PET) probes designed to target and image this receptor for disease diagnosis and staging. However, these probes have been restricted to small-molecule quinalizonones and peptide derivatives of the endogenous ligand ghrelin. We describe the design, synthesis and biological evaluation of a series of 4-fluorobenzoylated growth hormone secretagogues (GHSs) derived from peptidic (GHRP-1, GHPR-2 and GHRP-6) and peptidomimetic (G-7039, [1-Nal4]G-7039 and ipamorelin) families in order to test locations for the insertion of fluorine-18 for PET imaging. The peptidomimetic G-7039 was found to be the most suitable for 18F-radiolabelling as its non-radioactive 4-fluorobenzoylated analogue ([1-Nal4,Lys5(4-FB)]G-7039), had both a high binding affinity (IC50 = 69 nM) and promising in vitro efficacy (EC50 = 1.1 nM). Prosthetic group radiolabelling of the precursor compound [1-Nal4]G-7039 using N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) delivered the PET probe [1-Nal4,Lys5(4-[18F]-FB)]G-7039 in an average decay-corrected radiochemical yield of 48%, a radio-purity ≥ 99% and an average molar activity of >34 GBq/µmol. This compound could be investigated as a PET probe for the detection of diseases that are characterised by overexpression of the ghrelin receptor.

Viral nanoparticles decorated with novel EGFL7 ligands enable intravital imaging of tumor neovasculature.

Angiogenesis is a dynamic process fundamental to the development of solid tumors. Epidermal growth factor-like domain 7 (EGFL7) is a protein whose expression is restricted to endothelial cells undergoing active remodeling that has emerged as a key mediator of this process. EGFL7 expression is associated with poor outcome in several cancers, making it a promising target for imaging or therapeutic strategies. Here, EGFL7 is explored as a molecular target for active neovascularization. Using a combinatorial peptide screening approach, we describe the discovery and characterization of a novel high affinity EGFL7-binding peptide, E7p72, that specifically targets human endothelial cells. Viral nanoparticles decorated with E7p72 peptides specifically target tumor-associated neovasculature with high specificity as assessed by intravital imaging. This work highlights the value of EGFL7 as a target for angiogenic vessels and opens the door for novel targeted therapeutic approaches.

Binding of the radioligand SIL23 to α-synuclein fibrils in Parkinson disease brain tissue establishes feasibility and screening approaches for developing a Parkinson disease imaging agent.

Accumulation of α-synuclein (α-syn) fibrils in Lewy bodies and Lewy neurites is the pathological hallmark of Parkinson disease (PD). Ligands that bind α-syn fibrils could be utilized as imaging agents to improve the diagnosis of PD and to monitor disease progression. However, ligands for α-syn fibrils in PD brain tissue have not been previously identified and the feasibility of quantifying α-syn fibrils in brain tissue is unknown. We report the identification of the (125)I-labeled α-syn radioligand SIL23. [(125)I]SIL23 binds α-syn fibrils in postmortem brain tissue from PD patients as well as an α-syn transgenic mouse model for PD. The density of SIL23 binding sites correlates with the level of fibrillar α-syn in PD brain tissue, and [(125)I]SIL23 binding site densities in brain tissue are sufficiently high to enable in vivo imaging with high affinity ligands. These results identify a SIL23 binding site on α-syn fibrils that is a feasible target for development of an α-syn imaging agent. The affinity of SIL23 for α-syn and its selectivity for α-syn versus Aß and tau fibrils is not optimal for imaging fibrillar α-syn in vivo, but we show that SIL23 competitive binding assays can be used to screen additional ligands for suitable affinity and selectivity, which will accelerate the development of an α-syn imaging agent for PD.