Synthesis,radiolabeling and bioevaluation of a novel arylpiperazine derivative containing triazole as a 5-HT1A receptor imaging agents

IntroductionIt has been recognized that serotonin plays a main role in various pathological conditions such as anxiety, depression, aggressiveness, schizophrenia, suicidal behavior, panic and autism. 1-(2-Methoxyphenyl) piperazine pharmacophore, a fragment of the true 5-HT_1A antagonist WAY100635, is found in numerous selective 5-HT_1A imaging agents. In this paper, we have reported the synthesis of a novel derivative of 1-(2-methoxyphenyl) piperazine that is labeled with ^99mTc (CO)₃ via click chemistry.MethodsThe bidentate alkyne, propargylglycine was reacted with phenyl piperazine triazole derivative in the presence of a catalytic amount of Cu (I) to form tridentate ligand. The ligand was radiolabeled with the precursor [^99mTc] [(H₂O)₃ (CO)₃]⁺ and characterized by HPLC. The bioevaluation of radio labeled ligand was carried out in rats.ResultsTriazole complex was labeled by ^99mTc-tricarbonyl and its radiochemical yield was more than > 95% which was determined by HPLC. In vivo stability studies in human serum albumin show a 93% ratio of complex after a 24 h period. The calculated partition coefficient (logP) was 0.34 ± 0.02. Receptor binding assays indicated about 70% specific binding of radioligand to 5-HT_1A receptors. Biodistribution studies have shown brain hippocampus uptake of 0.40 ± 0.08 %ID/g at 30 min post injection.ConclusionsResults indicate that this ^99mTc-tricabonyl-arylpiperazine derivative has specific binding to 5-HT_1A receptors and presented suitable characters for its use as a CNS imaging agent.     

Synthesis and pharmacological evaluation of a new series of radiolabeled ligands for 5-HT7 receptor PET neuroimaging

IntroductionThe brain serotonin-7 receptor (5-HT₇) is the most recently discovered serotonin receptor. It is targeted by several drug-candidates in psychopharmacology and neuropharmacology. In these fields, positron emission tomography (PET) is a molecular imaging modality offering great promise for accelerating the development process from preclinical discovery to clinical phases. We recently described fluorinated 5-HT₇ radioligands, inspired by the structure of SB269970, the prototypical 5-HT₇ antagonist. Although these results were promising, it appeared that the radiotracer-candidates suffered, among other drawbacks, from too low a 5-HT₇ receptor affinity.MethodsIn the present study, seven structural analogs of SB269970 were synthesized using design strategies aiming to improve their radiopharmacological properties. Their 5-HT₇ binding properties were investigated by cellular functional assay. The nitro-precursors of the analogs were radiolabeled by [¹⁸ F-]nucleophilic substitution, and in vitro autoradiography was performed in rat brain, followed by in vivo microPET.ResultThe chemical and radiochemical purity of the fluorine radiotracers was > 99% with specific activity in the 40–129 GBq/μmol range. The seven derivatives presented heterogeneous binding affinities toward 5-HT₇ and 5-HT_1A receptors. While [¹⁸ F]2F3P3 had promising characteristics in vitro, it showed poor brain penetration in vivo, partially reversed after pharmacological inhibition of P-glycoprotein.ConclusionsThese results indicated that, while chemical modification of these series improved several radiotracer-candidates in terms of 5-HT₇ receptor affinity and specificity toward 5-HT_1A receptors, other physicochemical modulations would be required in order to increase brain penetration.     

Application of image-derived and venous input functions in major depression using [carbonyl-11C]WAY-100635

IntroductionImage-derived input functions (IDIFs) represent a promising non-invasive alternative to arterial blood sampling for quantification in positron emission tomography (PET) studies. However, routine applications in patients and longitudinal designs are largely missing despite widespread attempts in healthy subjects. The aim of this study was to apply a previously validated approach to a clinical sample of patients with major depressive disorder (MDD) before and after electroconvulsive therapy (ECT).MethodsEleven scans from 5 patients with venous blood sampling were obtained with the radioligand [carbonyl-¹¹C]WAY-100635 at baseline, before and after 11.0 ± 1.2 ECT sessions. IDIFs were defined by two different image reconstruction algorithms 1) OSEM with subsequent partial volume correction (OSEM + PVC) and 2) reconstruction based modelling of the point spread function (TrueX). Serotonin-1A receptor (5-HT_1A) binding potentials (BP_P, BP_ND) were quantified with a two-tissue compartment (2TCM) and reference region model (MRTM2).ResultsCompared to MRTM2, good agreement in 5-HT_1A BP_ND was found when using input functions from OSEM + PVC (R² = 0.82) but not TrueX (R² = 0.57, p < 0.001), which is further reflected by lower IDIF peaks for TrueX (p < 0.001). Following ECT, decreased 5-HT_1A BP_ND and BP_P were found with the 2TCM using OSEM + PVC (23%–35%), except for one patient showing only subtle changes. In contrast, MRTM2 and IDIFs from TrueX gave unstable results for this patient, most probably due to a 2.4-fold underestimation of non-specific binding.ConclusionsUsing image-derived and venous input functions defined by OSEM with subsequent PVC we confirm previously reported decreases in 5-HT_1A binding in MDD patients after ECT. In contrast to reference region modeling, quantification with image-derived input functions showed consistent results in a clinical setting due to accurate modeling of non-specific binding with OSEM + PVC.     

[18F]Altanserin and small animal PET: Impact of multidrug efflux transporters on ligand brain uptake and subsequent quantification of 5-HT2A receptor densities in the rat brain

IntroductionThe selective 5-hydroxytryptamine type 2a receptor (5-HT_2AR) radiotracer [¹⁸F]altanserin is a promising ligand for in vivo brain imaging in rodents. However, [¹⁸F]altanserin is a substrate of P-glycoprotein (P-gp) in rats. Its applicability might therefore be constrained by both a differential expression of P-gp under pathological conditions, e.g. epilepsy, and its relatively low cerebral uptake. The aim of the present study was therefore twofold: (i) to investigate whether inhibition of multidrug transporters (MDT) is suitable to enhance the cerebral uptake of [¹⁸F]altanserin in vivo and (ii) to test different pharmacokinetic, particularly reference tissue-based models for exact quantification of 5-HT_2AR densities in the rat brain.MethodsEighteen Sprague-Dawley rats, either treated with the MDT inhibitor cyclosporine A (CsA, 50 mg/kg, n = 8) or vehicle (n = 10) underwent 180-min PET scans with arterial blood sampling. Kinetic analyses of tissue time–activity curves (TACs) were performed to validate invasive and non-invasive pharmacokinetic models.ResultsCsA application lead to a two- to threefold increase of [¹⁸F]altanserin uptake in different brain regions and showed a trend toward higher binding potentials (BP_ND) of the radioligand.ConclusionsMDT inhibition led to an increased cerebral uptake of [¹⁸F]altanserin but did not improve the reliability of BP_ND as a non-invasive estimate of 5-HT_2AR. This finding is most probable caused by the heterogeneous distribution of P-gp in the rat brain and its incomplete blockade in the reference region (cerebellum). Differential MDT expressions in experimental animal models or pathological conditions are therefore likely to influence the applicability of imaging protocols and have to be carefully evaluated.     

Time-efficient and convenient synthesis of [18F]altanserin for human PET imaging by a new work-up procedure

[¹⁸F]Altanserin, an important PET radioligand for the in vivo imaging of the 5-HT_2A receptor, was synthesized from its precursor nitro-altanserin in DMF or DMSO at high temperatures of 150 °C in an overall radiochemical yield (EOB) of 23–25% after 75 min. A new solid phase work-up procedure involving the acidification of the crude reaction mixture and a C18-SepPak-solid phase separation preceded the final HPLC purification. This led to a significantly reduced synthesis time as a result of a stable and early elution from the HPLC column using improved HPLC conditions (MeOH/THF/NaOAc 0.05 N pH 5: 27/18/55, flow: 5 mL/min, Symetry Prep 7 μm C18 (Waters)). The synthesis was performed semi-automatically in a modified GE TracerLab synthesis module using an in-house-developed program. The synthesized [¹⁸F]altanserin was used in our ongoing human and animal PET imaging studies.     

Synthesis, biodistribution and PET studies in rats of (18)F-Labeled bridgehead fluoromethyl analogues of WAY-100635

IntroductionIn vitro screening of fluoromethyl bridge-fused ring (BFR) analogues of WAY-100635 (5a, 5b and 5c) has shown a high binding affinity and a good selectivity for the 5-HT_1A receptor. As these compounds were designed to provide PET ligands with high metabolic stability, they are now radiolabeled with fluorine-18 and investigated in vivo.MethodsBFR precursors were synthesized and reacted with fluorine-18 in dry MeCN in the presence of 2,2,2-kryptofix and K₂CO₃. In rats, biodistribution and PET studies were performed using [¹⁸F]5a, [¹⁸F]5b and [¹⁸F]5c. The binding specificity was determined by administration of non-labeled WAY-100635 prior to the radiolabeled ligands.Results[¹⁸F]5 ligands were synthesized in overall radiochemical yields of 24%–45%, respectively with a radiochemical purity of > 98%. Relatively good hippocampus to cerebellum ratios of 5.55, 4.79 and 5.45, respectively were reached at 45 min pi. However, PET studies indicated defluorination of the radioligands by showing high accumulation of radioactivity in the bones in the order of [¹⁸F]5a ≈ [¹⁸F]5b > [¹⁸F]5c.ConclusionAlso in vivo, the radioligands bind preferentially to the 5-HT_1A receptor. Unfortunately, no metabolic stability with regard to defluorination was observed in rats.     

Analysis of [11C]methyl-candesartan kinetics in the rat kidney for the assessment of angiotensin II type 1 receptor density in vivo with PET

IntroductionAngiotensin II type 1 (AT₁) receptors play a key role in the regulation of renal and cardiovascular functions and have been implicated in the pathogenesis of many diseases. The aim of this study was to assess binding of the novel radioligand [¹¹C]methyl-candesartan to AT₁ receptors in the rat kidney in vivo with PET.MethodsDynamic PET images were acquired for 60 min at baseline, with coinjection of candesartan (5 mg/kg), and after injection of PD123,319 (5 mg/kg). Volumes of distribution (R_C? V_T) were estimated with a two-compartment model, by Logan analysis, and by taking the tissue-to-plasma activity ratio at 50–60 min post-injection.ResultsThe two-compartment model did not describe the kinetics at baseline adequately and the baseline scans were too short to obtain accurate estimates of R_C? V_T with the Logan approach. Based on the tissue-to-plasma ratios, roughly one-third of V_T at baseline could be attributed to AT₁ receptor binding. There were no indications of AT₂ receptor binding in the rat kidney.ConclusionIt may be possible to detect changes in AT₁ receptor density in the rat kidney in vivo with [¹¹C]methyl-candesartan and PET. Imaging AT₁ receptors with PET may provide valuable information on the role of these receptors in the pathogenesis of diseases such as hypertension, diabetic nephropathy, ventricular remodeling, and heart failure.     

Synthesis and biological evaluation of 18F-Norfallypride in the rodent brain using PET imaging

Norfallypride (N-[(2-pyrolidinyl)methyl]-2,3-dimethoxy-5-(3’-fluoropropyl)benzamide), an analog of fallypride, has been synthesized and evaluated as a potential PET imaging agent for dopamine receptors with increased subtype selectivity. In order to synthesize ¹⁸F-Norfallypride, the substituted benzamide tosylate (S)-N-[(1-BOC-2-pyrolidinyl)methyl]-2,3-dimethoxy-5-(3’-tosyloxypropyl)-benzamide) was radiolabeled with ¹⁸F using Kryptofix and K₂CO₃ in acetonitrile and deprotected with trifluoroacetic acid to yield (S)-¹⁸F-Norfallypride in approx. 10% radiochemical yields. Norfallypride exhibited an IC₅₀ of 0.63 μM for displacing ¹⁸F-fallypride in rat brain slices. In vitro rat brain autoradiographic studies revealed weak binding of ¹⁸F-norfallypride to striatal regions. PET imaging in rats showed low brain uptake of ¹⁸F-norfallypride in the rat brain. Ex vivo brain PET analysis displayed binding of ¹⁸F-norfallypride in several brain regions. With respect to the cerebellum, ex vivo PET ratios were: striatum > 3; hypothalamus > 2; hippocampus ~ 2; cerebellar nuclei > 2 while autoradiographic ratios were 14, 9, 4 and 6 respectively. ¹⁸F-Norfallypride exhibited a unique binding profile to rat brain regions known to contain significant amounts of dopamine D3 and serotonin 5HT₃ receptors. Efforts are currently under way to increase brain permeability and fully characterize the binding of ¹⁸F-norfallypride in vivo.     

Novel fluorine-18 PET radiotracers based on flumazenil for GABAA imaging in the brain

IntroductionTwo 7-fluoroimidazobenzodiazepines (AH114726 and GEH120348), analogs of flumazenil, were labeled with fluorine-18 and evaluated as alternative radioligands for in vivo imaging of the GABA_A/benzodiazepine receptor by comparing them to [¹¹C]flumazenil in rhesus monkey.MethodsRadiotracers were prepared from the corresponding nitro-precursors in an automated synthesis module, and primate imaging studies were conducted on a Concorde MicroPET P4 scanner. The brain was imaged for 60 (12 × 5 min frames) or 90 min (18 × 5 min frames), and data was reconstructed using the 3D MAP algorithm. Specificity of [¹⁸F]AH114726 and [¹⁸F]GEH120348 was confirmed by displacement studies using unlabeled flumazenil.Results[¹⁸F]GEH120348 and [¹⁸F]AH114726 were obtained in 13–24% yields (end of synthesis) with high chemical (> 95%) and radiochemical (> 99%) purities, and high specific activities (2061 ± 985 Ci/mmol). The in vivo pharmacokinetics of [¹⁸F]AH114726 and [¹⁸F]GEH120348 were determined in a non-human primate and directly compared with [¹¹C]flumazenil. Both fluorine-18 radioligands showed time-dependent regional brain distributions that correlated with the distribution of [¹¹C]flumazenil and the known concentrations of GABA_A/benzodiazepine receptors in the monkey brain. [¹⁸F]AH114726 exhibited maximal brain uptake and tissue time-radioactivity curves that were most similar to [¹¹C]flumazenil. In contrast, [¹⁸F]GEH120348 showed higher initial brain uptake but very different pharmacokinetics with continued accumulation of radioactivity into the cortical regions of high GABA/benzodiazepine receptor concentrations and very little clearance from the regions of low receptor densities. Rapid washout of both radiotracers occurred upon treatment with unlabeled flumazenil.ConclusionThe ease of the radiochemical synthesis, together with in vivo brain pharmacokinetics most similar to [¹¹C]flumazenil, support that [¹⁸F]AH114726 is a suitable option for imaging the GABA_A receptor.     

Synthesis and 18F-labeling of the analogues of Omecamtiv Mecarbil as a potential cardiac myosin imaging agent with PET

IntroductionCardiac myosin is a potential molecular target for heart failure imaging since its changes can be used to assess the function of heart. In this study, two analogues of Omecamtiv Mecarbil, which is the first selective activator of cardiac myosin, were synthesized and radio-labeled with ¹⁸F. Then the radio-compounds were evaluated as potential cardiac myosin imaging agent.MethodsThe labeling precursor and the nonradioactive compounds were synthesized and characterized by IR, ¹H NMR, ¹³C NMR and MS analysis. By substituting bromo of precursors with ¹⁸F, the radiolabeled compounds [¹⁸F]8 and [¹⁸F]10 were prepared and further evaluated for their in vitro physicochemical properties, stabilities, protein binding assay and ex vivo biodistribution.ResultsStarting with [¹⁸F]F^- Kryptofix 2.2.2./K₂CO₃ solution, the total reaction time for [¹⁸F]8 and [¹⁸F]10 was about 40 min respectively, with final high-performance liquid chromatography purification included. Typical decay-corrected radiochemical yield stayed at 12.47% ± 3.30% (n = 8), the radiochemical purity, 98% or more. Their specific activity was estimated as 50 GBq/μmol. Both [¹⁸F]8 and [¹⁸F]10 could be stable after incubation in water at room temperature and in serum or binding buffer at 37 °C for 3 h. Biodistribution in normal mice showed that both [¹⁸F]8 and [¹⁸F]10 have good heart uptake at 2 min post-injection time. Compound [¹⁸F]10 has better heart retention and higher heart to background ratios than those of [¹⁸F]8. In vitro protein binding assay demonstrates that [¹⁸F]10 may have high affinity with myosin from bovine heart.Conclusion[¹⁸F]8 and [¹⁸F]10 were synthesized with good radiochemical yield and high radiochemical purity (> 98%). One of the compounds ([¹⁸F]10) has higher bovine heart myosin binding affinity and better heart/liver ratio. It will be further evaluated as a potent cardiac myosin imaging agent in normal and systolic heart failure model with positron emission tomography in the future.     

An improved radiosynthesis of the muscarinic M2 radiopharmaceutical, [18F]FP-TZTP

The radioligand 3-(4-(3-[¹⁸F]fluoropropylthio)-1,2,5-thiadiazol-3-yl)-1-methyl-1,2,5,6-tetrahydropyridine ([¹⁸F]FP-TZTP) is an agonist with specificity towards subtype 2 of muscarinic acetylcholine (M2) receptors. It is currently the only radiotracer available for imaging M2 receptors in human subjects with positron emission tomography. The present study reports on an improved method for the synthesis of [¹⁸F]FP-TZTP, automated using a GE TRACERlab™ FX_FN radiosynthesis module. A key facet was the use of a new precursor, 3-(4-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,5-thiadiazol-3-ylthio)propyl 4-methylbenzenesulfonate. The precursor was fluorinated via nucleophilic displacement of the tosyloxy group by potassium cryptand [¹⁸F]fluoride (K[¹⁸F]/K₂₂₂) in CH₃CN at 80 °C for 5 min, and purified by HPLC. Formulated [¹⁸F]FP-TZTP was prepared in an uncorrected radiochemical yield of 29±4%, with a specific activity of 138±41 GBq/μmol (3732±1109 mCi/μmol) at the end of synthesis (35 min; n=3). This methodology offers higher yields, faster synthesis times, an optimized precursor, and simpler automation than previously reported.     

Single-step radiosynthesis and in vivo evaluation of a novel fluorine-18 labeled hippurate for use as a PET renal agent

ObjectiveThe objective of this study was to investigate a new fluorine-18 labeled hippurate, m-cyano-p-[¹⁸ F]fluorohippurate ([¹⁸ F]CNPFH), as a potential radiopharmaceutical for evaluating renal function by PET.Methods[¹⁸ F]CNPFH was synthesized by a direct one-step nucleophilic aromatic substitution using an ¹⁸ F-for-[N(CH₃)₃]⁺-reaction. In vivo stability was determined by HPLC analysis of urine collected from a healthy rat at 30 min p.i. of [¹⁸ F]CNPFH. The plasma protein binding (PPB) and erythrocyte uptake of [¹⁸ F]CNPFH were determined using blood collected from healthy rats at 5 min p.i. Biodistribution studies were conducted in healthy rats at 10 min and 1 h p.i. of [¹⁸ F]CNPFH. Dynamic PET/CT imaging data were acquired in normal rats. For comparison, the same rats underwent an identical imaging study using the previously reported p-[¹⁸ F]fluorohippurate ([¹⁸ F]PFH) renal agent.Results[¹⁸ F]CNPFH demonstrated high in vivo stability with no metabolic degradation. The in vivo PPB and erythrocyte uptake of [¹⁸ F]CNPFH were found to be comparable to those of [¹⁸ F]PFH. Biodistribution and dynamic PET/CT imaging studies revealed a rapid clearance of [¹⁸ F]CNPFH primarily through the renal–urinary pathway. However, unlike [¹⁸ F]PFH, a minor (about 12%) fraction was eliminated via the hepatobiliary route. The PET-derived [¹⁸ F]CNPFH renograms revealed an average time-to-peak (T_max) of 3.2 ± 0.4 min which was similar to [¹⁸ F]PFH, but the average time-to-half-maximal activity (11.4 ± 2.8 min) was found to be higher than that of [¹⁸ F]PFH (7.1 ± 1.3 min).ConclusionsOur in vivo results indicate that [¹⁸ F]CNPFH has renogram characteristics similar to those of [¹⁸ F]PFH, however, the unexpected hepatobiliary elimination is adding undesirable background signal in the PET images.     

Comparison of two cross-bridged macrocyclic chelators for the evaluation of 64Cu-labeled-LLP2A,a peptidomimetic ligand targeting VLA-4-positive tumors

Integrin α₄β₁ (also called very late antigen-4 or VLA-4) plays an important role in tumor growth, angiogenesis and metastasis, and there has been increasing interest in targeting this receptor for cancer imaging and therapy. In this study, we conjugated a peptidomimetic ligand known to have good binding affinity for α₄β₁ integrin to a cross-bridged macrocyclic chelator with a methane phosphonic acid pendant arm, CB-TE1A1P. CB-TE1A1P-LLP2A was labeled with ⁶⁴Cu under mild conditions in high specific activity, in contrast to conjugates based on the “gold standard” di-acid cross-bridged chelator, CB-TE2A, which require high temperatures for efficient radiolabeling. Saturation binding assays demonstrated that ⁶⁴Cu-CB-TE1A1P-LLP2A had comparable binding affinity (1.2 nM vs 1.6 nM) but more binding sites (B_max = 471 fmol/mg) in B16F10 melanoma tumor cells than ⁶⁴Cu-CB-TE2A-LLP2A (B_max = 304 fmol/mg, p < 0.03). In biodistribution studies, ⁶⁴Cu-CB-TE1A1P-LLP2A had less renal retention but higher uptake in tumor (11.4 ± 2.3 %ID/g versus 3.1 ± 0.6 %ID/g, p < 0.001) and other receptor-rich tissues compared to⁶⁴Cu-CB-TE2A-LLP2A. At 2 h post-injection, ⁶⁴Cu-CB-TE1A1P-LLP2A also had significantly higher tumor:blood and tumor:muscle ratios than ⁶⁴Cu-CB-TE2A-LLP2A (CB-TE1A1P = 19.5 ± 3.0 and 13.0 ± 1.4, respectively, CB-TE2A = 4.2 ± 1.4 and 5.5 ± 0.9, respectively, p < 0.001). These data demonstrate that ⁶⁴Cu-CB-TE1A1P-LLP2A is an excellent PET radiopharmaceutical for the imaging of α₄β₁ positive tumors and also has potential for imaging other α₄β₁ positive cells such as those of the pre-metastatic niche.     

Evaluation of metabolism,plasma protein binding and other biological parameters after administration of (−)-[18 F]Flubatine in humans

Introduction(−)-[¹⁸ F]Flubatine is a PET tracer with high affinity and selectivity for the nicotinic acetylcholine α₄β₂ receptor subtype. A clinical trial assessing the availability of this subtype of nAChRs was performed. From a total participant number of 21 Alzheimer’s disease (AD) patients and 20 healthy controls (HCs), the following parameters were determined: plasma protein binding, metabolism and activity distribution between plasma and whole blood.MethodsPlasma protein binding and fraction of unchanged parent compound were assessed by ultracentrifugation and HPLC, respectively. The distribution of radioactivity (parent compound + metabolites) between plasma and whole blood was determined ex vivo at different time-points after injection by gamma counting after separation of whole blood by centrifugation into the cellular and non-cellular components. In additional experiments in vitro, tracer distribution between these blood components was assessed for up to 90 min.ResultsA fraction of 15% ± 2% of (−)-[¹⁸ F]Flubatine was found to be bound to plasma proteins. Metabolic degradation of (−)-[¹⁸ F]Flubatine was very low, resulting in almost 90% unchanged parent compound at 90 min p.i. with no significant difference between AD and HC. The radioactivity distribution between plasma and whole blood changed in vivo only slightly over time from 0.82 ± 0.03 at 3 min p.i. to 0.87 ± 0.03 at 270 min p.i. indicating the contribution of only a small amount of metabolites. In vitro studies revealed that (−)-[¹⁸ F]Flubatine was instantaneously distributed between cellular and non-cellular blood parts.Discussion(−)-[¹⁸ F]Flubatine exhibits very favourable characteristics for a PET radiotracer such as slow metabolic degradation and moderate plasma protein binding. Equilibrium of radioactivity distribution between plasma and whole blood is reached instantaneously and remains almost constant over time allowing both convenient sample handling and facilitated fractional blood volume contribution assessment.     

Synthesis and radiopharmacological evaluation of a high-affinity and metabolically stabilized 18F-labeled bombesin analogue for molecular imaging of gastrin-releasing peptide receptor-expressing prostate cancer

IntroductionBombesin (BBN) and BBN analogues have attracted much attention as high-affinity ligands for selective targeting of the gastrin-releasing peptide (GRP) receptor. GRP receptors are overexpressed in a variety of human cancers including prostate cancer. Radiolabeled BBN derivatives are promising diagnostic probes for molecular imaging of GRP receptor-expressing prostate cancer. This study describes the synthesis and radiopharmacological evaluation of various metabolically stabilized fluorobenzoylated bombesin analogues (BBN-1, BBN-2, BBN-3).MethodsThree fluorobenzoylated BBN analogues containing an aminovaleric (BBN-1, BBN-2), or an aminooctanoic acid linker (BBN-3) were tested in a competitive binding assay against ¹²⁵I-[Tyr⁴]-BBN for their binding potency to the GRP receptor. Intracellular calcium release in human prostate cancer cells (PC3) was measured to determine agonistic or antagonistic profiles of fluorobenzoylated BBN derivatives. Bombesin derivative BBN-2 displayed the highest inhibitory potency toward GRP receptor (IC₅₀ = 8.7 ± 2.2 nM) and was subsequently selected for radiolabeling with fluorine-18 (¹⁸F) through acylation with N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). The radiopharmacological profile of ¹⁸F-labeled bombesin [¹⁸F]BBN-2 was evaluated in PC3 tumor-bearing NMRI nude mice involving metabolic stability studies, biodistribution experiments and dynamic small-animal PET studies.ResultsAll fluorobenzoylated BBN derivatives displayed high inhibitory potency toward the GRP receptor (IC₅₀ = 8.7–16.7 nM), and all compounds exhibited antagonistic profiles as determined in an intracellular calcium release assay. The ¹⁸F-labeled BBN analogue [¹⁸F]BBN-2 was obtained in 30% decay-corrected radiochemical yield with high radiochemical purity > 95% after semi-preparative HPLC purification. [¹⁸F]BBN-2 showed high metabolic stability in vivo with 65% of the radiolabeled peptide remaining intact after 60 min p.i. in mouse plasma. Biodistribution experiments and dynamic small-animal PET studies demonstrated high tumor uptake of [¹⁸F]BBN-2 in PC3 xenografts (2.75 ± 1.82 %ID/g after 5 min and 2.45 ± 1.25 %ID/g after 60 min p.i.). Specificity of radiotracer uptake in PC3 tumors was confirmed by blocking experiments.ConclusionThe present study demonstrates that ¹⁸F-labeled BBN analogue [¹⁸F]BBN-2 is a suitable PET radiotracer with favorable metabolic stability in vivo for molecular imaging of GRP receptor-positive prostate cancer.     

[18 F]-(fluoromethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)propan-2-ol ([18 F FPTC) a novel PET-ligand for cerebral beta-adrenoceptors

Cerebral β‐adrenergic receptors (β‐ARs) play important roles in normal brain and changes of β-AR expression are associated with several neuropsychiatric illnesses. Given the high density of β‐AR in several brain regions, quantification of β‐AR levels using PET is feasible. However, there is a lack of radiotracers with suitable biological properties and meeting safety requirements for use in humans. We developed a PET tracer for β‐AR by ¹⁸ F‐fluorination of 1-((9H-carbazol-4-yl)oxy)-3-4(4-((2-(2-(fluoromethoxy)-ethoxy)methyl)-1H-1,2,3-triazol-1-yl)propan-2-ol (¹⁸ F-FPTC).Methods[¹⁸ F] FPTC was synthesized by Cu(I)-catalyzed alkyne-azide cycloaddition. First, ¹⁸ F‐PEGylated alkyne was prepared by ¹⁸ F‐fluorination of the corresponding tosylate. Next ¹⁸ F‐PEGylated alkyne was reacted with an azidoalcohol derivative of 4‐hydroxycarbazol in the presence of the phosphoramidite Monophos as a ligand and Cu(I) as a catalyst. After purification with radio‐HPLC, the binding properties of [¹⁸ F FPTC were tested in β‐AR‐expressing C6‐glioma cells in vitro and in Wistar rats in vivo using microPET.ResultsThe radiochemical yield of ¹⁸ F‐PEGylated alkyne was 74%–89%. The click reaction to prepare [¹⁸ F]FPTC proceeded in 10 min with a conversion efficiency of 96%. The total synthesis time was 55 min from the end of bombardment. Specific activities were > 120 GBq/μmol. Propranolol strongly and dose-dependently inhibited the binding of both [¹²⁵I]-ICYP and [¹⁸ F]FPTC to C6 glioma cells, with IC₅₀ values in the 50–60 nM range. However, although both FPTC and propranolol inhibited cellular [¹²⁵I]ICYP binding, FPTC decreased [¹²⁵I]ICYP uptake by only 25%, whereas propranolol reduced it by 83%. [¹⁸ F]FPTC has the appropriate lipophilicity to penetrate the blood brain barrier (logP + 2.48). The brain uptake reached a maximum within 2 min after injection of 20–25 MBq [¹⁸ F]FPTC. SUV values ranged from 0.4 to 0.6 and were not reduced by propranolol. Cerebral distribution volume of the tracer (calculated from a Logan plot) was increased rather than decreased after propranolol treatment.Conclusion‘Click chemistry’ was successfully applied to the synthesis of [¹⁸ F]FPTC resulting in high radiochemical yields. [¹⁸ F]FPTC showed specific binding in vitro, but not in vivo. Based on the logP value and its ability to block [¹²⁵I]ICYP binding to C6 cells, FPTC may be a lead to suitable cerebral β-AR ligands.     

In vitro and in vivo evaluation of the effects of aluminum [18F]fluoride radiolabeling on an integrin αvβ6-specific peptide

IntroductionIncorporation of fluorine-18 (¹⁸F) into radiotracers by capturing ionic [¹⁸F]-species can greatly accelerate and simplify radiolabeling for this important positron emission tomography (PET) radioisotope. Among the different strategies, the incorporation of aluminum [¹⁸F]fluoride (Al[¹⁸F]^2 +) into NOTA chelators has recently emerged as a robust approach to peptide radiolabeling. This study presents Al[¹⁸F]^2 +-radiolabeling of an α_vβ₆ integrin-targeted peptide (NOTA-PEG₂₈-A20FMDV2) and its in vitro and in vivo evaluation.MethodsAluminum [¹⁸F]fluoride was prepared at r.t. from [¹⁸F]fluoride (40 MBq–11 GBq) and introduced into NOTA-PEG₂₈-A20FMDV2 (1) in sodium acetate (pH 4.1; 100°C, 15 min). The radiotracer Al[¹⁸F] NOTA-PEG₂₈-A20FMDV2 (2) was purified by HPLC, formulated in PBS and evaluated in vitro (stability; binding and internalization in α_vβ₆(+) and α_vβ₆(−) cells) and in vivo (paired α_vβ₆(+) and α_vβ₆(−) xenograft mice: PET/CT, biodistribution, tumor autoradiography and metabolites).ResultsThe radiotracer 2 was prepared in 90 ± 6 min (incl. formulation; n = 3) in 19.3 ± 5.4% decay corrected radiochemical yield (radiochemical purity: > 99%; specific activity: 158 ± 36 GBq/μmol) and was stable in PBS and serum (2 h). During in vitro cell binding studies, 2 showed high, α_vβ₆-targeted binding (α_vβ₆(+): 42.4 ± 1.2% of total radioactivity, ratio (+)/(−) = 8.4/1) and internalization (α_vβ₆(+): 28.3 ± 0.5% of total radioactivity, (+)/(−) = 11.7/1). In vivo, 2 maintained α_vβ₆-targeted binding (biodistribution; 1 h: α_vβ₆(+): 1.74 ± 0.38% ID/g, (+)/(−) = 2.72/1; 4 h: α_vβ₆(+): 1.21 ± 0.56% ID/g, (+)/(−) = 4.0/1; 11% intact 2 in tumor at 1 h), with highest uptake around the tumor edge (autoradiography). Most of the radioactivity cleared rapidly in the urine within one hour, but a significant fraction remained trapped in the kidneys (4 h: 229 ± 44% ID/g).ConclusionThe Al[¹⁸F]/NOTA-based radiolabeling was rapid and efficient, and the radiotracer 2 showed good α_vβ₆-selectivity in vitro and in vivo. However, in contrast to A20FMDV2 labeled with covalently bound [¹⁸F]-prosthetic groups (e.g., [¹⁸F]fluorobenzoic acid), 2 demonstrated significant trapping in kidneys, similar to radiometal-labeled chelator-analogs of 2.     

A fully automated one-pot synthesis of [carbonyl-11C]WAY-100635 for clinical PET applications

A fully automated synthesis of the important 5HT_1A receptor radioligand, [carbonyl-¹¹C]WAY-100635 (I), was developed based on the optimized one-pot “wet” synthesis procedure. A modern automated apparatus was constructed from commercially available components and operated via LabView software. In average, (906±525) MBq (n=94) of (I) was obtained from 40 min bombardment at 50 μA beam current within 50 min synthesis time. The specific radioactivity (SA) at the time of injection was (50.5±29.3) GBq/μmol (n=94).     

Effect of co-ligands on chemical and biological properties of 99mTc(III) complexes [99mTc(L)(CDO)(CDOH)2BMe] (L = Cl,F, SCN and N3; CDOH2 = cyclohexanedione dioxime)

Introduction^99mTc-Teboroxime ([^99mTcCl(CDO)(CDOH)₂BMe]) is a member of the BATO (boronic acid adducts of technetium dioximes) class of ^99mTc(III) complexes. This study sought to explore the impact of co-ligands on solution stability, heart uptake and myocardial retention of [^99mTc(L)(CDO)(CDOH)₂BMe] (^99mTc-Teboroxime: L = Cl; ^99mTc-Teboroxime(F): L = F; ^99mTc-Teboroxime(SCN): L = SCN; and ^99mTc-Teboroxime(N₃): L = N₃).MethodsRadiotracers ^99mTc-Teboroxime(L) (L = F, SCN and N₃) were prepared by reacting ^99mTc-Teboroxime with NaF, NaSCN and NaN₃, respectively. Biodistribution and imaging studies were carried out in Sprague–Dawley rats. Image quantification was performed to compare their heart retention and liver clearance kinetics.ResultsComplexes ^99mTc-Teboroxime(L) (L = F, SCN and N₃) were prepared in high yield with high radiochemical purity. All new radiotracers were stable for > 6 h in the kit matrix. In its HPLC chromatogram, ^99mTc-Teboroxime showed one peak at ~ 15.5 min, which was shorter than that of ^99mTc-Teboroxime(F) (~ 16.4 min). There were two peaks for ^99mTc-Teboroxime(SCN) at 16.5 and 18.3 min. ^99mTc-Teboroxime(N₃) appeared as a single peak at 18.4 min. Their heart retention and liver clearance curves were best fitted to the bi-exponential decay function. The half-times of fast/slow components were 1.6 ± 0.4/60.7 ± 8.9 min for ^99mTc-Teboroxime, 0.8 ± 0.2/101.7 ± 20.7 min for ^99mTc-Teboroxime(F), 1.2 ± 0.3/84.8 ± 16.6 min for ^99mTc-Teboroxime(SCN), and 2.9 ± 0.9/51.6 ± 5.0 min for ^99mTc-Teboroxime(N₃). The 2-min heart uptake followed the order of ^99mTc-Teboroxime (3.00 ± 0.37%ID/g) > ^99mTc-Teboroxime(N₃) (2.66 ± 0.01 %ID/g) ≈ ^99mTc-Sestamibi (2.55 ± 0.46 %ID/g) > ^99mTcN-MPO (2.38 ± 0.15 %ID/g). ^99mTc-Teboroxime remains the best in first-pass extraction. The best image acquisition window is 0–5 min for ^99mTc-Teboroximine and 0–15 min for ^99mTc-Teboroximine(N₃).ConclusionCo-ligands had significant impact on the heart uptake and myocardial retention of complexes [^99mTc(L)(CDO)(CDOH)₂BMe] (L = Cl, F, SCN and N₃). Future studies should be directed towards minimizing the liver uptake and radioactivity accumulation in the blood vessels while maintaining their high heart uptake.     

Evaluation of an integrin αvβ6-specific peptide labeled with [18F]fluorine by copper-free,strain-promoted click chemistry

IntroductionClick chemistry, particularly the Huisgen 1,3-dipolar cycloaddition of an alkyne with an azide, has quickly become popular for site-specific radiolabeling. Recently, strain-promoted click chemistries have been developed, eliminating the need for potentially toxic copper catalysts. This study presents radiolabeling of an α_vβ₆ integrin targeting peptide (A20FMDV2) via strain-promoted click using a fluorine-18 prosthetic group, and in vitro and in vivo evaluation.MethodsThe radiotracer [¹⁸F]FBA-C₆-ADIBON₃-PEG₇-A20FMDV2 (1) was prepared from [¹⁸F]FBA-C₆-ADIBO (2) and N₃-PEG₇-A20FMDV2 (ethanol; 10 min; 35–45 °C). HPLC-purified and formulated radiotracer 1 was evaluated in vitro by cell binding (DX3puroβ6, α_vβ₆-positive; and DX3puro, α_vβ₆-negative control) and serum stability, and in vivo using PET/CT imaging and biodistribution studies in mice.ResultsThe radiotracer 1 was readily prepared and purified (from 2: 40 ± 4 min including HPLC, 11.9 ± 3.2% decay corrected isolated radiochemical yield, > 99% radiochemical purity, n = 4) and displayed good stability (1 h: > 99%, saline; 94.6%, serum). Strong α_vβ₆-targeted binding was observed in vitro (DX3puroβ6 cells, 15 min: 43.2% binding, > 6:1 for DX3puroβ6:DX3puro). In the mouse model DX3puroβ6-tumor binding was low (1 h: 0.47 ± 0.28% ID/g, 4 h: 0.14 ± 0.09% ID/g) and clearing from the bloodstream was via the renal and hepatobiliary routes (urine: 167 ± 84% ID/g at 1 h, 10.3 ± 4.8% ID/g at 4 h; gall bladder: 95 ± 33% ID/g at 1 h, 63 ± 11% ID/g at 4 h).ConclusionCopper-free, strain-promoted click chemistry is an attractive, straightforward approach to radiolabeling. Although the [¹⁸F]FBA-C₆-ADBIO-based prosthetic group did not interfere with α_vβ₆-targeted binding in vitro, it did influence the pharmacokinetics, possibly due to its size and lipophilic nature.