Synthesis and preclinical evaluation of the radiolabeled P-glycoprotein inhibitor [11C]MC113

ObjectivesWith the aim to develop a PET tracer to visualize P-glycoprotein (Pgp) expression levels in different organs, the Pgp inhibitor MC113 was labeled with ¹¹C and evaluated using small-animal PET.Methods[¹¹C]MC113 was synthesized by reaction of O-desmethyl MC113 with [¹¹C]methyl triflate. Small-animal PET was performed with [¹¹C]MC113 in FVB wild-type and Mdr1a/b^(-/-) mice (n = 3 per group) and in a mouse model of high (EMT6Ar1.0) and low (EMT6) Pgp expressing tumor grafts (n = 5). In the tumor model, PET scans were performed before and after administration of the reference Pgp inhibitor tariquidar (15 mg/kg).ResultsBrain uptake of [¹¹C]MC113, expressed as area under the time-activity curve from time 0 to 60 min (AUC_0-60), was moderately but not significantly increased in Mdr1a/b^(-/-) compared with wild-type mice (mean ± SD AUC_0-60, Mdr1a/b^(-/-): 88 ± 7 min, wild-type: 62 ± 6 min, P = 0.100, Mann Whitney test). In the tumor model, AUC_0-60 values were not significantly different between EMT6Ar1.0 and EMT6 tumors. Neither in brain nor in tumors was activity concentration significantly changed in response to tariquidar administration. Half-maximum effect concentrations (IC₅₀) for inhibition of Pgp-mediated rhodamine 123 efflux from CCRFvcr1000 cells were 375 ± 60 nM for MC113 versus 8.5 ± 2.5 nM for tariquidar.Conclusion[¹¹C]MC113 showed higher brain uptake in mice than previously described Pgp PET tracers, suggesting that [¹¹C]MC113 was only to a low extent effluxed by Pgp. However, [¹¹C]MC113 was found unsuitable to visualize Pgp expression levels presumably due to insufficiently high Pgp binding affinity of MC113 in relation to Pgp densities in brain and tumors.     

Synthesis,metabolite analysis,and in vivo evaluation of [11C]irinotecan as a novel positron emission tomography (PET) probe

IntroductionIrinotecan is a semisynthetic derivative of camptothecin that exerts potent antitumor activity by inhibiting topoisomerase I. Despite much research into the complex pharmacokinetic profile and pharmacodynamic effects of irinotecan, unpredictable and severe side effects are still commonly observed. In this study, we synthesized [¹¹C]irinotecan as a positron emission tomography (PET) probe, performed the metabolite analysis, and evaluated the biodistribution and kinetics of [¹¹C]irinotecan using small animal PET.Methods[¹¹C]Irinotecan was synthesized by two routes using [¹¹C]phosgene and [¹¹C]carbon dioxide fixation. Metabolites in the plasma of mice following injection of [¹¹C] irinotecan were investigated using a combination of column-switching high-performance liquid chromatography (HPLC) and on-line solid-phase extraction (SPE). Whole-body PET studies were conducted in wild-type mice and P-glycoprotein and breast cancer resistance protein (Pgp/Bcrp) knockout mice.Results[¹¹C]Irinotecan was successfully synthesized by the two abovementioned routes. Decay-corrected radiochemical yields based on [¹¹C]carbon dioxide using [¹¹C]phosgene and [¹¹C]carbon dioxide fixation were 8.8 ± 2.0% (n = 8) and 16.9 ± 2.9 % (n = 5), respectively. Metabolite analysis of the plasma of mice following injection of [¹¹C]irinotecan was successfully performed using the column-switching HPLC and on-line SPE combination resulting in greater than 87 % recovery of radioactivity from HPLC. In the PET study in mice, the radioactivity levels in the brain, liver, and small intestine were slightly increased by inhibition of the Pgp/Bcrp function for more than 30 min after [¹¹C]irinotecan injection. This result demonstrated that in vivo behavior of [¹¹C] irinotecan and radioactive metabolites are influenced by the Pgp/Bcrp function.ConclusionPET studies using [¹¹C]irinotecan combined with metabolite analysis may be a useful tool for evaluating irinotecan pharmacokinetics and toxicity.     

Synthesis and preclinical evaluation of [11C-carbonyl]PF-04457845 for neuroimaging of fatty acid amide hydrolase

IntroductionFatty acid amide hydrolase (FAAH) has a significant role in regulating endocannabinoid signaling in the central nervous system. As such, FAAH inhibitors are being actively sought for pain, addiction, and other indications. This has led to the recent pursuit of positron emission tomography (PET) radiotracers targeting FAAH. We report herein the preparation and preclinical evaluation of [¹¹C-carbonyl]PF-04457845, an isotopologue of the potent irreversible FAAH inhibitor.MethodsPF-04457845 was radiolabeled at the carbonyl position via automated [¹¹C]CO₂-fixation. Ex vivo brain biodistribution of [¹¹C-carbonyl]PF-04457845 was carried out in conscious rats. Specificity was determined by pre-administration of PF-04457845 or URB597 prior to [¹¹C-carbonyl]PF-04457845. In a separate experiment, rats injected with the title radiotracer had whole brains excised, homogenized and extracted to examine irreversible binding to brain parenchyma.ResultsThe title compound was prepared in 5 ± 1% (n = 4) isolated radiochemical yield based on starting [¹¹C]CO₂ (decay uncorrected) within 25 min from end-of-bombardment in > 98% radiochemical purity and a specific activity of 73.5 ± 8.2 GBq/μmol at end-of-synthesis. Uptake of [¹¹C-carbonyl]PF-04457845 into the rat brain was high (range of 1.2–4.4 SUV), heterogeneous, and in accordance with reported FAAH distribution. Saturable binding was demonstrated by a dose-dependent reduction in brain radioactivity uptake following pre-treatment with PF-04457845. Pre-treatment with the prototypical FAAH inhibitor, URB597, reduced the brain radiotracer uptake in all regions by 71–81%, demonstrating specificity for FAAH. The binding of [¹¹C-carbonyl]PF-04457845 to FAAH at 40 min post injection was irreversible as 98% of the radioactivity in the brain could not be extracted.Conclusions[¹¹C-carbonyl]PF-04457845 was rapidly synthesized via an automated radiosynthesis. Ex vivo biodistribution studies in conscious rodents demonstrate that [11C PF-04457845 is a promising candidate radiotracer for imaging FAAH in the brain with PET. These results coupled with the known pharmacology and toxicology of PF-04457845 should facilitate clinical translation of this radiotracer.     

Radiation dosimetry and biodistribution of the translocator protein radiotracer [11C]DAA1106 determined with PET/CT in healthy human volunteers

IntroductionWhen microglia become activated (an integral part of neuroinflammation), cellular morphology changes and expression of translocator protein (TSPO) 18 kDa is increased. Over the past several years, [¹¹C]DAA1106 has emerged as a reliable radiotracer for labeling TSPO with high affinity during positron emission tomography (PET) scanning. While [¹¹C]DAA1106 PET scanning has been used in several research studies, a radiation dosimetry study of this radiotracer in humans has not yet been published.MethodsTwelve healthy participants underwent full body dynamic [¹¹C]DAA1106 PET scanning, with 8 sequential whole body scans (approximately 12 bed positions each), following a single injection. Regions of interest were drawn manually, and time activity curves (TACs) were obtained for 15 organs. OLINDA/EXM 1.1 was used to compute radiation absorbed doses to the target organs, as well as effective dose (ED) and effective dose equivalent (EDE).ResultsThe ED and EDE were 4.06 ± 0.58 μSv/MBq and 5.89 ± 0.83 μSv/MBq, respectively. The highest absorbed doses were to the heart wall, kidney, liver, pancreas, and spleen. TACs revealed that peak dose rates are during the first scan (at 6 min) for all organs other than the urinary bladder wall, which had its peak dose rate during the fourth scan (at 30 min).ConclusionsThe recently developed radiotracer [¹¹C]DAA1106 has its EDE and target-organ absorbed dose such that, for a single administration, its radiation dosimetry is well within the U.S. FDA guidelines for basic research studies in adults. This dose level implies that the dosimetry for multiple [¹¹C]DAA1106 scans within a given year also falls within FDA guidelines, and this favorable property makes this radiotracer suitable for examining microglial activation repeatedly over time, which may in the future be useful for longitudinal tracking of disease progression and monitoring of therapy response in conditions marked by neuroinflammation (e.g., head trauma and multiple sclerosis).     

Radiosynthesis and in vivo evaluation of fluorinated huprine derivates as PET radiotracers of acetylcholinesterase

IntroductionDeveloping positron emission tomography (PET) radiotracers for non-invasive study of the cholinergic system is crucial to the understanding of neurodegenerative diseases. Although several acetylcholinesterase (AChE) PET tracers radiolabeled with carbon-11 exist, no fluorinated radiotracer is currently used in clinical imaging studies. The purpose of the present study is to describe the first fluorinated PET radiotracer for this brain enzyme.MethodsThree structural analogs of huprine, a specific AChE inhibitor presenting high affinity towards AChE in vitro, were synthesized and labeled with fluorine-18 via a mesylate/fluoro-nucleophilic aliphatic substitution: ([¹⁸ F]-FHUa, [¹⁸ F]-FHUb and [¹⁸ F]-FHUc). Initial biological evaluation included in vitro autoradiography in rat with competition with an AChE inhibitor at different concentrations, and microPET-scan on anesthetized rats. In vivo PET studies in anesthetized cat focused on [¹⁸ F]-FHUa.Results and ConclusionsAlthough radiosynthesis of these huprine analogs was straightforward, they showed poor brain penetration potential, partially reversed after pharmacological inhibition of P-glycoprotein. These results indicated that current huprine analogs are not suitable for PET mapping of brain AChE receptors, but require physicochemical modulation in order to increase brain penetration.     

(R)-[11C]verapamil is selectively transported by murine and human P-glycoprotein at the blood–brain barrier,and not by MRP1 and BCRP

IntroductionPositron emission tomography (PET) with [¹¹C]verapamil, either in racemic form or in form of the (R)-enantiomer, has been used to measure the functional activity of the adenosine triphosphate-binding cassette (ABC) transporter P-glycoprotein (Pgp) at the blood–brain barrier (BBB). There is some evidence in literature that verapamil inhibits two other ABC transporters expressed at the BBB, i.e. multidrug resistance protein 1 (MRP1) and breast cancer resistance protein (BCRP). However, previous data were obtained with micromolar concentrations of verapamil and do not necessarily reflect the transporter selectivity of verapamil at nanomolar concentrations, which are relevant for PET experiments. The aim of this study was to assess the selectivity of verapamil, in nanomolar concentrations, for Pgp over MRP1 and BCRP.MethodsConcentration equilibrium transport assays were performed with [³H]verapamil (5 nM) in cell lines expressing murine or human Pgp, human MRP1, and murine Bcrp1 or human BCRP. Paired PET scans were performed with (R)-[¹¹C]verapamil in female FVB/N (wild-type), Mrp1^(?/?), Mdr1a/b^(?/?), Bcrp1^(?/?) and Mdr1a/b^(?/?)Bcrp1^(?/?) mice, before and after Pgp inhibition with 15 mg/kg tariquidar.ResultsIn vitro transport experiments exclusively showed directed transport of [³H]verapamil in Mdr1a- and MDR1-overexpressing cells which could be inhibited by tariquidar (0.5 μM). In PET scans acquired before tariquidar administration, brain-to-blood ratio (K_b,brain) of (R)-[¹¹C]verapamil was low in wild-type (1.3 ± 0.1), Mrp1^(?/?) (1.4 ± 0.1) and Bcrp1^(?/?) mice (1.8 ± 0.1) and high in Mdr1a/b^(?/?) (6.9 ± 0.8) and Mdr1a/b^(?/?)Bcrp1^(?/?) mice (7.9 ± 0.5). In PET scans after tariquidar administration, K_b,brain was significantly increased in Pgp-expressing mice (wild-type: 5.0 ± 0.3-fold, Mrp1^(?/?): 3.2 ± 0.6-fold, Bcrp1^(?/?): 4.3 ± 0.1-fold) but not in Pgp knockout mice (Mdr1a/b^(?/?) and Mdr1a/b^(?/?)Bcrp1^(?/?)).ConclusionOur combined in vitro and in vivo data demonstrate that verapamil, in nanomolar concentrations, is selectively transported by Pgp and not by MRP1 and BCRP at the BBB, which supports the use of (R)-[¹¹C]verapamil or racemic [¹¹C]verapamil as PET tracers of cerebral Pgp function.     

[11C]-Acetoacetate PET imaging: a potential early marker for cardiac heart failure

The ketone body acetoacetate could be used as an alternate nutrient for the heart, and it also has the potential to improve cardiac function in an ischemic–reperfusion model or reduce the mitochondrial production of oxidative stress involved in cardiotoxicity. In this study, [¹¹C]-acetoacetate was investigated as an early marker of intracellular damage in heart failure.MethodsA rat cardiotoxicity heart failure model was induced by doxorubicin, Dox(+). [¹⁴C]-Acetoacetate, a non-positron (β −) emitting radiotracer, was used to characterize the arterial blood input function and myocardial mitochondrial uptake. Afterward, [¹¹C]-acetoacetate (β +) myocardial PET images were obtained for kinetic analysis and heart function assessment in control Dox(−) (n = 15) and treated Dox(+) (n = 6) rats. The uptake rate (K₁) and myocardial clearance rate (k₂ or k_mono) were extracted.Results[¹⁴C]-Acetoacetate in the blood was increased in Dox(+), from 2 min post-injection until the last withdrawal point when the heart was harvested, as well as the uptake in the heart and myocardial mitochondria (unpaired t-test, p < 0.05). PET kinetic analysis of [¹¹C]-acetoacetate showed that rate constants K₁, k₂ and k_mono were decreased in Dox(+) (p < 0.05) combined with a reduction of 24% of the left ventricular ejection fraction (p < 0.001).ConclusionRadioactive acetoacetate ex vivo analysis [¹⁴C], and in vivo kinetic [¹¹C] studies provided evidence that [¹¹C]-acetoacetate can assess heart failure Dox(+). Contrary to myocardial flow reserve (rest–stress protocol), [¹¹C]-acetoacetate can be used to assess reduced kinetic rate constants without requirement of hyperemic stress response. The proposed [¹¹C]-acetoacetate cardiac radiotracer in the investigation of heart disease is novel and paves the way to a potential role for [¹¹C]-acetoacetate in cardiac pathophysiology.     

Equivalence of arterial and venous blood for [11C]CO2-metabolite analysis following intravenous administration of 1-[11C]acetate and 1-[11C]palmitate

PurposeSampling of arterial blood for metabolite correction is often required to define a true radiotracer input function in quantitative modeling of PET data. However, arterial puncture for blood sampling is often undesirable. To establish whether venous blood could substitute for arterial blood in metabolite analysis for quantitative PET studies with 1-[¹¹C]acetate and 1-[¹¹C]palmitate, we compared the results of [¹¹C]CO₂-metabolite analyses performed on simultaneously collected arterial and venous blood samples.MethodsPaired arterial and venous blood samples were drawn from anesthetized pigs at 1, 3, 6, 8, 10, 15, 20, 25 and 30 min after i.v. administration of 1-[¹¹C]acetate and 1-[¹¹C]palmitate. Blood radioactivity present as [¹¹C]CO₂ was determined employing a validated 10-min gas-purge method. Briefly, total blood ¹¹C radioactivity was counted in base-treated [¹¹C]-blood samples, and non-[¹¹C]CO₂ radioactivity was counted after the [¹¹C]-blood was acidified using 6 N HCl and bubbled with air for 10 min to quantitatively remove [¹¹C]CO₂.ResultsAn excellent correlation was found between concurrent arterial and venous [¹¹C]CO₂ levels. For the [¹¹C]acetate study, the regression equation derived to estimate the venous [¹¹C]CO₂ from the arterial values was: y = 0.994x + 0.004 (r² = 0.97), and for the [¹¹C]palmitate: y = 0.964x ? 0.001 (r² = 0.9). Over the 1–30 min period, the fraction of total blood ¹¹C present as [¹¹C]CO₂ rose from 4% to 64% for acetate, and 0% to 24% for palmitate. The rate of [¹¹C]CO₂ appearance in venous blood appears similar for the pig model and humans following i.v. [¹¹C]-acetate administration.ConclusionVenous blood [¹¹C]CO₂ values appear suitable as substitutes for arterial blood samples in [¹¹C]CO₂ metabolite analysis after administration of [¹¹C]acetate or [¹¹C]palmitateAdvances in Knowledge and Implications for Patient CareQuantitative PET studies employing 1-[¹¹C]acetate and 1-[¹¹C]palmitate can employ venous blood samples for metabolite correction of an image-derived tracer arterial input function, thereby avoiding the risks of direct arterial blood sampling.     

Radiosynthesis and ex vivo evaluation of [11C-carbonyl]carbamate- and urea-based monoacylglycerol lipase inhibitors

IntroductionMonoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) are the two primary enzymes that regulate the tone of endocannabinoid signaling. Although new PET radiotracers have been discovered for imaging FAAH in vivo, no such radiotracer exists for imaging MAGL. Here we report the radiosynthesis of five candidate MAGL radiotracers and their ex vivo evaluations in mice and rats.MethodsCandidate carbamate and urea MAGL inhibitors were radiolabeled at the carbonyl position by [¹¹C]CO₂ fixation. Radiotracers were administered (tail-vein injection) to rodents and brain uptake of radioactivity measured at early and late time points ex vivo. Specificity of uptake was explored by pretreatment with unlabeled inhibitors (2 mg/kg, ip) 30 min prior to radiotracer administration.ResultsAll five candidate MAGL radiotracers were prepared in high specific activity (> 65 GBq/μmol) and radiochemical purity (> 98%). Moderate brain uptake (0.2–0.8 SUV) was observed for each candidate while pretreatment did not reduce uptake for four of the five tested. For two candidates ([¹¹C]12 and [¹¹C]14), high retention of radioactivity was observed in the blood (ca. 10 and 4 SUV at 40 min) which was blocked by pretreatment with unlabeled inhibitors. The most promising candidate, [¹¹C]18, demonstrated moderate brain uptake (ca. 0.8 SUV) which showed circa 50% blockade by pretreatment with unlabeled 18.ConclusionOne putative and four reported potent and selective MAGL inhibitors have been radiolabeled via [¹¹C]CO₂ fixation as radiotracers for this enzyme. Despite the promising in vitro pharmacological profile, none of the five candidate radiotracers exhibited in vivo behavior suitable for PET neuroimaging.     

Synthesis and evaluation of 2-amino-5-(4-[18F]fluorophenyl)pent-4-ynoic acid ([18F]FPhPA): A novel 18F-labeled amino acid for oncologic PET imaging

Introduction¹⁸ F-labeled amino acids are important PET radiotracers for molecular imaging of cancer. This study describes synthesis and radiopharmacological evaluation of 2-amino-5-(4-[¹⁸ F]fluorophenyl)pent-4-ynoic acid ([¹⁸ F]FPhPA) as a novel amino acid radiotracer for oncologic imaging.Methods¹⁸ F]FPhPA was prepared using Pd-mediated Sonogashira cross-coupling reaction between 4-[¹⁸ F]fluoroiodobenzene ([¹⁸ F]FIB) and propargylglycine. The radiopharmacological profile of [¹⁸ F]FPhPA was evaluated in comparison with O-(2-[¹⁸ F]fluoroethyl)-L-tyrosine ([¹⁸ F]FET) using the murine breast cancer cell line EMT6 involving cellular uptake studies, radiotracer uptake competitive inhibition experiments and small animal PET imaging.Results¹⁸ F]FPhPA was prepared in 42 ± 10% decay-corrected radiochemical yield with high radiochemical purity >95% after semi-preparative HPLC purification. Cellular uptake of L-[¹⁸ F]FPhPA reached a maximum of 58 ± 14 % radioactivity/mg protein at 90 min. Lower uptake was observed for racemic and D-[¹⁸ F]FPhPA.Radiotracer uptake inhibition studies by synthetic and naturally occurring amino acids suggested that Na⁺-dependent system ASC, especially ASCT2, and Na⁺-independent system L are important amino acid transporters for [¹⁸ F]FPhPA uptake into EMT6 cells. Small animal PET studies demonstrated similar high tumor uptake of [¹⁸ F]FPhPA in EMT6 tumor-bearing mice compared to [¹⁸ F]FET reaching a maximum standardized uptake value (SUV) of 1.35 after 60 min p.i.. Muscle uptake of [¹⁸ F]FPhPA was higher (SUV_30min = 0.65) compared to [¹⁸ F]FET (SUV_30min = 0.40), whereas [¹⁸ F]FPhPA showed a more rapid uptake and clearance from the brain compared to [¹⁸ F]FET.ConclusionL-[¹⁸ F]FPhPA is the first ¹⁸ F-labeled amino acid prepared through Pd-mediated cross-coupling reaction.Advances in Knowledge and Implications for patient CareL-[¹⁸ F]FPhPA displayed promising properties as a novel amino acid radiotracer for molecular imaging of system ASC and system L amino acid transporters in cancer.     

Synthesis and evaluation of new radioligands [11C]A-833834 and [11C]A-752274 for positron-emission tomography of α7-nicotinic acetylcholine receptors

Introductionα7-nicotinic acetylcholine receptor (α7-nAChR) is one of the major neuronal nAChR subtypes. α7-nAChR is involved in variety of neuronal processes and disorders including schizophrenia and Alzheimer's disease. A number of α7-nAChR PET radioligands have been developed, but a quality radiotracer remains to be discovered.MethodsHigh binding affinity α7-nAChR ligands A-833834 and A-752274 were radiolabeled with ¹¹C. Baseline and blockade biodistribution studies in the mouse brain of [¹¹C]A-833834 (5-(6-(5-[¹¹C]methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)pyridazin-3-yl)-1H-indole) and [¹¹C]A-752274 (2-(6-[¹¹C]methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)-7-(6-methyl-3,6-diazabicyclo[3.2.0]heptan-3-yl)-9H-fluoren-9-one) were performed. [¹¹C]A-752274 was evaluated in a baseline baboon PET study.Results[¹¹C]A-833834 and [¹¹C]A-752274 were synthesized by radiomethylation of corresponding des-methyl precursors. The radioligands were prepared with radiochemical yield of 12%–32%, high specific radioactivity (330–403 GBq/μmol) and radiochemical purity > 95%. Dissection studies with [¹¹C]A-833834 demonstrated low specific α7-nAChR binding in the mouse brain. [¹¹C]A-752274 specifically (~ 50%) labeled α7-nAChR in the mouse thalamus. However, [¹¹CA-752274 exhibited low brain uptake in baboon (%SUV < 100).ConclusionTwo novel α7-nAChR ligands radioligands were synthesized and studied in animals. Specific binding of [¹¹C]A-833834 in the mouse brain is low due to the insufficient binding affinity of the radioligand. The very high binding affinity [¹¹C]A-752274 exhibited good specific binding in the α7-nAChR-rich mouse brain regions. The low uptake of [¹¹C]A-752274 in the baboon brain is due to its high hydrophilicity, rapid metabolism or other properties. Future development of α7-nAChR PET radioligands will be based on compounds with high binding affinities and good blood–brain barrier permeability.     

Assessment of cerebral P-glycoprotein expression and function with PET by combined [11C]inhibitor and [11C]substrate scans in rats

IntroductionThe adenosine triphosphate-binding cassette (ABC) transporter P-glycoprotein (Pgp) protects the brain from accumulation of lipophilic compounds by active efflux transport across the blood–brain barrier. Changes in Pgp function/expression may occur in neurological disorders, such as epilepsy, Alzheimer’s or Parkinson’s disease. In this work we investigated the suitability of the radiolabeled Pgp inhibitors [¹¹C]elacridar and [¹¹C]tariquidar to visualize Pgp density in rat brain with PET.MethodsRats underwent a first PET scan with [¹¹C]elacridar (n = 5) or [¹¹C]tariquidar (n = 6) followed by a second scan with the Pgp substrate (R)-[¹¹C]verapamil after administration of unlabeled tariquidar at a dose which half-maximally inhibits cerebral Pgp (3 mg/kg). Compartmental modeling using an arterial input function and Logan graphical analysis were used to estimate rate constants and volumes of distribution (V_T) of radiotracers in different brain regions.ResultsBrain PET signals of [¹¹C]elacridar and [¹¹C]tariquidar were very low (~ 0.5 standardized uptake value, SUV). There was a significant negative correlation between V_T and K₁ (i.e. influx rate constant from plasma into brain) values of [¹¹C]elacridar or [¹¹C]tariquidar and V_T and K₁ values of (R)-[¹¹C]verapamil in different brain regions which was consistent with binding of [¹¹C]inhibitors to Pgp and efflux of (R)-[¹¹C]verapamil by Pgp.ConclusionThe small Pgp binding signals obtained with [¹¹C]elacridar and [¹¹C]tariquidar limit the applicability of these tracers to measure cerebral Pgp density. PET tracers with higher (i.e. subnanomolar) binding affinities will be needed to visualize the low density of Pgp in brain.     

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.     

[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.     

Synthesis and evaluation of 11C-labeled naphthalene derivative as a novel non-peptidergic probe for the β-secretase (BACE1) imaging in Alzheimer's disease brain

IntroductionAs a first trial for in vivo imaging of β-secretase (BACE1) in Alzheimer's disease brain, we applied a novel non-peptidergic small molecule which has high affinity to the enzyme, naphthalene-1-carboxylic acid (3′-chloro-4′-fluoro-4-piperazin-1-yl-biphenyl-3-yl)amide (NCFB) into positron emission tomography (PET) probe. In the current study, N-¹¹C-methylated compound of NCFB, [¹¹C]Me-NCFB was synthesized and evaluated for the visualization of BACE1 in brain.MethodsBACE1 inhibitory constant was measured by FRET assay. [¹¹C]Me-NCFB was synthesized from NCFB with [¹¹C]methyl triflate. To evaluate properties of [¹¹C]Me-NCFB, log P value, stability in mouse plasma and brain uptake index were measured. The biodistribution in 6-week-old ddY mice was also studied.ResultsBACE1 inhibitory constant showed an affinity of Me-NCFB to the enzyme (IC₅₀ = 2.3 ± 0.80 μM). [¹¹C]Me-NCFB was synthesized in a 3.0% ± 0.55% decay-corrected radiochemical yield. [¹¹C]Me-NCFB showed high lipophilicity, high stability in mouse plasma and blood–brain barrier (BBB) permeability. Injected to 6-week-old ddY mice, [¹¹C]Me-NCFB penetrated BBB and was retained in the brain (0.79% ± 0.22% ID/g at 2 min and 0.75% ± 0.08% ID/g at 60 min after injection, respectively), moreover, rapid blood clearance was observed.Conclusion[¹¹C]Me-NCFB could have a potential as a PET probe for the imaging of BACE1 in the brain.     

Detection of activated platelets in a mouse model of carotid artery thrombosis with 18F-labeled single-chain antibodies

IntroductionActivated platelets are key players in thrombosis and inflammation. We previously generated single-chain antibodies (scFv) against ligand-induced binding sites (LIBS) on the highly abundant platelet glycoprotein integrin receptor IIb/IIIa. The aim of this study was the construction and characterisation of a novel ¹⁸F PET radiotracer based on this antibody.MethodsScFv_anti-LIBS and control antibody mut-scFv were reacted with N-succinimidyl-4-[¹⁸F]fluorobenzoate (S[¹⁸F]FB). Radiolabeled scFv was incubated with in vitro formed platelet clots and injected into mice with FeCl₃ induced thrombus in the left carotid artery. Clots were imaged in the PET scanner and amount of radioactivity measured using an ionization chamber and image analysis. Assessment of vessel injury as well as the biodistribution of the radiolabeled scFv was studied.ResultsAfter incubation with increasing concentrations of ¹⁸F-scFv_anti-LIBS clots had retained significantly higher amounts of radioactivity compared to clots incubated with radiolabeled ¹⁸F-mut-scFv (13.3 ± 3.8 vs. 3.6 ± 1 KBq, p < 0.05, n = 9, decay corrected). In the in vivo experiments we found an high uptake of the tracer in the injured vessel compared with the non-injured vessel, with 12.6 ± 4.7% injected dose per gram (ID/g) uptake in the injured vessel and 3.7 ± 0.9% ID/g in the non-injured vessel 5 minutes after injection (p < 0.05, n = 6).ConclusionsOur results show that the novel antibody radiotracer ¹⁸F-scFv_anti-LIBS is useful for the sensitive detection of activated platelets and thrombosis.Advances in knowledge and implications for patient careWe describe the first ¹⁸F variant of a scFv_anti-LIBS against activated platelets. This diagnostic agent could provide a powerful tool for the assessment of acute thrombosis and inflammation in patients in the future.     

Synthesis and evaluation of [11C]PyrATP-1, a novel radiotracer for PET imaging of glycogen synthase kinase-3β (GSK-3β)

IntroductionThe dysfunction of glycogen synthase kinase-3β (GSK-3β) has been implicated in a number of diseases, including Alzheimer’s disease. The ability to non-invasively quantify GSK-3β activity in vivo is therefore of critical importance, and this work is focused upon development of inhibitors of GSK-3β radiolabeled with carbon-11 to examine quantification of the enzyme using positron emission tomography (PET) imaging.Methods¹¹C PyrATP-1 was prepared from the corresponding desmethyl-piperazine precursor in an automated synthesis module. In vivo rodent and primate imaging studies were conducted on a Concorde MicroPET P4 scanner to evaluate imaging properties and in vitro autoradiography studies with rat brain samples were carried out to examine specific binding.Results2035 ± 518 MBq (55 ± 14 mCi) of [¹¹C]PyrATP-1 was obtained (1%–2% non-corrected radiochemical yield at end-of-synthesis based upon [¹¹C]CO₂) with high chemical (> 95%) and radiochemical (> 99%) purities, and good specific activities (143 ± 52 GBq/μmol (3874 ± 1424 Ci/mmol)), n = 5. In vivo microPET imaging studies revealed poor brain uptake in rodents and non-human primates. Pretreatment of rodents with cyclosporin A resulted in moderately increased brain uptake suggesting Pgp transporter involvement. Autoradiography demonstrated high levels of specific binding in areas of the rodent brain known to be rich in GSK-3β.Conclusion¹¹C PyrATP-1 is readily synthesized using standard carbon-11 radiochemistry. However the poor brain uptake in rodents and non-human primates indicates that the radiotracer is not suitable for the purposes of quantifying GSK-3β in neurological and psychiatric disorders.     

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.     

Longitudinal observation of [11C]4DST uptake in turpentine-induced inflammatory tissue

IntroductionLongitudinal changes of 4′-[methyl-¹¹C]thiothymidine ([¹¹C]4DST) uptake were evaluated in turpentine-induced inflammation.MethodsTurpentine (0.1 ml) was injected intramuscularly into the right hind leg of male Wistar rats. Longitudinal [¹¹C]4DST uptake was evaluated by the tissue dissection method at 1, 2, 4, 7, and 14 days after turpentine injection (n = 5). The tumor selectivity index was calculated using the previously published biodistribution data in C6 glioma-bearing rats. Dynamic PET scan was performed on day 4 when maximum [¹¹C]4DST uptake was observed during the longitudinal study. Histopathological analysis and Ki-67 immunostaining were also performed.ResultsThe uptake of [¹¹C]4DST in inflammatory tissue was significantly increased on days 2–4 after turpentine injection, and then decreased. On day 14, tracer uptake returned to the day 1 level. The maximum SUV of inflamed muscle was 0.6 and was 3 times higher than that of the contralateral healthy muscle on days 2–4 after turpentine injection. However, tumor selectivity index remains very high (> 10) because of the low inflammation uptake. A dynamic PET scan showed that the radioactivity in inflammatory tissues peaked at 5 min after [¹¹C]4DST injection, and then washed out until 20 min. At intervals > 20 min, radioactivity levels were constant and double that of healthy muscle. The changes in Ki-67 index were paralleled with those of [¹¹C]4DST uptake, indicating cell proliferation-dependent uptake of [¹¹C]4DST in inflammatory tissues.ConclusionIn our animal model, low but significant levels of [¹¹C]4DST uptake were observed in subacute inflammation.     

Radiosynthesis and radiopharmacological evaluation of [N-methyl-11C]Org 34850 as a glucocorticoid receptor (GR)-binding radiotracer

The radiosynthesis of [N-methyl-¹¹C]Org 34850 as a potential brain glucocorticoid receptor (GR)-binding radiotracer is described. The radiosynthesis was accomplished via N-methylation of the corresponding desmethyl precursor with [¹¹C]methyl triflate in a remotely controlled synthesis module to give the desired compound in a radiochemical yield of 23±5% (decay-corrected, based upon [¹¹C]CO₂) at a specific activity of 47±12 GBq/μmol (n=15) at the end-of-synthesis (EOS). The radiochemical purity after semi-preparative HPLC purification exceeded 95%. The total synthesis time was 35–40 min after end-of-bombardment (EOB).The radiotracer is rapidly metabolized in rat plasma leading to the formation of two more hydrophilic metabolites as the major metabolites. Radiopharmacological evaluation involving biodistribution and small animal PET imaging in normal Wistar rats showed that the compound [N-methyl-¹¹C]Org 34850 is not able to sufficiently penetrate the blood–brain barrier. Therefore, compound [N-methyl-¹¹C]Org 34850 seems not to be a suitable PET radiotracer for imaging rat brain GRs. However, involvement of Pgp or species differences requires further clarification to establish whether the radiotracer [N-methyl-¹¹C]Org 34850 may still represent a suitable candidate for imaging GRs in humans.