Search

Introduction (−)-[ 18 F]Flubatine is a PET tracer with high affinity and selectivity for the nicotinic acetylcholine α 4 β 2 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. Methods Plasma 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 90min. Results A 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 90min 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 3min p.i. to 0.87±0.03 at 270min 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.

The influence of different additives (PEG 300, PEG 400, PG) to the product solution of [11C]PiB was investigated with regard to tracer retention for a number of commonly used sterile filters for aseptic manufacturing of PET-tracers. The effect of the amount of additive with regard to tracer retention and the resulting viscosity of the filtration solution was determined. Recommendations for the individual combinations of filters and amounts of additives suitable for the different filtration methods that are implemented in commercially available synthesis modules are given as well.

[18F]Florbetaben ([18F]BAY 94-9172) is a promising β-amyloid (Aβ) targeted PET-tracer currently in late stage clinical development. [18F]Florbetaben can assist in the more accurate diagnosis of Alzheimer’s Disease (AD) by non-invasive, in vivo detection of Aβ in the brain. To determine the arterial input function of the PET tracer—as part of a proof of mechanism (PoM) study—arterial samples were drawn from all subjects at predefined time points post injection (p.i.), and the proportion of unchanged tracer [18F]Florbetaben was determined by HPLC analysis. Plasma metabolite profiles were investigated following intravenous administration of 300 MBq (±60 MBq) of [18F]Florbetaben to both, patients with AD and healthy controls (HCs), and various methods for processing the blood samples were evaluated. Addition of acetonitrile to plasma samples (obtained from whole blood by centrifugation) and precipitation of proteins resulted in a recovery of more than 90% of the initial radioactivity in the supernatants. High Performance Liquid Chromatography using a polymer-based column (PRP-1) in conjunction with gradient elution was found to be a suitable method of metabolite analysis of [18F]Florbetaben. HPLC analyses indicated that [18F]Florbetaben is rapidly metabolized in vivo with an estimated initial half-life of about 6 min. A polar metabolite fraction, consisting presumably of more than one component, and (to a smaller extent) of the demethylated derivative of [18F]Florbetaben were time-dependently detected in plasma.

Quantum dots functionalized on the outer surface with either amino- or carboxyl functions were labelled with [18F]fluoroethyltosylate and [11C]methyliodide in order to use the positron emitter-labelled fluorescence agents for multimodality imaging techniques, i.e. fluorescence imaging and positron emission tomography. 18F-Labelling of both compounds was realized with yields up to 5% as determined by size exclusion chromatography, which is twice as much as reported in literature before [1]. 11C-Labelling of amino- and carboxyl-QDs proceeded with good yields (up to 45 and 35%, respectively) under optimized reaction conditions. In general for both QD-types and both labelling agents the labelling yield increased with the amount of QDs used in the reaction as well as with reaction time and reaction temperature.

Functional changes of nicotinic acetylcholine receptors (nAChR) are important during age-related neuronal degeneration. Recent studies demonstrate the applicability of the nAChR ligand 2-[(18)F]F-A-85380 for neuroimaging of patients with dementias. However, its binding kinetics demands a 7-h acquisition time limiting its practicality for clinical PET studies. Thus, the authors developed [(18)F]norchloro-fluoro-homoepibatidine ([(18)F]NCFHEB) for nAChR imaging. The kinetics of the two enantiomers of [(18)F]NCFHEB were compared with 2-[(18)F]F-A85380 in porcine brain to evaluate their potential for human neuroimaging. Twenty-four juvenile female pigs were studied with PET using [(18)F]NCFHEB. Nine animals received an additional i.v. injection (1 mg/kg) of the nAChR agonist A81418 before radiotracer administration followed by infusion (2 mg/kg/7h) thereafter. Several compartment models were applied for quantification. (-)- and (+)-[(18)F]NCFHEB showed a twofold to threefold higher brain uptake than 2-[(18)F]F-A-85380. All three radiotracers displayed spatially heterogeneous binding kinetics in regions with high, moderate, or low specific binding. The equilibrium of specific binding of (-)-[(18)F]NCFHEB was reached earlier than that of (+)-[(18)F]NCFHEB or 2-[(18)F]F-A85380. Continuous administration of the nAChR agonist A81418 inhibited the specific binding of (-)- and (+)-[(18)F]NCFHEB but not of 2-[(18)F]F-A85380. The peripheral metabolism of (+)-[(18)F]NCFHEB proceeded somewhat slower than that of the other radiotracers. Both enantiomers of [(18)F]NCFHEB are appropriate radiotracers for neuroimaging of nAChR in pigs. Their binding profile in vivo appears to be more selective than that of 2-[(18)F]F-A85380. (-)-[(18)F]NCFHEB offers a faster equilibrium of specific binding than 2-[(18)F]F-A85380.

Introduction To determine the availability of nicotinic acetylcholine receptors in different human brain regions using the positron emission tomography (PET) radioligand 2-[ 18 F]fluoro-A-85380 (2-[ 18 F]FA) and invasive approaches for quantification, it is important to correct the arterial input function as well for plasma protein binding (PPB) of the radioligand as for radiolabeled metabolites accumulating in blood. This study deals with some aspects of PPB of 2-[ 18 F]FA. Methods Patients with different neurological disorders ( n =72), such as Parkinson's disease, Alzheimer's disease and multiple sclerosis, and a group of healthy volunteers ( n =15) subjected for PET imaging were analyzed for their PPB level of 2-[ 18 F]FA using ultrafiltration. Protein gel electrophoresis of plasma samples was performed to identify the binding protein of 2-[ 18 F]FA. The dependency of PPB on time and on free ligand concentration was analyzed to obtain the binding parameters B max and K d . Results Albumin was identified to be the binding protein of 2-[ 18 F]FA. PPB of 2-[ 18 F]FA was low at 17±4% and did not show significant differences between the groups of patients. Corresponding to this, a narrow range of plasma albumin of 0.62±0.05 mM was observed. B max was determined as twice the albumin concentration, which indicates two binding sites for 2-[ 18 F]FA on the protein. No time dependence of the PPB could be observed. By relating PPB to B max , an average K d value of 6.0±1.5 mM was obtained. Conclusion This study shows the dependency of PPB of 2-[ 18 F]FA on human albumin plasma concentration. An equation utilizing B max and K d to easily estimate PPB is presented.

Summary A new promising galanthamine derivative, 10-[ 3 H]methylgalanthamine iodide, was synthesized for binding studies to nicotinic acetylcholine receptors expressed in Torpedo electric ray electroplaques. Galanthamine was reacted with [ 3 H]methyl iodide to yield 10-[ 3 H]methylgalanthamine iodide with a radiochemical yield of >70% and a specific activity of 32 Ci/mmol after purification via solid phase extraction. To test the ligand properties of the radioligand, calcium imaging and electrophysiology of the non-radioactive analogue were performed to obtain an EC50 of 270 nM, a Hill coefficient of 1.9 and the induced cell current. Copyright # 2003 John Wiley & Sons, Ltd.

A fully automatized radiosynthesis of (+)- and (−)-[ 18 F]Flubatine ((+)- and (−)NCFHEB) by means of a commercially available synthesis module (TRACERlab FX FN) under GMP conditions is reported. Radiochemical yields of 30% within an overall synthesis time of 40 min were achieved in more than 70 individual syntheses. Specific activities were approximately 3000 GBq/μmol and radiochemical purity was determined to be at least 97%.

Cholinergic neurotransmission depends on the integrity of nicotinic acetylcholine receptors (nAChRs), and impairment of both is characteristic for various neurodegenerative diseases. Visualization of specific receptor subtypes by positron emission tomography (PET) has potential to assist with diagnosis of such neurodegenerative diseases and with design of suitable therapeutic approaches. The goal of our study was to evaluate in vivo the potential of 18 F-labelled (+)- and (−)-norchloro-fluoro-homoepibatidine ([ 18 F]NCFHEB) in comparison to 2-[ 18 F]F-A-85380 as PET tracers. In the brains of NMRI mice, highest levels of radioactivity were detected at 20 min post-injection of (+)-[ 18 F]NCFHEB, (−)-[ 18 F]NCFHEB, and 2-F-[ 18 F]-A-85380 (7.45, 5.60, and 3.2% ID/g tissue, respectively). No marked pharmacological adverse effects were observed at 25 μg NCFHEB/kg. Uptake studies in RBE4 cells and in situ perfusion studies suggest an interaction of epibatidine and NCFHEB with the carrier-mediated choline transport at the blood-brain barrier. The data indicate that (+)- and (−)-[ 18 F]NCFHEB have potential for further development as PET tracers.

2-[ 18 F]Fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[ 18 F]F-A-85380) was among the first subtype selective radioligands to visualise the in vivo distribution of α 4 β 2-containing neuronal nicotinic acetylcholine receptors (nAChRs) in human brain. We developed a one-pot synthesis for the preparation of 2-[ 18 F]F-A-85380 in a commercially available TRACERlab FX F−N synthesis module. The synthesis comprises a nucleophilic substitution followed by hydrolysis of a t -butyloxycarbonyl (BOC)-protected intermediate. After formulation for intravenous application up to 20 GBq 2-[ 18 F]F-A-85380 were produced from a starting activity of 100 GBq [ 18 F]fluoride in 60 min with a specific activity of about 4·10 5 GBq/mmol and a mean radiochemical purity of more than 99%.

2-[(18)F]fluoro-A-85380 (2-[(18)F]FA) is a new radioligand for noninvasive imaging of alpha4beta2* nicotinic acetylcholine receptors (nAChRs) by positron emission tomography (PET) in human brain. In most cases, quantification of 2-[(18)F]FA receptor binding involves measurement of free nonmetabolized radioligand concentration in blood. This requires an efficient and reliable method to separate radioactive metabolites from the parent compound. In the present study, three analytical methods, thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and solid phase extraction (SPE) have been tested. Reversed-phase TLC of deproteinized aqueous samples of plasma provides good estimates of 2-[(18)F]FA and its metabolites. However, because of the decreased radioactivity in plasma samples, this method can be used in humans over the first 2 h after radioligand injection only. Reliable quantification of the parent radioligand and its main metabolites was obtained using reversed-phase HPLC, followed by counting of eluted fractions in a well gamma counter. Three main and five minor metabolites of 2-[(18)F]FA were detected in human blood using this method. On average, the unchanged 2-[(18)F]FA fraction in plasma of healthy volunteers measured at 14, 60, 120, 240 and 420 min after radioligand injection was 87.3+/-2.2%, 74.4+/-3%, 68.8+/-5%, 62.3+/-8% and 61.0+/-8%, respectively. In patients with neurodegenerative disorders, the values corresponding to the three last time points were significantly lower. The fraction of nonmetabolized 2-[(18)F]FA in plasma determined using SPE did not differ significantly from that obtained by HPLC (+gamma counting) (n=73, r=.95). Since SPE is less time-consuming than HPLC and provides comparable results, we conclude that SPE appears to be the most suitable method for measurement of 2-[(18)F]FA parent fraction during PET investigations.

Noninvasive molecular imaging of subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) will provide information on the role of these receptors in neurodegenerative diseases. The binding of the positron emission tomography ligand 2-[ 1 8 F]F-A-85380 to nAChRs was investigated in the porcine brain by quantitative autoradiography in vitro. The high-affinity binding of 2-[ 1 8 F]F-A-85380 to each of the investigated 12 brain areas was saturable and apparently monophasic (e.g., apparent K D value of 1.72 nM in the thalamus). The highest density of specific binding sites was observed in the thalamus (1,158 fmol/mg protein), and the lowest density was measured in the cerebellar gray matter (11 fmol/mg protein). An attempt to assess nAChR subtype specificity of 2-[ 1 8 F]F-A-85380 was made by competitive autoradiography. Binding of 2-[ 1 8 F]F-A-85380 coincubated with 2-F-A-85380, epibatidine, cytisine, or methyllycaconitine, respectively, indicated a specificity of 2-[ 1 8 F]F-A-85380 to β2-containing nAChRs in the porcine brain. The autoradiographic data confirmed the suitability of swine as a model for the evaluation of radioligands designed for imaging of nAChR subtypes in the living brain. Synapse 59:201-210, 2006. 02005 Wiley-Liss, Inc.