Imaging of dopamine transporter with [18F]LBT-999: initial evaluation in healthy volunteers.

BACKGROUND: The aim of this study was to evaluate in healthy human brain the distribution, uptake, and kinetics of [18F]LBT-999, a PET ligand targeting the dopamine transporter, to assess its ability to explore dopaminergic innervation, using a shorter protocol, more convenient for patients than currently with [123I]ioflupane. METHODS: After intravenous injection of [18F]LBT-999, 8 healthy subjects (53-80y) underwent a dynamic PET-scan. Venous samples were concomitantly obtained for metabolites analysis. Time activity curves (TACs) were generated for several ROIs (caudate, putamen, occipital cortex, substantia nigra and cerebellum). Cerebellum was used as reference region to calculate binding potentials (BPND). RESULTS: No adverse events or detectable pharmacological effects were reported. [18F]LBT-999 PET revealed a good cerebral distribution, with an intense and symmetric uptake in both putamen and caudate (BPND of 6.75±1.17 and 6.30±1.17, respectively), without other brain abnormal tracer accumulation. Regional TACs showed a plateau from the maximal uptake, 20min pi, to the end of the acquisition for both caudate and putamen, whereas uptake in substantia nigra decreased progressively. A faster clearance and lowest BPND values were observed in both cortex and cerebellum. Ratios to the cerebellum exhibit value of about 3 in substantia nigra, close to 10 for both caudate and putamen, and remained around the value of 1 in cortex. The parent fraction of [18F]LBT-999 in plasma was 80%, 60% and 45% at 15, 30 and 45 min pi, respectively. CONCLUSIONS: These findings support the usefulness of [18F]LBT-999 for a quantitative clinical evaluation of presynaptic dopaminergic innervation.

Fully automated radiosynthesis of [18F]LBT999 on TRACERlab FXFN and AllinOne modules, a PET radiopharmaceutical for imaging the dopamine transporter in human brain.

BACKGROUND: Fluorine labelled 8-((E)-4-fluoro-but-2-enyl)-3ß-p-tolyl-8-aza-bicyclo[3.2.1]octane-2ß-carboxylic acid methyl ester ([18F]LBT999) is a selective radioligand for the in vivo neuroimaging and quantification of the dopamine transporter by Positron Emission Tomography (PET). [18F]LBT999 was produced on a TRACERlab FXFN for the Phase I study but for Phase III and a potent industrial production transfer, production was also implemented on an AllinOne (AIO) system requiring a single use cassette. Both production methods are reported herein. RESULTS: Automation of [18F]LBT999 radiosynthesis on FXFN was carried out in 35% yield (decay-corrected) in 65 min (n = 16), with a radiochemical purity higher than 99% and a molar activity of 158 GBq/µmol at the end of synthesis. The transfer to the AIO platform followed by optimizations allowed the production of [18F]LBT999 in 32.7% yield (decay-corrected) within 48 min (n = 5), with a radiochemical purity better than 98% and a molar activity above 154 GBq/µmol on average at the end of synthesis. Quality controls of both methods met the specification for clinical application. CONCLUSION: Both modules allow efficient and reproducible radiosynthesis of [18F]LBT999 with good radiochemical yields and a reasonable synthesis time. The developments made on AIO, such as its ability to meet pharmaceutical criteria and to more easily comply with GMP requirements, make it an optimal approach for the potent industrial production of [18F]LBT999 and future wider use.

Usefulness of PET With [18F]LBT-999 for the Evaluation of Presynaptic Dopaminergic Neuronal Loss in a Clinical Environment.

Purpose: The density of the neuronal dopamine transporter (DAT) is directly correlated with the presynaptic dopaminergic system injury. In a first study, we evaluated the brain distribution and kinetics of [18F]LBT-999, a DAT PET radioligand, in a group of eight healthy subjects. Taking into account the results obtained in healthy volunteers, we wanted to evaluate whether the loss of presynaptic striatal dopaminergic fibers could be estimated, under routine clinical conditions, using [18F]LBT-999 and a short PET acquisition. Materials and methods: Six patients with Parkinson's disease (PD) were compared with eight controls. Eighty-nine minutes of dynamic PET following an intravenous injection of [18F]LBT-999 were acquired. Using regions of interest for striatal nuclei, substantia nigra (SN), cerebellum, and occipital cortex, defined over each T1 3D MRI, time-activity curves (TACs) were obtained. From TACs, binding potential (BPND) using the simplified reference tissue model and distribution volume ratios (DVRs) using Logan graphical analysis were calculated. Ratios obtained for a 10-min image, acquired between 30 and 40 min post-injection, were also calculated. Cerebellum activity was used as non-specific reference region. Results: In PD patients and as expected, striatal uptake was lower than in controls which is confirmed by BPND, DVR, and ratios calculated for both striatal nuclei and SN, significantly inferior in PD patients compared with controls (p < 0.001). Conclusions: PET with [18F]LBT-999 could be an alternative to assess dopaminergic presynaptic injury in a clinical environment using a single 10 min acquisition.

Bis(het)aryl-1,2,3-triazole quinuclidines as α7 nicotinic acetylcholine receptor ligands: Synthesis, structure affinity relationships, agonism activity, [18F]-radiolabeling and PET study in rats.

In this paper we describe the design and synthesis of bis(Het)Aryl-1,2,3-triazole quinuclidine α7R ligands using an efficient three-step sequence including a Suzuki-Miyaura cross coupling reaction with commercially available and home-made boron derivatives. The exploration of SAR required the preparation of uncommon boron derivatives. Forty final drugs were tested for their ability to bind the target and nine of them exhibited Ki values below nanomolar concentrations. The best scores were always obtained when the 5-phenyl-2-thiophenyl core was attached to the triazole. The selectivity of these compounds towards the nicotinic α4ß2 and serotoninergic 5HT3 receptors was assessed and their brain penetration was quantified by the preparation and in vivo evaluation of two [18F] radiolabelled derivatives. It can be expected from our results that some of these compounds will be suitable for further developments and will have effects on cognitive disorders.

The Story of the Dopamine Transporter PET Tracer LBT-999: From Conception to Clinical Use.

The membrane dopamine transporter (DAT) is involved in a number of brain disorders and its exploration by positron emission tomography (PET) imaging is highly relevant for the early and differential diagnosis, follow-up and treatment assessment of these diseases. A number of carbon-11 and fluor-18 labeled tracers are to date available for this aim, the majority of them being derived from the chemical structure of cocaine. The development of such a tracer, from its conception to its use, is a long process, the expected result being to obtain the best radiopharmaceutical adapted for clinical protocols. In this context, the cocaine derivative (E)-N-(4-fluorobut-2-enyl)2ß-carbomethoxy-3ß-(4'-tolyl)nortropane, or LBT-999, has passed all the required stages of the development that makes it now a highly relevant imaging tool, particularly in the context of Parkinson's disease. This review describes the different steps of the development of LBT-999 which initially came from its non-fluorinated derivative (E)-N-(3-iodoprop-2-enyl)-2-carbomethoxy-3-(4-methylphenyl) nortropane, or PE2I, because of its high promising properties. [18F]LBT-999 has been extensively characterized in rodent and non-human primate models, in which it demonstrated its capability to explore in vivo the DAT localized at the dopaminergic nerve endings as well as at the mesencephalic cell bodies, in physiological conditions. In lesion-induced rat models of Parkinson's disease, [18F]LBT-999 was able to precisely quantify in vivo the dopaminergic neuron loss, and to assess the beneficial effects of therapeutic approaches such as pharmacological treatment and cell transplantation. Finally recent clinical data demonstrated the efficiency of [18F]LBT-999 in the diagnosis of Parkinson's disease.

Design of selective COX-2 inhibitors in the (aza)indazole series. Chemistry, in vitro studies, radiochemistry and evaluations in rats of a [18F] PET tracer.

A series of novel derivatives exhibiting high affinity and selectivity towards the COX-2 enzyme in the (aza) indazole series was developed. A short synthetic route involving a bromination/arylation sequence under microwave irradiation and direct C-H activation were established in the indazole and azaindazole series respectively. In vitro assays were conducted and structural modifications were carried out on these scaffolds to furnish compound 16 which exhibited effective COX-2 inhibitory activity, with IC50 values of 0.409 µM and an excellent selectivity versus COX-1. Radiolabeling of this most potent derivative [18F]16 was achieved after boron ester release and the tracer was evaluated in vivo in a rat model of neuroinflammation. All chemistry, radiochemistry and biological experimental data are discussed.

Extensive exploration of a novel rat model of Parkinson's disease using partial 6-hydroxydopamine lesion of dopaminergic neurons suggests new therapeutic approaches.

Parkinson's disease (PD) is characterized by the degeneration of dopaminergic (DA) neurons constituting the nigrostriatal pathway. Neuroinflammation, related to microglial activation, plays an important role in this process. Exploration of animal models of PD using neuroimaging modalities allows to better understand the pathophysiology of the disease. Here, we fully explored a moderate lesion model in the rat in which 6-hydroxydopamine was unilaterally delivered in three sites along the striatum. The degenerative process was assessed through in vivo Positron Emission Tomography (PET) imaging and in vitro autoradiographic quantitation of the striatal dopamine transporter (DAT) and immunostaining of tyrosine hydroxylase (TH). The microglial activation was studied through in vitro autoradiographic quantitation of the 18 kDa translocator protein (TSPO) in the striatum and CD11b staining in the SN. In addition, a targeted metabolomics exploration was performed in both these structures using mass spectrometry coupled to HPLC. Our results showed a reproducible decrease in the striatal DAT density associated with a reduction in the number of TH-positive cells in the SN and striatum, reflecting a robust moderate degeneration of nigrostriatal DA neurons. In addition, we observed strong microglia activation in both the striatum and SN ipsilateral to the lesion, highlighting that this moderate degeneration of DA neurons was associated with a marked neuroinflammation. Our metabolomics studies revealed alterations of specific metabolites and metabolic pathways such as carnitine, arginine/proline, and histidine metabolisms. These results bring new insights in the PD mechanism knowledge and new potential targets for future therapeutic strategies.

Design of α7 nicotinic acetylcholine receptor ligands using the (het)Aryl-1,2,3-triazole core: Synthesis, in vitro evaluation and SAR studies.

We report here the synthesis of a large library of 1,2,3-triazole derivatives which were in vitro tested as α7 nAchR ligands. The SAR study revealed that several crucial factors are involved in the affinity of these compounds for α7 nAchR such as a (R) quinuclidine configuration and a mono C-3 quinuclidine substitution. The triazole ring was substituted by a phenyl ring bearing small OMe/CH2F groups or fluorine atom and by several heterocycles such as thiophenes, furanes, benzothiophenes or benzofuranes. Among the 30 derivatives tested, the two derivatives 10 and 39 with Ki in the nanomolar range were identified (2.3 and 3 nM respectively). They exhibited a strict selectivity toward the α4ß2 nicotinic receptor (up to 1 µM) but interacted with the 5HT3 receptors with Ki around 3 nM. Synthesis, SAR studies and a full description of the derivatives are reported.

Synthesis, radiolabeling and preliminary in vivo evaluation of multimodal radiotracers for PET imaging and targeted radionuclide therapy of pigmented melanoma.

Melanin pigment represents an attractive target to address specific treatment to melanoma cells, such as cytotoxic radionuclides. However, less than half of the patients have pigmented metastases. Hence, specific marker is required to stratify this patient population before proceeding with melanin-targeted radionuclide therapy. In such a context, we developed fluorinated analogues of a previously studied melanin-targeting ligand, N-(2-diethylaminoethyl)-6-iodoquinoxaline-2-carboxamide (ICF01012). These latter can be labeled either with (18)F or (131)I/(125)I for positron emission tomography imaging (melanin-positive patient selection) and targeted radionuclide therapy purposes. Here we describe the syntheses, radiosyntheses and preclinical evaluations on melanoma-bearing mice model of several iodo- and fluoro(hetero)aromatic derivatives of the ICF01012 scaffold. After preliminary planar gamma scintigraphic and positron emission tomography imaging evaluations, [(125)I]- and [(18)F]-N-[2-(diethylamino)ethyl]-4-fluoro-3-iodobenzamides ([(125)I]4, [(18)F]4) were found to be chemically and biologically stable with quite similar tumor uptakes at 1 h p.i. (9.7 ± 2.6% ID/g and 6.8 ± 1.9% ID/g, respectively).

Design of α7 nicotinic acetylcholine receptor ligands in quinuclidine, tropane and quinazoline series. Chemistry, molecular modeling, radiochemistry, in vitro and in rats evaluations of a [(18)F] quinuclidine derivative.

In this report, we describe the synthesis of a novel library of α7 nAChR ligands based on the modulation of the quinuclidine, quinazoline and tropane moieties. Spirane derivatives were newly synthesized under stereo specific 1,3 dipolar cylcoadditions. Only amide derivatives bonded efficiently to the receptor with Ki measured between 14 and 133 nM. The best fluorinated candidate was selected and radiolabeled. The potent [(18)F]4 PET tracer was evaluated in rats and its brain accumulation quantified.

In vivo PET quantification of the dopamine transporter in rat brain with [¹8F]LBT-999.

INTRODUCTION: We examined whether [(18)F]LBT-999 ((E)-N-(4-fluorobut-2-enyl)2ß-carbomethoxy-3ß-(4'-tolyl)nortropane) is an efficient positron emission tomography (PET) tracer for the quantification of the dopamine transporter (DAT) in the healthy rat brain. METHODS: PET studies were performed using several experimental designs, i.e. test-retest, co-injection with different doses of unlabelled LBT, displacement with GBR12909 and pre-injection of amphetamine. RESULTS: The uptake of [(18)F]LBT-999 confirmed its specific binding to the DAT. The non-displaceable uptake (BP(ND)) in the striatum, between 5.37 and 4.39, was highly reproducible and reliable, and was decreased by 90% by acute injection of GBR12909. In the substantia nigra/ventral tegmental area (SN/VTA), the variability was higher and the reliability was lower. Pre-injection of amphetamine induced decrease of [(18)F]LBT-999 BP(ND) of 50% in the striatum. CONCLUSIONS: [(18)F]LBT-999 allows the quantification of the DAT in living rat brain with high reproducibility, sensitivity and specificity. It could be used to quantify the DAT in rodent models, thereby allowing to study neurodegenerative and neuropsychiatric diseases.

Fully automated radiosynthesis of 2-[18F]fludarabine for PET imaging of low-grade lymphoma.

PURPOSE: An efficient and fully automated radiosynthesis of 2-[(18)F]fluoro-9-ß-D-arabinofuranosyl-adenine (2-[(18)F]fludarabine, [(18)F]-5) based on a GE TRACERlab™ FX-FN module has been developed. PROCEDURES: A 2-nitro purine derivative 3 was developed as precursor for labeling with fluorine-18. The radiosynthesis of [(18)F]-5 was performed in two steps in a single reactor with an intermediary purification on Sep-Pak® silica which involved the addition of a three-way valve on the original module. After hydrolysis, [(18)F]-5 was purified by semi-preparative high-pressure liquid chromatography (HPLC) and a quality control was established. RESULTS: The labeling precursor 3 was obtained in 45% overall yield. Nucleophilic substitution with K(18)F/K2.2.2 afforded protected 2-[(18)F]fludarabine ([(18)F]-4) in 73 ± 4%, radiochemical yield (decay corrected to the end of bombardment (EOB)) and based on the initial [(18)F]F(-) activity. An aqueous ammonia/methanol solution was used for the deprotection reaction and gave the desired [(18)F]-5 in 67 ± 3% yield after 20 min at 70 °C based on HPLC profile. CONCLUSIONS: The process afforded pure 2-[(18)F]fludarabine in 48 ± 3% yield (decay corrected to the EOB) in 85 min, with a specific activity of 310 ± 72 GBq/µmol at the end of synthesis (EOS) and a radiochemical purity up to 99%.

Imaging of the striatal and extrastriatal dopamine transporter with (18)F-LBT-999: quantification, biodistribution, and radiation dosimetry in nonhuman primates.

UNLABELLED: The aim of this study was to evaluate the quantification, biodistribution, and radiation dosimetry of the novel dopamine transporter (DAT) radioligand (18)F-(2S,3S)-methyl 8-((E)-4-fluorobut-2-en-1-yl)-3-(p-tolyl)-8-azabicyclo[3.2.1]octane-2-carboxylate ((18)F-LBT-999) in nonhuman primates. METHODS: The brain study was conducted in 4 female rhesus monkeys. PET measurements were conducted for 243 min using the high-resolution research tomograph (HRRT) with the measurement of the metabolite-corrected arterial input function and protein binding. Quantification was performed with kinetic analysis using 2-tissue- and 1-tissue-compartment models, with Logan graphical analysis and with different reference tissue models. The outcome measures were total distribution volume (V(T)), nondisplaceable distribution volume (V(ND)), binding potential relative to the free concentration of radioligand in plasma (BP(F)), and binding potential relative to the concentration of nondisplaceable radioligand in tissue (BP(ND)) = V(T) - V(ND)/V(ND) using the cerebellum as a reference region. For the biodistribution and radiation dosimetry, 2 female cynomolgus monkeys were studied. Whole-body PET scans were obtained using a PET/CT system for approximately 250 min. Estimates of the absorbed radiation dose in humans were calculated using OLINDA/EXM software. RESULTS: (18)F-LBT-999 showed good brain uptake (300% standardized uptake value) and regional distribution according to known DAT density. The 2-tissue-compartment model was the preferred model for the quantification. Late peak equilibrium (120-140 min) and slow washout were observed in the striatum, with high variability of V(T), BP(F), and BP(ND). When the different models were compared with the 2-tissue-compartment model, the underestimation of V(T) or BP(ND) was larger in the caudate and putamen than in the midbrain and thalamus. The reference tissue models were suitable for the quantification. The whole-body distribution study showed that the main routes of excretion of (18)F-LBT-999 were the urinary and gastrointestinal systems, with the bladder being the critical organ. Accumulation of (18)F-LBT-999 was found in the bone and skull, with a relatively high dose estimated for the osteogenic cells. The range of calculated effective dose was 0.021-0.022 mSv/MBq. CONCLUSION: (18)F-LBT-999 seemed to be a suitable PET radioligand for the DAT quantification, particularly for extrastriatal regions. The skull uptake did not seem to be a limitation for brain imaging. The calculated dosimetry estimates based on data in nonhuman primates seemed comparable with those of other clinically used (18)F-labeled radioligands, for example, (18)F-FDG (0.024-0.027 mSv/MBq).

[11C]LBT-999: a suitable radioligand for investigation of extra-striatal dopamine transporter with PET.

A new tropane derivative, (E)-N-(4-fluorobut-2-enyl)-2beta-carbomethoxy-3beta-(4'-tolyl)nortropane (LBT-999), was evaluated in baboons as a carbon-11 radioligand for studies of the dopamine transporter (DAT) using positron emission tomography (PET). Brain uptake was high in the striatum (17 and 13% ID/100 mL tissue in the putamen and the caudate, respectively), moderate in the midbrain and thalamus (5 and 3% ID/100 mL tissue, respectively), and low in the cortex and cerebellum (2% ID/100 mL tissue) at 30 min post injection. The striatum-to-cerebellum ratio was high (30 at 110 min post injection). Specific binding was completely blocked following pretreatment with the DAT antagonists GBR12909 (5 mg/kg i.v.) or PE2I (1 mg/kg i.v.). The [(11)C]LBT-999 uptake was decreased by these antagonists in the putamen (-79 and -92%, respectively), caudate (-80 and -91%, respectively), midbrain (-73 and -78%, respectively), and thalamus (-34 and -46%, respectively). The serotonin transporter (SERT) antagonist citalopram (5 mg/kg i.v.) or the norepinephrine transporter antagonist maprotiline (5 mg/kg i.v.) had no effect on LBT specific binding. Pharmacological challenge with PE2I (1 mg/kg i.v.) induced a rapid and almost complete decrease of the specific binding in the putamen (-97%), caudate (-96%), midbrain (-96%), and thalamus (-81%), confirming the reversibility of [(11)C]LBT-999 binding. The high brain uptake of [(11)C]LBT-999 together with its low nonspecific binding (reflected by the very high brain structure-to-cerebellum ratio) indicate that this radiotracer is an excellent candidate for in vivo quantification of the DAT, especially in extrastriatal structures, such as the midbrain.

Synthesis, radiosynthesis and in vivo preliminary evaluation of [11C]LBT-999, a selective radioligand for the visualisation of the dopamine transporter with PET.

LBT-999 (8-((E)-4-fluoro-but-2-enyl)-3beta-p-tolyl-8-aza-bicyclo[3.2.1]octane-2beta-carboxylic acid methyl ester), a cocaine derivative belonging to a new generation of highly selective dopamine transporter (DAT) ligands, and its corresponding carboxylic acid derivative, the latter used as precursor for labelling both with tritium and the positron-emitter carbon-11 (half-life: 20.38 min), were synthesized from (R)-cocaine. [(3)H]LBT-999 (>99% radiochemically pure, specific radioactivity of 3.1 TBq/mmol) was prepared from [(3)H]methyl iodide, allowing its in vitro pharmacological evaluation (K(D): 9 nM for DAT and IC(50) > 1000 nM for SERT and NET). Routine production batches of 4.5-9.0 GBq of iv injectable solutions of [(11)C]LBT-999 (with specific radioactivities ranging from 30 to 45 GBq/mumol) were prepared in 25-30 min (HPLC purification and formulation included) using the efficient methylation reagent [(11)C]methyl triflate. The preliminary in vivo pharmacological evaluation of [(11)C]LBT-999, using both biodistributions in rats and brain imaging in monkeys with positron emission tomography (PET), clearly illustrates that this ligand is an excellent candidate for quantification with PET of DAT in humans.

Pharmacological characterization of (E)-N-(4-fluorobut-2-enyl)-2beta-carbomethoxy-3beta-(4'-tolyl)nortropane (LBT-999) as a highly promising fluorinated ligand for the dopamine transporter.

In the aim to develop an efficient fluorinated probe for positron emission tomography (PET) exploration of the dopamine transporter (DAT), we studied several in vitro and in vivo characteristics of the phenyltropane derivative (E)-N-(4-fluorobut-2-enyl)-2beta-carbomethoxy-3beta-(4'-tolyl)nortropane (LBT-999). In vitro on rat striatal membrane, [(3)H]LBT-999 bound to a single site with a K(d) of 9 nM, B(max) of 17 pmol/mg protein, and a very high selectivity for the DAT [IC(50) for 1-{2-[bis-(4-fluorophenyl)-methoxy]ethyl}-4-(3-phenylpropyl)piperazine (GBR 12909) and (E)-N-(3-iodoprop-2-enyl)-2beta-carbomethoxy-3beta-(4'-methylphenyl)nortropane (PE2I): 2.4 and 18 nM, respectively; IC(50) for paroxetine, citalopram, N,N-dimethyl-2-(2-amino-4-methylphenyl thio)benzylamine, nisoxetine, and desipramine >1 muM]. In vitro on post-mortem human brain sections, LBT-999 bound with high intensity to the caudate-putamen, weakly to the thalamus, and not in the neocortex and cerebellum. This binding was totally abolished in the presence of PE2I. Ex vivo cerebral biodistribution of [(11)C]LBT-999 in rats showed striatum/cerebellum radioactivity ratios of 18 and 25 at 30 and 60 min postinjection, respectively. This accumulation was strongly prevented by preinjection of GBR 12909, whereas paroxetine and nisoxetine had no effect. An in vivo kinetic PET study in three baboons showed a fast and very high uptake in the striatum, with a plateau at 30 min postinjection and a maximal putamen/cerebellum ratio of 30. Taken together, these findings demonstrate that LBT-999 is a highly promising agent for in vivo exploration of the DAT. This probe is currently labeled with (18)F for further characterizations.