Toward the Optimization of (+)-[11C]PHNO Synthesis: Time Reduction and Process Validation.

(+)-[11C]PHNO, a dopamine D2/3 receptor agonistic radiotracer, is applied for investigating the dopaminergic system via positron emission tomography (PET). An improved understanding of neuropsychiatric disorders associated with dysfunctions in the dopamine system and the underlying mechanism is a necessity in order to promote the development of new potential therapeutic drugs. In contrast to other broadly applied 11C-radiopharmaceuticals, the production of this radiotracer requires a challenging four-step radiosynthesis involving harsh reaction conditions and reactants as well as an inert atmosphere. Consequently, the production is prone to errors and troubleshooting after failed radiosyntheses remains time consuming. Hence, we aimed to optimize the radiosynthesis of (+)-[11C]PHNO for achieving better activity yields without loss of product quality. Therefore, we synthesized (+)-[11C]PHNO and omitted all heating and cooling steps leading to higher activity yields. As a result, radiosynthesis fully conducted at room temperature led to a time-reduced production procedure that saves about 5 min, which is an appreciable decay-prevention of around 15% of the activity yield. Additionally, we established a troubleshooting protocol by investigating reaction intermediates, byproducts, and impurities. Indeed, partial runs enabled the assignment of byproducts to their associated error source. Finally, we were able to generate a decision tree facilitating error detection in (+)-[11C]PHNO radiosynthesis.

Development of a radiolabeled caninized anti-EGFR antibody for comparative oncology trials.

Due to large homology of human and canine EGFR, dogs suffering from spontaneous EGFR+ cancer can be considered as ideal translational models. Thereby, novel immunotherapeutic compounds can be developed for both human and veterinary patients. This study describes the radiolabeling of a canine anti-EGFR IgG antibody (can225IgG) with potential diagnostic and therapeutic value in comparative clinical settings. Can225IgG was functionalized with DTPA for subsequent chelation with the radionuclide 99mTc. Successful coupling of 10 DTPA molecules per antibody on average was proven by significant mass increase in MALDI-TOF spectroscopy, gel electrophoresis and immunoblots. Following functionalization and radiolabeling, 99mTc-DTPA-can225IgG fully retained its binding capacity towards human and canine EGFR in flow cytometry, immuno- and radioblots, and autoradiography. The affinity of radiolabeled can225IgG was determined to KD 0.8 ±0.0031 nM in a real-time kinetics assay on canine carcinoma cells by a competition binding technique. Stability tests of the radiolabeled compound identified TRIS buffered saline as the ideal formulation for short-term storage with 87.11 ±6.04% intact compound being still detected 60 minutes post radiolabeling. High stability, specificity and EGFR binding affinity pinpoint towards 99mTc-radiolabeled can225IgG antibody as an ideal lead compound for the first proof-of-concept diagnostic and therapeutic applications in canine cancer patients.

[(18)F]FMeNER-D2: A systematic in vitro analysis of radio-metabolism.

INTRODUCTION: The norepinephrine transporter (NET) presents an important target for therapy and diagnosis of ADHD and other neurodegenerative and psychiatric diseases. Thus, PET is the diagnostic method of choice, using radiolabeled NET-ligands derived from reboxetine. So far, [(18)F]FMeNER-D2 showed best pharmacokinetic and -dynamic properties. However, the disadvantage of reboxetine derived PET tracers is their high metabolic cleavage-resulting in impeding signals in the PET scans, which hamper a proper quantification of the NET in cortical areas. METHODS: Metabolic stability testing was performed in vitro using a plethora of human and murine enzymes. RESULTS: No metabolism was observed using monoamine oxidase A and B or catechol-O-methyl transferase. Incubation of [(18)F]FMeNER-D2 with CYP450-enzymes, predominantly located in the liver, led to a significant and fast metabolism of the tracer. Moreover, the arising three radiometabolites were found to be more polar than [(18)F]FMeNER-D2. Surprisingly, definitely no formation of free [(18)F]fluoride was observed. CONCLUSION: According to our in vitro data, the interfering uptake in cortical regions might be attributed to these emerging radiometabolites but does not reflect bonding in bone due to defluorination. Further research on these radiometabolites is necessary to elucidate the in vivo situation. This might include an analysis of human blood samples after injection of [(18)F]FMeNER-D2, to enable a better correction of the PET-input function.

Effects of norepinephrine transporter gene variants on NET binding in ADHD and healthy controls investigated by PET.

Attention deficit hyperactivity disorder (ADHD) is a heterogeneous disorder with a strong genetic component. The norepinephrine transporter (NET) is a key target for ADHD treatment and the NET gene has been of high interest as a possible modulator of ADHD pathophysiology. Therefore, we conducted an imaging genetics study to examine possible effects of single nucleotide polymorphisms (SNPs) within the NET gene on NET nondisplaceable binding potential (BPND ) in patients with ADHD and healthy controls (HCs). Twenty adult patients with ADHD and 20 HCs underwent (S,S)-[18F]FMeNER-D2 positron emission tomography (PET) and were genotyped on a MassARRAY MALDI-TOF platform using the Sequenom iPLEX assay. Linear mixed models analyses revealed a genotype-dependent difference in NET BPND between groups in the thalamus and cerebellum. In the thalamus, a functional promoter SNP (-3081 A/T) and a 5'-untranslated region (5'UTR) SNP (-182 T/C), showed higher binding in ADHD patients compared to HCs depending on the major allele. Furthermore, we detected an effect of genotype in HCs, with major allele carriers having lower binding. In contrast, for two 3'UTR SNPs (*269 T/C, *417 A/T), ADHD subjects had lower binding in the cerebellum compared to HCs depending on the major allele. Additionally, symptoms of hyperactivity and impulsivity correlated with NET BPND in the cerebellum depending on genotype. Symptoms correlated positively with cerebellar NET BPND for the major allele, while symptoms correlated negatively to NET BPND in minor allele carriers. Our findings support the role of genetic influence of the NE system on NET binding to be pertubated in ADHD.

Radiosynthesis and first preclinical evaluation of the novel norepinephrine transporter pet-ligand [(11)C]ME@HAPTHI.

BACKGROUND: The norepinephrine transporter (NET) has been demonstrated to be relevant to a multitude of neurological, psychiatric and cardiovascular pathologies. Due to the wide range of possible applications for PET imaging of the NET together with the limitations of currently available radioligands, novel PET tracers for imaging of the cerebral NET with improved pharmacological and pharmacodynamic properties are needed. METHODS: The present study addresses the radiosynthesis and first preclinical evaluation of the novel NET PET tracer [(11)C]Me@HAPTHI by describing its affinity, selectivity, metabolic stability, plasma free fraction, blood-brain barrier (BBB) penetration and binding behaviour in in vitro autoradiography. RESULTS: [(11)C]Me@HAPTHI was prepared and displayed outstanding affinity and selectivity as well as excellent in vitro metabolic stability, and it is likely to penetrate the BBB. Moreover, selective NET binding in in vitro autoradiography was observed in human brain and rat heart tissue samples. CONCLUSIONS: All preclinical results and radiosynthetic key-parameters indicate that the novel benzothiadiazole dioxide-based PET tracer [(11)C]Me@HAPTHI is a feasible and improved NET radioligand and might prospectively facilitate clinical NET imaging.

Interaction between 5-HTTLPR and 5-HT1B genotype status enhances cerebral 5-HT1A receptor binding.

Serotonergic neurotransmission is thought to underlie a dynamic interrelation between different key structures of the serotonin system. The serotonin transporter (SERT), which is responsible for the reuptake of serotonin from the synaptic cleft into the neuron, as well as the serotonin-1A (5-HT1A) and -1B (5-HT1B) receptors, inhibitory auto-receptors in the raphe region and projection areas, respectively, are likely to determine serotonin release. Thereby, they are involved in the regulation of extracellular serotonin concentrations and the extent of serotonergic effects in respective projection areas. Complex receptor interactions can be assessed in vivo with positron emission tomography (PET) and single-nucleotide-polymorphisms, which are thought to alter protein expression levels. Due to the complexity of the serotonergic system, gene × gene interactions are likely to regulate transporter and receptor expression and therefore subsequently serotonergic transmission. In this context, we measured 51 healthy subjects (mean age 45.5 ± 12.9, 38 female) with PET using [carbonyl-(11)C]WAY-100635 to determine 5-HT1A receptor binding potential (5-HT1A BPND). Genotyping for rs6296 (HTR1B) and 5-HTTLPR (SERT gene promoter polymorphism) was performed using DNA isolated from whole blood. Voxel-wise whole-brain ANOVA revealed a positive interaction effect of genotype groups (5-HTTLPR: LL, LS+SS and HTR1B: rs6296: CC, GC+GG) on 5-HT1A BPND with peak t-values in the bilateral parahippocampal gyrus. More specifically, highest 5-HT1A BPND was identified for individuals homozygous for both the L-allele of 5-HTTLPR and the C-allele of rs6296. This finding suggests that the interaction between two major serotonergic structures involved in serotonin release, specifically the SERT and 5-HT1B receptor, results in a modification of the inhibitory serotonergic tone mediated via 5-HT1A receptors.

Synthesis and in silico evaluation of novel compounds for PET-based investigations of the norepinephrine transporter.

Since the norepinephrine transporter (NET) is involved in a variety of diseases, the investigation of underlying dysregulation-mechanisms of the norepinephrine (NE) system is of major interest. Based on the previously described highly potent and selective NET ligand 1-(3-(methylamino)-1-phenylpropyl)-3-phenyl-1,3-dihydro-2H-benzimidaz- ol-2-one (Me@APPI), this paper aims at the development of several fluorinated methylamine-based analogs of this compound. The newly synthesized compounds were computationally evaluated for their interactions with the monoamine transporters and represent reference compounds for PET-based investigation of the NET.

Effects of Silexan on the serotonin-1A receptor and microstructure of the human brain: a randomized, placebo-controlled, double-blind, cross-over study with molecular and structural neuroimaging.

BACKGROUND: Recently, Silexan, a patented active substance comprised of an essential oil produced from Lavandula angustifolia flowers, has been authorized in Germany as a medicinal product for the treatment of states of restlessness related to anxious mood. Its efficacy has been shown in several forms of anxiety disorders. Findings from preclinical and clinical studies attribute a major role to the serotonin-1A receptor in the pathogenesis and treatment of anxiety. METHODS: To elucidate the effect of Silexan on serotonin-1A receptor binding, 17 healthy men underwent 2 positron emission tomography measurements using the radioligand [carbonyl-(11)C]WAY-100635 following the daily intake of 160 mg Silexan or placebo for a minimum of 8 weeks (randomized, double-blind, cross-over design). Additionally, structural magnetic resonance imaging and voxel-based morphometry analysis was performed to determine potential effects on gray matter microstructure. RESULTS: Serotonin-1A receptor binding potential was shown to be significantly reduced following the intake of Silexan compared with placebo in 2 large clusters encompassing the temporal gyrus, the fusiform gyrus and the hippocampus on one hand as well as the insula and anterior cingulate cortex on the other hand. No effects of Silexan on gray matter volume could be detected in this investigation. CONCLUSION: This positron emission tomography study proposes an involvement of the serotonin-1A receptor in the anxiolytic effects of Silexan. The study was registered in the International Standard Randomized Controlled Trial Number Register as ISRCTN30885829 (http://www.controlled-trials.com/isrctn/).

The norepinephrine transporter in attention-deficit/hyperactivity disorder investigated with positron emission tomography.

IMPORTANCE: Attention-deficit/hyperactivity disorder (ADHD) research has long focused on the dopaminergic system's contribution to pathogenesis, although the results have been inconclusive. However, a case has been made for the involvement of the noradrenergic system, which modulates cognitive processes, such as arousal, working memory, and response inhibition, all of which are typically affected in ADHD. Furthermore, the norepinephrine transporter (NET) is an important target for frequently prescribed medication in ADHD. Therefore, the NET is suggested to play a critical role in ADHD. OBJECTIVE: To explore the differences in NET nondisplaceable binding potential (NET BPND) using positron emission tomography and the highly selective radioligand (S,S)-[18F]FMeNER-D2 [(S,S)-2-(α-(2-[18F]fluoro[2H2]methoxyphenoxy)benzyl)morpholine] between adults with ADHD and healthy volunteers serving as controls. DESIGN, SETTING, AND PARTICIPANTS: Twenty-two medication-free patients with ADHD (mean [SD] age, 30.7 [10.4] years; 15 [68%] men) without psychiatric comorbidities and 22 age- and sex-matched healthy controls (30.9 [10.6] years; 15 [68%] men) underwent positron emission tomography once. A linear mixed model was used to compare NET BPND between groups. MAIN OUTCOMES AND MEASURES: The NET BPND in selected regions of interest relevant for ADHD, including the hippocampus, putamen, pallidum, thalamus, midbrain with pons (comprising a region of interest that includes the locus coeruleus), and cerebellum. In addition, the NET BPND was evaluated in thalamic subnuclei (13 atlas-based regions of interest). RESULTS: We found no significant differences in NET availability or regional distribution between patients with ADHD and healthy controls in all investigated brain regions (F1,41<0.01; P=.96). Furthermore, we identified no significant association between ADHD symptom severity and regional NET availability. Neither sex nor smoking status influenced NET availability. We determined a significant negative correlation between age and NET availability in the thalamus (R2=0.29; P<.01 corrected) and midbrain with pons, including the locus coeruleus (R2=0.18; P<.01 corrected), which corroborates prior findings of a decrease in NET availability with aging in the human brain. CONCLUSIONS AND RELEVANCE: Our results do not indicate involvement of changes in brain NET availability or distribution in the pathogenesis of ADHD. However, the noradrenergic transmitter system may be affected on a different level, such as in cortical regions, which cannot be reliably quantified with this positron emission tomography ligand. Alternatively, different key proteins of noradrenergic neurotransmission might be affected.

Exploring the impact of BDNF Val66Met genotype on serotonin transporter and serotonin-1A receptor binding.

BACKGROUND: The brain-derived neurotrophic factor (BDNF) Val66Met polymorphism (rs6265) may impact on the in-vivo binding of important serotonergic structures such as the serotonin transporter (5-HTT) and the serotonin-1A (5-HT1A) receptor. Previous positron emission tomography (PET) studies on the association between Val66Met and 5-HTT and 5-HT1A binding potential (BPND) have demonstrated equivocal results. METHODS: We conducted an imaging genetics study investigating the effect of Val66Met genotype on 5-HTT or 5-HT1A BPND in 92 subjects. Forty-one subjects (25 healthy subjects and 16 depressive patients) underwent genotyping for Val66Met and PET imaging with the 5-HTT specific radioligand [11C]DASB. Additionally, in 51 healthy subjects Val66Met genotypes and 5-HT1A binding with the radioligand [carbonyl-11C]WAY-100635 were ascertained. Voxel-wise and region of interest-based analyses of variance were used to examine the influence of Val66Met on 5-HTT and 5-HT1A BPND. RESULTS: No significant differences of 5-HTT nor 5-HT1A BPND between BDNF Val66Met genotype groups (val/val vs. met-carrier) were detected. There was no interaction between depression and Val66Met genotype status. CONCLUSION: In line with previous data, our work confirms an absent effect of BDNF Val66Met on two major serotonergic structures. These results could suggest that altered protein expression associated with genetic variants, might be compensated in vivo by several levels of unknown feedback mechanisms. In conclusion, Val66Met genotype status is not associated with changes of in-vivo binding of 5-HTT and 5-HT1A receptors in human subjects.

Effects of hormone replacement therapy on cerebral serotonin-1A receptor binding in postmenopausal women examined with [carbonyl-¹¹C]WAY-100635.

Preclinical research points to a strong modulatory influence of gonadal hormones on the serotonin system. However, human data corroborating this association remains scarce. The aim of this study was to examine the effects of hormone replacement therapy on 5-HT1A receptor binding in postmenopausal women using positron emission tomography (PET) and the radioligand [carbonyl-(11)C]WAY-100635. In this randomized, double-blind, longitudinal study, 30 postmenopausal women underwent treatment with either a combination of oral 17ß-estradiol valerate and micronized progesterone (group 1, n=10), oral 17ß-estradiol valerate (group 2, n=10), or placebo (group 3, n=10). Two PET measurements were performed, one the day before treatment start and the second after at least eight weeks of treatment. Plasma levels of estradiol (E2), progesterone (P4), sex hormone-binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), follicle stimulating hormone (FSH) and luteinizing hormone (LH) were collected prior to PET measurements. As expected, hormone replacement therapy led to a significant increase in E2 and P4 plasma levels in group 1 and to a significant increase in E2 levels in group 2. The 5-HT1A receptor binding did not change significantly after estrogen, combined estrogen/progesterone treatment or placebo in any of the investigated brain regions. There were no significant correlations between changes in E2 or P4 values and changes in 5-HT1A receptor binding. Although we were not able to confirm effects of gonadal hormone treatment on 5-HT1A receptor binding, our data do not preclude associations between sex steroid levels and serotonin, the neurotransmitter implicated most strongly in the pathogenesis of affective and anxiety disorders. ClinicalTrials.gov Identifier: NCT00755963.

Synthesis, radiosynthesis and first in vitro evaluation of novel PET-tracers for the dopamine transporter: [(11)C]IPCIT and [(18)F]FE@IPCIT.

INTRODUCTION: Present data indicate that merging beneficial structural elements from previously published DAT-ligands highest DAT affinity, selectivity and a suitable metabolic profile should be achieved. This combination led to the development of IPCIT and FE@IPCIT. METHODS: Precursor synthesis was done starting from cocaine in a six step reaction. O-[(11)C]-methylation was established using [(11)C]methyl iodide, optimized and subsequently automated. Small scale (18)F-fluroroethylation as well as optimization of reaction parameters and automation were performed. Affinity and selectivity of the candidate substances were tested in standard binding experiments on human membranes. Metabolic stability and blood-brain-barrier (BBB) penetration were determined. RESULTS: Precursor compound, IPCITacid, and reference compounds, IPCIT and FE@IPCIT, were obtained in 4.9%, 12.7% and 4.1% yield, respectively. Automated radiosynthesis of [(11)C]IPCIT yielded 1.9 ± 0.7 GBq (12.5 ± 4%, corrected for decay). Optimum parameters for (18)F-fluoroethylation were 110 °C for 15 min under TBAH catalysis, yielding 67 ± 16 % radiochemical incorporation. Affinity was determined as 1.7 ± 0.6 nM for IPCIT, 1.3 ± 0.2 nM for FE@IPCIT and 37 ± 13 nM for the precursor molecule, IPCIT-acid. Results from in vitro and in silico evaluations revealed high stability but also high lipophilicity. CONCLUSION: Present data indicate high affinity and stability of both IPCIT and FE@IPCIT. Radiolabelling, optimization of reaction parameters and automation succeeded. On the other hand, data concerning BBB-penetration are not promising.

[18F]FMeNER-D2: reliable fully-automated synthesis for visualization of the norepinephrine transporter.

PURPOSE: In neurodegenerative diseases and neuropsychiatric disorders dysregulation of the norepinephrine transporter (NET) has been reported. For visualization of NET availability and occupancy in the human brain PET imaging can be used. Therefore, selective NET-PET tracers with high affinity are required. Amongst these, [(18)F]FMeNER-D2 is showing the best results so far. Furthermore, a reliable fully automated radiosynthesis is a prerequisite for successful application of PET-tracers. The aim of this work was the automation of [(18)F]FMeNER-D2 radiolabelling for subsequent clinical use. The presented study comprises 25 automated large-scale syntheses, which were directly applied to healthy volunteers and adult patients suffering from attention deficit hyperactivity disorder (ADHD). PROCEDURES: Synthesis of [(18)F]FMeNER-D2 was automated within a Nuclear Interface Module. Starting from 20-30 GBq [(18)F]fluoride, azeotropic drying, reaction with Br2CD2, distillation of 1-bromo-2-[(18)F]fluoromethane-D2 ([(18)F]BFM) and reaction of the pure [(18)F]BFM with unprotected precursor NER were optimized and completely automated. HPLC purification and SPE procedure were completed, formulation and sterile filtration were achieved on-line and full quality control was performed. RESULTS: Purified product was obtained in a fully automated synthesis in clinical scale allowing maximum radiation safety and routine production under GMP-like manner. So far, more than 25 fully automated syntheses were successfully performed, yielding 1.0-2.5 GBq of formulated [(18)F]FMeNER-D2 with specific activities between 430 and 1707 GBq/µmol within 95 min total preparation time. CONCLUSIONS: A first fully automated [(18)F]FMeNER-D2 synthesis was established, allowing routine production of this NET-PET tracer under maximum radiation safety and standardization.

Reliable set-up for in-loop ¹¹C-carboxylations using Grignard reactions for the preparation of [carbonyl-¹¹C]WAY-100635 and [¹¹C]-(+)-PHNO.

Aim of this work was the implementation of a generalized in-loop synthesis for (11)C-carboxylations and subsequent (11)C-acylations on the TRACERlab FxC Pro platform. The set-up was tested using [carbonyl-(11)C]WAY-100635 and, for the first time, [(11)C]-(+)-PHNO. Its general applicability could be demonstrated and both [carbonyl-(11)C]WAY-100635 and [(11)C]-(+)-PHNO were prepared with high reliability and satisfying outcome.