Positron emission tomography and ex vivo and in vitro autoradiography studies on dopamine D2-like receptor degeneration in the quinolinic acid-lesioned rat striatum: comparison of [11C]raclopride, [11C]nemonapride and [11C]N-methylspiperone

With [11C]raclopride,[11C]nemonapride and [11C]N-methylspiperone, degeneration of dopamine D2-like receptors in the unilaterally quinolinic acid-lesioned rats was evaluated by positron emission tomography (PET) and ex vivo and in vitro autoradiography. PET showed a decreased uptake of [11C]raclopride in the lesioned striatum, but an increased uptake of [11C]nemonapride and [11C]N-methylspiperone despite a decreased binding in vitro. Ex vivo autoradiography showed an increased accumulation of the three ligands in the cortical region overlying the injured striatum, probably enlarging PET signals. PET has the limited potential for evaluating the receptor degeneration in the present animal model.     

Upregulation of putaminal dopamine D2 receptors in early Parkinson's disease: a comparative PET study with [11C] raclopride and [11C]N-methylspiperone.

Dopamine D2 receptor function was assessed in a PET study with 2 dopamine D2 receptor PET ligands, [11C]raclopride (RAC) and [11C]N-methylspiperone (NMSP), in early Parkinson's disease. METHODS: Seven patients with early Parkinson's disease and 5 healthy volunteers were studied. Each underwent PET both with reversible [11C]RAC and with irreversible [11C]NMSP. RESULTS: Upregulation of dopamine D2 receptors in the putamen contralateral to the predominant symptoms of Parkinson's disease was confirmed using both [11C]RAC and [11C]NMSP. Uptake of [11C]RAC in the contralateral putamen was 105% of uptake in the opposite putamen (P = 0.020). For [11C]NMSP, uptake in the contralateral putamen was 105% of uptake in the ipsilateral putamen (P = 0.011). No significant differences between Parkinson's disease patients and healthy volunteers were detected in any of the studied brain regions using either [11C]RAC or [11C]NMSP. No significant differences between [11C]RAC and [11C]NMSP uptake were detected in the striatum, whereas in the extrastriatal regions, [11C]NMSP showed significantly higher uptake than [11C]RAC both in healthy volunteers and in Parkinson's disease patients. CONCLUSION: This study confirms an increase in dopamine D2 receptors in the putamen contralateral to the predominant symptoms, compared with the ipsilateral putamen, in early Parkinson's disease. This increase was seen both with reversible ligand [11C]RAC and with irreversible ligand [11C]NMSP and thus does not seem a consequence of depleted endogenous dopamine.     

Test-retest reproducibility of dopamine D2/3 receptor binding in human brain measured by PET with [11C]MNPA and [11C]raclopride

Purpose

Dopamine D_2/3 receptors (D_2/3Rs) have two affinity states for endogenous dopamine, referred to as high-affinity state (D_2/3 ^HIGH), which has a high affinity for endogenous dopamine, and low-affinity state (D_2/3 ^LOW). The density of D_2/3 ^HIGH can be measured with (R)-2-¹¹CH₃O-N-n-propylnorapomorphine ([¹¹C]MNPA), while total density of D_2/3 ^HIGH and D_2/3 ^LOW (D_2/3Rs) can be measured with [¹¹C]raclopride using positron emission tomography (PET). Thus, the ratio of the binding potential (BP) of [¹¹C]MNPA to that of [¹¹C]raclopride ([¹¹C]MNPA/[¹¹C]raclopride) may reflect the proportion of the density of D_2/3 ^HIGH to that of D_2/3Rs. In the caudate and putamen, [¹¹C]MNPA/[¹¹C]raclopride reflects the proportion of the density of D₂ ^HIGH to that of D₂Rs. To evaluate the reliability of the PET paradigm with [¹¹C]MNPA and [¹¹C]raclopride, we investigated the test-retest reproducibility of non-displaceable BP (BP _ND) measured with [¹¹C]MNPA and of [¹¹C]MNPA/[¹¹C]raclopride in healthy humans.

Methods

Eleven healthy male volunteers underwent two sets of PET studies on separate days that each included [¹¹C]MNPA and [¹¹C]raclopride scans. BP _ND values in the caudate and putamen were calculated. Test-retest reproducibility of BP _ND of [¹¹C]MNPA and [¹¹C]MNPA/[¹¹C]raclopride was assessed by intra-subject variability (absolute variability) and test-retest reliability (intraclass correlation coefficient: ICC).

Results

The absolute variability of [¹¹C]MNPA BP _ND was 5.30?±?3.96 % and 12.3?±?7.95 % and the ICC values of [¹¹C]MNPA BP _ND were 0.72 and 0.82 in the caudate and putamen, respectively. The absolute variability of [¹¹C]MNPA/[¹¹C]raclopride was 6.11?±?3.68 % and 11.60?±?5.70 % and the ICC values of [¹¹C]MNPA/[¹¹C]raclopride were 0.79 and 0.80 in the caudate and putamen, respectively.

Conclusion

In the present preliminary study, the test-retest reproducibility of BP _ND of [¹¹C]MNPA and of [¹¹C]MNPA/[¹¹C]raclopride was reliable in the caudate and putamen.     

Quantitative ex vivo and in vitro receptor autoradiography using 11C-labeled ligands and an imaging plate: a study with a dopamine D2-like receptor ligand [11C]nemonapride.

Ex vivo and in vitro autoradiography (ARG) with radioluminography is a useful technique to characterize newly developed 11C-labeled positron emission tomography (PET) tracers and to apply them to biological and pharmacological studies. In this report, we have described a method of evaluating the radioactivity distribution quantitatively in ex vivo and in vitro ARG using imaging plates and a dopamine D2-like receptor ligand [11C]nemonapride as a model compound. The photo-stimulated luminescence (PSL) values of the rat brain section provided by the imaging plates showed an excellent linear relationship with the radioactivity in a wide range under constant slice-thickness, although the PSL values slightly decreased with increasing slice-thickness both in ex vivo and in vitro ARG. The injection dose of 11C-tracers for ex vivo ARG was also discussed. We found saturable binding sites of [11C]nemonapride in the cortex besides the striatum both ex vivo and in vitro.     

A preliminary PET evaluation of the new dopamine D2 receptor agonist [11C]MNPA in cynomolgus monkey

This study describes the preliminary positron emission tomography (PET) evaluation of a dopamine D(2)-like receptor agonist, (R)-2-(11)CH(3)O-N-n-propylnorapomorphine ([(11)C]MNPA), as a potential new radioligand for in vivo imaging of the high-affinity state of the dopamine D(2) receptor (D(2)R). MNPA is a selective D(2)-like receptor agonist with a high affinity (K(i)=0.17 nM). [(11)C]MNPA was successfully synthesized by direct O-methylation of (R)-2-hydroxy-NPA using [(11)C]methyl iodide and was evaluated in cynomolgus monkeys. This study included baseline PET experiments and a pretreatment study using unlabeled raclopride (1 mg/kg). High uptake of radioactivity was seen in regions known to contain high D(2)R, with a maximum striatum-to-cerebellum ratio of 2.23+/-0.21 at 78 min and a maximum thalamus-to-cerebellum ratio of 1.37+/-0.06 at 72 min. The pretreatment study demonstrated high specific binding to D(2)R by reducing the striatum-to-cerebellum ratio to 1.26 at 78 min. This preliminary study indicates that the dopamine agonist [(11)C]MNPA has potential as an agonist radioligand for the D(2)-like receptor and has potential for examination of the high-affinity state of the D(2)R in human subjects and patients with neuropsychiatric disorders.     

Measurement of dopamine D2 receptors in living human brain using [11C]raclopride with ultra-high specific radioactivity

IntroductionHigh specific radioactivity is preferable in the measurement of neuroreceptor bindings with positron emission tomography (PET) because receptor occupancy by mixed cold ligand hampers the accurate estimation of receptor binding. Recently, we succeeded in synthesizing [¹¹C]raclopride, a dopamine D₂ receptor ligand, with ultra-high specific radioactivity, i.e., several thousand GBq/μmol. In the present study, we compared the [¹¹C]raclopride bindings to dopamine D₂ receptors between radioligands with ultra-high specific radioactivity and ordinary high specific radioactivity in healthy human subjects.MethodsTwo PET studies using [¹¹C]raclopride with ultra-high specific radioactivity (4302–7222 GBq/μmol) or ordinary high specific radioactivity (133-280 GBq/μmol) were performed on different days in 14 healthy men. Binding potential (BP) was calculated by the simplified reference tissue method, peak equilibrium method, and area-under-the-curve method for each region-of-interest using time-activity data in the cerebellum as a reference brain region.ResultsBP values for radioligands with ultra-high specific radioactivity and ordinary high specific radioactivity calculated by the simplified reference tissue method were 4.06±0.29 and 4.10±0.25 in the putamen, 0.44±0.07 and 0.47±0.07 in the thalamus and 0.37±0.06 and 0.38±0.06 in the temporal cortex, respectively (mean±S.D.). No significant difference in BP was observed between ultra-high specific radioactivity and ordinary high specific radioactivity in any of the brain regions.ConclusionBP values of [¹¹C]raclopride with ultra-high specific radioactivity did not differ from those with ordinary high specific radioactivity in the measured brain regions, including striatal and extrastriatal regions.     

[C]NNC 687 and [C]NNC 756, dopamine D-1 receptor ligands. Preparation, autoradiography and PET investigation in monkey

NNC 687 and NNC 756 [(+)-5-(2,3-dihydrobenzofuran-7-yl)-7-hydroxy-3-methyl-8-nitro-2,3,4,5-tetrahydro-1H-3-benzazepine and (+)-8-chloro-5-(2,3-dihydrobenzofuran-7-yl)-7-hydroxy-3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine] are two new potent dopamine D-1 receptor antagonists. [¹¹C]NNC 687 and [¹¹C]NNC 756 were both prepared by N-methylation of the corresponding desmethyl compounds with [¹¹C]methyl iodide. The reactions were performed in acetone with subsequent normal-phase semi-preparative HPLC and resulting in 50–60% radiochemical yield (from EOB and decay-corrected) with a total synthesis time of 30–35 min and a radiochemical purity higher than 99%. The specific radioactivity obtained at time of injection was about 1500 Ci/mmol (55 GBq/μmol). Autoradiographic examination of [¹¹C]NNC 687 and [¹¹C]NNC 756 binding in post-mortem human brain sections showed specific binding in the striatum, a region with high density of dopamine D-1 receptors. PET examination of the radioligands in a Cynomolgus monkey demonstrated accumulation of radioactivity predominantly in the striatum. The ratio between radioactivities in the striatum and the cerebellum was about 2 and 8 for [¹¹C]NNC 687 and [¹¹C]NNC 756 after 60 min. [¹¹C]NNC 756 should have potential as PET ligand for examination of central dopamine D-1 receptors in man.     

An improved synthesis of dopamine D2/D3 receptor radioligands [11C]fallypride and [18F]fallypride

Improved syntheses of dopamine D₂/D₃ receptor radioligands [¹¹C]Fallypride and [¹⁸F]Fallypride are reported. The phenolic precursor (9) for C-11 labeling and the Fallypride (10) reference standard were synthesized from the starting material 2-hydroxy-3-methoxy-5-(2-propenyl)benzoic acid methyl ester (1) in 7 and 8 steps with 16% and 5% overall chemical yields, respectively. The tosylated precursor (15) for F-18 labeling was synthesized from compound 1 in 5 steps with 32% overall chemical yield. An alternate synthetic approach for Fallypride has been developed using the same starting material 1 in 5 steps with 26% overall chemical yield. [¹¹C]Fallypride ([¹¹C]10) was prepared by O-[¹¹C]methylation of the phenolic precursor with [¹¹C]methyl triflate and purified with a semi-preparative HPLC method in 50–60% radiochemical yield, decay corrected to end of bombardment (EOB), based on [¹¹C]CO₂, and 370±185 GBq/μmol specific radioactivity at EOB. [¹⁸F]Fallypride ([¹⁸F]10) was prepared by nucleophilic substitution of the tosylated precursor with K[¹⁸F]F/Kryptofix 2.2.2 and HPLC combined with solid-phase extraction (SPE) purification in variable (up to 50%) decay corrected radiochemical yield from K[¹⁸F]F and 111–222 GBq/μmol specific activity at EOB.     

Enhanced radiosyntheses of [11C]raclopride and [11C]DASB using ethanolic loop chemistry

IntroductionTo improve the synthesis and quality control of carbon-11 labeled radiopharmaceuticals, we report the fully automated loop syntheses of [¹¹C]raclopride and [¹¹C]DASB using ethanol as the only organic solvent for synthesis module cleaning, carbon-11 methylation, HPLC purification, and reformulation.MethodsEthanolic loop chemistry is fully automated using a GE TRACERLab FX_C-Pro synthesis module, and is readily adaptable to any other carbon-11 synthesis apparatus. Precursors (1 mg) were dissolved in ethanol (100 μL) and loaded into the HPLC loop. [¹¹C]MeOTf was passed through the HPLC loop and then the labeled products were purified by semi-preparative HPLC and reformulated into ethanolic saline.ResultsBoth [¹¹C]raclopride (3.7% RCY; > 95% RCP; SA = 20831 Ci/mmol; n = 64) and [¹¹C]DASB, both with (3.0% RCY; > 95% RCP; SA = 15152 Ci/mmol; n = 9) and without (3.0% RCY; > 95% RCP; SA = 10931 Ci/mmol; n = 3) sodium ascorbate, have been successfully prepared using the described methodology. Doses are suitable for human use and the described methods are now employed for routine clinical production of both radiopharmaceuticals at the University of Michigan.ConclusionsEthanolic loop chemistry is a powerful technique for preparing [¹¹C]raclopride and [¹¹C]DASB, and we are in the process of adapting it for other carbon-11 radiopharmaceuticals prepared in our laboratories ([¹¹C]PMP, [¹¹C]PBR28 etc.).     

Synthesis of [11C]SCH 23390 for dopamine D1 receptor studies

The optimal conditions for the synthesis of ¹¹C-labeled SCH 23390 by radio-methylation of its desmethyl precursor, SCH 24518, with [¹¹C]iodomethane are described. Isocratic reversed phase HPLC was used for the purification of [¹¹C]SCH 23390. The specific activity range in 30 runs was 10–235 Ci/mmol and average radiochemical yield was 72% based on [¹¹C]iodomethane. Mean synthesis time was 40–60 min from the end of bombardment. Preliminary animal studies indicate that [¹¹C]SCH 23390 would be useful in visualizing D1 receptors in a living brain by positron tomography.     

Measurement of extrastriatal D2-like receptor binding with [11C]FLB 457--a test-retest analysis

[11C]FLB 457 is a radioligand for positron emission tomography (PET) that possesses high affinity to D2/D3 receptors. It has been suggested to be useful for quantification of low-density dopamine D2 receptor populations, e.g. in cortical and limbic brain areas. We explored the reproducibility of five methods for measuring extrastriatal D2-like receptor binding potential with [11C]FLB 457. Seven healthy male volunteers were examined twice with [11C]FLB 457 (high specific radioactivity) on the same day, at least 3 h apart. Four brain areas, frontal cortex, nucleus thalamus, temporal cortex and cerebellar cortex, were examined. Binding potentials (BPs) were derived from (1) a target to cerebellum distribution volume ratio, (2/3) two reversible reference tissue compartment models and (4) a transient equilibrium approach. For comparison, BP values were also calculated with the standard three-compartment kinetic model that does not assume a receptor-free reference region. The use of the standard three-compartment model did not result in reproducible BP estimates. The distribution volume (DV) ratio, reference tissue compartment models and the transient equilibrium method all had good to excellent intraclass correlation coefficients (ICCs) in the studied brain areas ranging from 0.56 to 0.93. Absolute variability was also relatively low, ranging from 5.3% to 10.4%. There were no marked differences in the ICC or absolute and relative variability between the four methods based on a reference tissue (cerebellum). In addition, we did not observe systematic differences in the BP between the first and the second scan. These data indicate that the reproducibility of the DV ratio, reference tissue models and the transient equilibrium method is good or excellent. However, each of these methods includes assumptions affecting their validity. Thus, the choice of method will be critically dependent on the purpose of the study.     

Effects of reserpine treatment on the dopamine receptor binding of [3H/11C]nemonapride in the mouse and rat brain

We investigated the effect of reserpine treatment on the striatal uptake of a radiolabeled dopamine D₂-like receptor ligand nemonapride (NEM). In mice, the uptake of the [³H]NEM in the striatum, cortex and cerebellum was enhanced by the reserpine pretreatment. Neither the ratio of striatum to cerebellum nor that to cortex was affected by the reserpine pretreatment. In rats,ex vivo autoradiography showed no effect of the reserpine treatment on the striatal uptake of [¹¹C]NEM or the striatum to cortex ratio. The results suggest that the receptor binding of NEM was not significantly influenced by reserpine-induced depletion of endogenous dopamine probably because of its high affinity for the receptors.     

Sigma1 and dopamine D2 receptor occupancy in the mouse brain after a single administration of haloperidol and two dopamine D2-like receptor ligands

We investigated sigma(1) and dopamine D(2) receptor occupancy in mouse brain after a single injection of haloperidol, nemonapride, or spiperone using [(11)C]SA4503 and [(11)C]raclopride, respectively. Co-injection of the three compounds significantly blocked the uptake of each radioligand. Six hours later, only haloperidol blocked [(11)C]SA4503 uptake, while all three reduced [(11)C]raclopride uptake. Sigma(1) receptor occupancy by haloperidol was reduced to 19% at day 2 when D(2) receptor occupancy disappeared. [(11)C]SA4503 would be applicable to the investigation of sigma(1) receptor occupancy of antispychotic drugs using PET.     

Synthesis and biological evaluation of [11C]talopram and [11C]talsupram: candidate PET ligands for the norepinephrine transporter

PET and SPECT ligands for the norepinephrine transporter (NET) will be important tools for studying the physiology, pathophysiology and pharmacology of the CNS noradrenergic system in vivo. A series of candidate NET ligands were synthesized and characterized in terms of their affinity for human monoamine transporters. The two most promising compounds, talopram and talsupram, were radiolabeled with carbon-11 and evaluated through biodistribution studies in rats and PET imaging studies in a rhesus monkey. Although both compounds displayed high affinity and selectivity for the human NET in vitro, these compounds did not enter the CNS in adequate amounts to be used in PET imaging studies.     

Biodistribution and radiation dosimetry of [11C]raclopride in healthy volunteers

Purpose This study reports on the whole-body biodistribution and radiation dosimetry of [¹¹C]raclopride, a dopamine D₂ receptor antagonist.Methods In three healthy male volunteers, whole-body scans were performed up to 2 h following i.v. injection of 320±65 MBq [¹¹C]raclopride. Transmission scans (3 min per step, eight or nine steps according to the height of the subject) in 2D mode were used for subsequent attenuation correction of emission scans. Emission scans (1 min per step, eight or nine steps) were acquired over 2 h. Venous blood samples and urine were collected up to 2 h after injection of the radiotracer. For each subject, the percentage of injected activity measured in regions of interest over brain, intestine, lungs, kidneys and liver was fitted to a mono-exponential model, as an uptake phase followed by a mono-exponential washout, for urinary bladder to generate time–activity curves. Using the MIRD method, several source organs were considered in estimating residence time and mean effective radiation absorbed doses.Results Blood pressure and ECG findings remained unchanged after tracer injection. The analysed blood and urine pharmacological parameters did not change significantly after [¹¹C]raclopride injection. The primary routes of clearance were renal and intestinal. Ten minutes after injection, high activities were observed in the gall-bladder, kidneys and liver. High activity was observed in the gall-bladder during the whole study. The kidneys, urinary bladder wall, liver and gall-bladder received the highest absorbed doses. The average effective dose of [¹¹C]raclopride was estimated to be 6.7±0.4 Sv/MBq.Conclusion The amount of [¹¹C]raclopride required for adequate dopamine D₂ receptor imaging results in an acceptable effective dose equivalent, permitting two or three repeated clinical PET imaging studies, with the injection of 222 MBq for each study.     

Preclinical evaluation of [11C]NE40, a type 2 cannabinoid receptor PET tracer

IntroductionUp-regulation of the type 2 cannabinoid receptor (CB₂R) has been reported in (neuro)inflammatory diseases. In this study, we report the preclinical evaluation of [¹¹C]NE40 as positron emission tomography (PET) radioligand for visualization of the CB₂R.MethodsThe selectivity of NE40 for CB₂R and its toxicity and mutagenicity were determined. [¹¹C]NE40 was evaluated by biodistribution and autoradiography studies in normal rats and a microPET study in normal mice, rats and a rhesus monkey. Specific in vivo binding of [¹¹C]NE40 to human CB₂R (hCB₂R) was studied in a rat model with hCB₂R overexpression.Results[¹¹C]NE40 shows specific CB₂R binding in the spleen and blood of normal rats and high brain uptake in rhesus monkey. [¹¹C]NE40 showed specific and reversible binding to hCB₂R in vivo in a rat model with local hCB₂R overexpression.Conclusions[¹¹C]NE40 shows favorable characteristics as radioligand for in vivo visualization of the CB₂R and is a promising candidate for hCB₂R PET imaging.     

Measurement of dopamine release with continuous infusion of [11C]raclopride: optimization and signal-to-noise considerations.

PET studies with [11C]raclopride provide an indirect measure of changes in synaptic dopamine. Previously, we used the bolus-plus-infusion (B/I) method to assess dopamine response from the percentage change in binding potential (deltaBP) before and after administration of amphetamine. The goal of this work is to optimize the measurement of changes in neurotransmitter with the B/I method by choosing the optimal timing for pre- and poststimulus scanning. METHODS: Two sources of variability in deltaBP were considered: within-subject and between-subject noise. A noise model based on a phantom study and human data was used to evaluate the within-subject noise. For between-subject noise, simulated time--activity curves were generated from measured [11C]raclopride input functions. Optimal timing to measure deltaBP was determined and applied to human data. RESULTS: According to the simulation study, the optimal scan times for pre-and poststimulus scans were 39-50 and 58-100 min, respectively. The optimal timing resulted in a 28% noise reduction compared with the original timing. By applying the optimal timing to human studies, the statistical significance of the difference in deltaBP between patients with schizophrenia and healthy volunteers increased from P = 0.038 to 0.012. CONCLUSION: Careful assessment of the sources of noise in receptor imaging studies can increase the sensitivity of the B/I method for the detection of biologic signals.     

Radiosynthesis of [2-11C-carbonyl]dantrolene using [11C]phosgene for PET

Automated radiosynthesis of [2-¹¹C-carbonyl]dantrolene, the substrate of breast cancer resistance protein (BCRP/ABCG2), was performed for the first time through a multi-step/one-pot labeling sequence that started with ethyl 2-{2-[5-(4-nitrophenyl)furfurylidene]hydrazino}acetate and used [¹¹C]phosgene as a labeling agent. After optimization of the automated synthesis conditions and parameters, [2-¹¹C-carbonyl]dantrolene was obtained at a radiochemical yield of 34.0±8.4% (decay-corrected). The radiochemical purity was greater than 98% and the specific activity was 46.8±15.2 GBq/μmol at the end of the synthesis.     

An alternative synthesis of [11C]raclopride for routine use

The standard method of [11C]raclopride synthesis requires a large amount of its desmethyl precursor. We prepared [11C]raclopride by methylation of a small amount of desmethyl derivative (0.3-0.5 mg) with [11C]methyl iodide in a DMF solution containing NaH, with a decay-corrected radiochemical yield of 11-14% based on [11C]methyl iodide and with a specific activity of 48 TBq/mmol for 25 min from EOB. The reaction was reproducible and reliable.     

Correction of head movement by frame-to-frame image realignment for receptor imaging in positron emission tomography studies with [11C]raclopride and [11C]FLB 457

Annals of Nuclear Medicine - Positron emission tomography (PET) scans of imaging receptors require 60–90-min dynamic acquisition for quantitative analysis. Head movement is often observed...