Muscarinic acetylcholine receptors (mAChR), including M4, draw attention as therapeutic targets for several neurodegenerative diseases including Alzheimer's disease (AD). PET imaging of M4 positive ...allosteric modulator (PAM) allows qualification of the distribution as well as the expression of this receptor under physiological conditions and thereby helps to assess the receptor occupancy (RO) of a drug candidate. In this study, our aims were (a) to synthesize a novel M4 PAM PET radioligand 11CPF06885190 (b) to evaluate the brain distribution of
CPF06885190 in nonhuman primates (NHP) and (c) to analyze its radiometabolites in the blood plasma of NHP. Radiolabeling of
CPF06885190 was accomplished via N-methylation of the precursor. Six PET measurements were performed using two male cynomolgus monkeys, where three PET measurements were at baseline, two after pretreatment with a selective M4 PAM compound CVL-231 and one after pretreatment with donepezil. The total volume of distribution (
) of
CPF06885190 was examined using Logan graphical analysis with arterial input function. Radiometabolites were analyzed in monkey blood plasma using gradient HPLC system. Radiolabeling of
CPF06885190 was successfully accomplished and the radioligand was found to be stable in the formulation, with radiochemical purity exceeding 99% 1 h after the end of the synthesis.
CPF06885190 was characterized in the cynomolgus monkey brain where a moderate brain uptake was found at the baseline condition. However, it showed fast wash-out as it dropped to half of the peak at around 10 min. Change of
from baseline was around -10% after pretreatment with a M4 PAM, CVL-231. Radiometabolite studies showed relatively fast metabolism. Although sufficient brain uptake of
CPF06885190 was observed, these data suggest that
CPF06885190 might have too low specific binding in the NHP brain to be further applied in PET imaging.
Purpose
Beta-secretase 1 (BACE1) enzyme is implicated in the pathophysiology of Alzheimer’s disease.
18
FPF-06684511 is a positron emission tomography (PET) radioligand for imaging BACE1. Despite ...favorable brain kinetic properties, the effective dose (ED) of
18
FPF-06684511 estimated in non-human primates was relatively high. This study was therefore designed to evaluate the whole-body distribution, dosimetry, quantification, and test-retest reliability of imaging brain BACE1 with
18
FPF-06684511 in healthy volunteers.
Methods
Five subjects were studied for the dosimetry study. Whole-body PET was performed for 366 min with 4 PET-CT sessions. Estimates of the absorbed radiation dose were calculated using the male adult model. Eight subjects participated in the test-retest study. Brain PET measurements were conducted for 123 min with an interval of 5 to 19 days between test and retest conditions. The total distribution volume (
V
T
) was estimated with one-tissue (1T), two-tissue (2T), compartment model (CM), and graphical analysis. Test-retest variability (TRV) and intraclass correlation coefficient (ICC) of
V
T
were calculated as reliability measures.
Results
In the dosimetry study, the highest uptake was found in the liver (25.2 ± 2.3 %ID at 0.5 h) and the largest dose was observed in the pancreas (92.9 ± 52.2 μSv/MBq). The calculated ED was 24.7 ± 0.8 μSv/MBq. In the test-retest study, 2TCM described the time-activity curves well.
V
T
(2TCM) was the highest in the anterior cingulate cortex (6.28 ± 1.09 and 6.85 ± 0.81) and the lowest in the cerebellum (4.23 ± 0.88 and 4.20 ± 0.75). Mean TRV and ICC of
V
T
(2TCM) were 16.5% (12.4–20.5%) and 0.496 (0.291–0.644).
Conclusion
The ED of
18
FPF-06684511 was similar to other
18
F radioligands, allowing repeated PET measurements. 2TCM was the most appropriate quantification method. TRV of
V
T
was similar to other radioligands without a reference region, albeit with lower ICC. These data indicated that
18
FPF-06684511 is a suitable radioligand to measure BACE1 level in the human brain.
Trial registration
EudraCT 2016-001110-19 (registered 2016-08-08)
Background
The G-protein-coupled receptor 44 (GPR44) is a beta cell-restricted target that may serve as a marker for beta cell mass (BCM) given the development of a suitable PET ligand.
Methods
The ...binding characteristics of the selected candidate, AZ12204657, at human GPR44 were determined using in vitro ligand binding assays. AZ12204657 was radiolabeled using
11
C- or
3
H-labeled methyl iodide (
11
C/
3
HCH
3
I) in one step, and the conversion of
11
C/
3
HCH
3
I to the radiolabeled product
11
C/
3
HAZ12204657 was quantitative. The specificity of radioligand binding to GPR44 and the selectivity for beta cells were evaluated by in vitro binding studies on pancreatic sections from human and non-human primates as well as on homogenates from endocrine and exocrine pancreatic compartments.
Results
The radiochemical purity of the resulting radioligand
11
CAZ12204657 was > 98%, with high molar activity (MA), 1351 ± 575 GBq/μmol (
n
= 18). The radiochemical purity of
3
HAZ12204657 was > 99% with MA of 2 GBq/μmol. Pancreatic binding of
11
C/
3
HAZ12204657 was co-localized with insulin-positive islets of Langerhans in non-diabetic individuals and individuals with type 2 diabetes (T2D). The binding of
11
CAZ12204657 to GPR44 was > 10 times higher in islet homogenates compared to exocrine homogenates. In human islets of Langerhans GPR44 was co-expressed with insulin, but not glucagon as assessed by co-staining and confocal microscopy.
Conclusion
We radiolabeled
11
CAZ12204657, a potential PET radioligand for the beta cell-restricted protein GPR44. In vitro evaluation demonstrated that
3
HAZ12204657 and
11
CAZ12204657 selectively target pancreatic beta cells.
11
CAZ12204657 has promising properties as a marker for human BCM.
Abstract Introduction The positron emission tomography (PET) tracer 9-18 Ffluoroethyl-(+)-dihydrotetrabenazine (18 F-FE-(+)-DTBZ) is a potential candidate for quantifying beta-cell mass in vivo. The ...purpose was to investigate in vitro and in vivo utility of this tracer for the assessment of beta-cell mass. Methods Three pigs were intravenously administered 18 F-FE-(+)-DTBZ and examined by PET/computed tomography. Binding parameters were estimated by kinetic modeling. In vitro kD and Bmax were determined by saturation binding studies of endocrine and exocrine human tissue homogenates. In vitro pancreatic uptake was determined by tissue autoradiography with pancreases from patients with types 1 (T1DM) and 2 diabetes mellitus (T2DM) and healthy controls. Results 18 F-FE-(+)-DTBZ had a kD of 3.5±1.0 nM, a Bmax of 382±108 fmol/mg protein and a specificity of 89±1.8% in islet homogenates. The total exocrine uptake was lower and 65% was nondisplaceable. No uptake difference was observed in pancreatic tissue slices from patients with T1DM, T2DM or healthy controls. The in vivo porcine pancreatic uptake reached a peak of standardized uptake value (SUV) of 2.8 with a low distribution volume ratio in all animals. Moderate to high tracer uptake was identified in the bile system and in bone. Conclusions 18 F-FE-(+)-DTBZ binds to vesicular monoamine transporter 2 (VMAT2) with high specificity in pure islet tissue in vitro. However, there is high nondisplaceable binding to exocrine tissue. In addition, in vivo tracer metabolism and dehalogenation result in severe underestimation of porcine pancreatic VMAT2 expression and BCM. The results do not support 18 F-FE-(+)-DTBZ as a suitable tracer for in vivo beta-cell imaging.
The radioligand 18FFPEB, used for PET imaging of the brain's metabotropic glutamate receptor subtype 5 (mGluR5), undergoes a thorough validation process to ensure its safety, efficacy, and quality ...for clinical use. The process starts by optimizing the synthesis of 18FFPEB to achieve high radiochemical yield and purity. This study focuses on optimizing the radiolabeling process using an aryl‐chloro precursor and validating the GMP production for clinical applications. Fully automated radiolabeling was achieved via one‐step nucleophilic substitution reaction. 18FFPEB was produced and isolated in high radioactivity and radiochemical purity. Throughout the validation process, thorough quality control measures are implemented. Radiopharmaceutical batch release criteria are established, including testing for physical appearance, filter integrity, pH, radiochemical purity, molar activity, radiochemical identity, chemical impurity, structural identity, stability, residual solvent, sterility, and endotoxin levels. In conclusion, the validation of 18FFPEB involved a comprehensive process of synthesis optimization, quality control, which ensure the safety, efficacy, and quality of 18FFPEB, enabling its reliable use in clinical PET. Here, we successfully radiolabeled and validated 18FFPEB using aryl‐chloro precursor according to GMP production for clinical application.
Validation of mGluR5 PET tracer 18FFPEB using aryl‐chloro precursor.
The vesicular monoamine transporter type 2 (VMAT2) is believed to be responsible for the uptake of monoamines into the vesicles of the synaptic terminals. Two VMAT2 radioligands 11CDTBZ and ...18FFP-DTBZ have been used to assess the degree of nigrostriatal deficit in Parkinson’s disease (PD) using positron emission tomography (PET). 18FFE-DTBZ-d4, the nondeuterated analogue of 18FFE-DTBZ showed similar imaging properties with better stability against defluorination. Therefore, 18FFE-DTBZ-d4 draws attention to be investigated as an imaging marker for VMAT2 in the brain. The aim of this study was to investigate the brain kinetics and quantification of 18FFE-DTBZ-d4 in nonhuman primates (NHPs), with comparison to 11CDTBZ and 18FFE-DTBZ. Radiolabeling was successfully achieved either by one-step 11C-methylation or by a two-step fluorine-18 nucleophilic substitution reaction. The stability and radiochemical yield were analyzed with high-performance liquid chromatography (HPLC). Three female cynomolgus monkeys were included in the study and underwent a total of 12 positron emission tomography (PET) measurements. Each monkey was examined with each tracer. In addition, two pretreatment and one displacement PET measurements with tetrabenazine (2.0 mg/kg) were performed for 18FFE-DTBZ-d4. All PET measurements were conducted using a high-resolution research tomograph (HRRT) system. Radiometabolites were measured in monkey plasma using gradient radio-HPLC. 18FFE-DTBZ-d4 (SUV: 4.28 ± 1.01) displayed higher brain uptake compared to both 18FFE-DTBZ (SUV: 3.43 ± 0.54) and 11CDTBZ (SUV: 3.06 ± 0.32) and faster washout. Binding potential (BPND) values of 18FFE-DTBZ-d4 in different brain regions (putamen: 5.5 ± 1.4; caudate: 4.4 ± 1.1; midbrain: 1.4 ± 0.4) were higher than those of 11CDTBZ and 18FFE-DTBZ. 18FFE-DTBZ showed faster radiometabolism in plasma compared to 11CDTBZ and 18FFE-DTBZ-d4. 18FFE-DTBZ-d4 is a suitable radioligand for quantification of VMAT2 in the nonhuman primate brain, with better imaging properties than 11CDTBZ and 18FFE-DTBZ. A preliminary comparison suggests that 18FFE-DTBZ-d4 has increased stability against defluorination compared to the nondeuterated analogue.
GPR44 expression has recently been described as highly β-cell selective in the human pancreas and constitutes a tentative surrogate imaging biomarker in diabetes. A radiolabeled small-molecule GPR44 ...antagonist,
CAZ12204657, was evaluated for visualization of β-cells in pigs and nonhuman primates by positron emission tomography as well as in immunodeficient mice transplanted with human islets under the kidney capsule. In vitro autoradiography of human and animal pancreatic sections from subjects without and with diabetes, in combination with insulin staining, was performed to assess β-cell selectivity of the radiotracer. Proof of principle of in vivo targeting of human islets by
CAZ12204657 was shown in the immunodeficient mouse transplantation model. Furthermore,
CAZ12204657 bound by a GPR44-mediated mechanism in pancreatic sections from humans and pigs without diabetes, but not those with diabetes. In vivo
CAZ12204657 bound specifically to GPR44 in pancreas and spleen and could be competed away dose-dependently in nondiabetic pigs and nonhuman primates.
CAZ12204657 is a first-in-class surrogate imaging biomarker for pancreatic β-cells by targeting the protein GPR44.
Neutrophils are part of the innate immune system and function as a first line of defense against invading microorganisms. Overactivity of the immune system may result in a devastating ...immuno-inflammation with extensive damage to tissue leading to organ damage and/or failure. The literature suggests several human diseases in which neutrophil elastase (NE) is postulated to be important in the pathophysiology including inflammatory bowel disease (IBD), chronic obstructive pulmonary disorder (COPD), abdominal aortic aneurysms (AAA), breast and lung cancer, and recently also in Sars-cov-2 virus infection (Covid-19). In particular, the lungs are affected by the destructive power of the protease neutrophil elastase (NE). In this paper, we report the pre-clinical development of a selective and specific positron emission tomography (PET) tracer, 11CGW457427, as an in vivo biomarker for the study of NE, now available for human studies.
11CGW457427 was produced by methylation of GW447631 using 11Cmethyl triflate and GMP validated production and quality control methods were developed. Chemical purity was high with no traces of the precursor GW611437 or other uv-absorbing compounds. A method for the determination of intact 11CGW457427 in plasma was developed and the binding characteristics were evaluated in vitro and in vivo. An animal model for lung inflammation was used to investigate the specificity and sensitivity of the 11CGW457427 tracer for neutrophil elastase (NE) in pulmonary inflammation, verified by blockade using two structurally different elastase inhibitors.
11CGW457427 was obtained in approximately 45% radiochemical yield and with a radiochemical purity higher than 98%. Molar activity was in the range 130–360 GBq/μmol. Binding to NE was shown to be highly specific both in vitro and in vivo and a significantly higher uptake of tracer was found in a lipopolysaccharide mouse model of pulmonary inflammation compared with control animals. The uptake in lung tissue measured as standardized uptake value (SUV) strongly correlated with tissue NE content as measured by ELISA. In vitro studies also showed specific tracer binding in aortic tissue of patients with abdominal aorta aneurysm (AAA). The rate of metabolism in rats was appropriate considering the critical balance between available tracer for binding and requirement for blood clearance with about 40% and 20% intact 11CGW457427 in plasma at 5 and 40 min, respectively. Radioactivity was cleared from blood and organs in control animals with mainly hepatobiliary excretion with distribution in the intestines and the urinary bladder; but without retention of the tracer in healthy organs of interests such as the lung, liver, kidneys or in the cardiovascular system. A dosimetry study in rat indicated that the whole-body effective dose was 2.2 μSv/MBq with bone marrow as the limiting organ. It is estimated that up to five PET-CT investigations could be performed in humans without exceeding a total dose of 10 mSv.
11CGW457427 is a promising in vivo PET-biomarker for NE with high specific binding demonstrated both in vitro and in vivo. A GMP validated production method including quality control has been developed and a microdosing toxicity study performed with no adverse signs. 11CGW457427 is currently being evaluated in a First-In-Man PET study.
The metabotropic glutamate receptor subtype mGluR5 has been proposed as a potential drug target for CNS disorders such as anxiety, depression, Parkinson's disease, and epilepsy. The AstraZeneca ...compound AZD9272 has previously been labeled with carbon-11 and used as a PET radioligand for mGluR5 receptor binding. The molecular structure of AZD9272 allows one to label the molecule with fluorine-18 without altering the structure. The aim of this study was to develop a fluorine-18 analogue of AZD9272 and to examine its binding distribution in the nonhuman primate brain
as well as to obtain whole body radiation dosimetry.
F-AZD9272 was successfully synthesized from a nitro precursor. The radioligand was stable, with a radiochemical purity of >99% at 2 h after formulation in a sterile phosphate buffered solution (pH = 7.4). After injection of
F-AZD9272 in two cynomolgus monkeys, the maximum whole brain radioactivity concentration was 4.9-6.7% of the injected dose (
= 2) and PET images showed a pattern of regional radioactivity consistent with that previously obtained for
C-AZD9272. The percentage of parent radioligand in plasma was 59 and 64% (
= 2) at 120 min after injection of
F-AZD9272, consistent with high metabolic stability. Two whole body PET scans were performed in nonhuman primates for a total of 231 min after injection of
F-AZD9272. Highest uptakes were seen in liver and small intestine, followed by brain and kidney. The estimated effective dose was around 0.017 mSv/MBq.
F-AZD9272 shows suitable properties as a PET radioligand for
imaging of binding in the primate brain.
F-labeled AZD9272 offers advantages over
C-AZD9272 in terms of higher image resolution, combined with a longer half-life. Moreover, based on the distribution and the estimated radiation burden, imaging of
F-AZD9272 could be used as an improved tool for quantitative assessment and characterization of AZD9272 binding sites in the human brain by using PET.
The metabotropic glutamate receptor subtype mGluR5 has been proposed as a potential drug target for CNS disorders such as anxiety, depression, Parkinson's disease, and epilepsy. The AstraZeneca ...compound AZD9272 has previously been labeled with carbon-11 and used as a PET radioligand for mGluR5 receptor binding. The molecular structure of AZD9272 allows one to label the molecule with fluorine-18 without altering the structure. The aim of this study was to develop a fluorine-18 analogue of AZD9272 and to examine its binding distribution in the nonhuman primate brain in vivo as well as to obtain whole body radiation dosimetry. F-18-AZD9272 was successfully synthesized from a nitro precursor. The radioligand was stable, with a radiochemical purity of >99% at 2 h after formulation in a sterile phosphate buffered solution (pH = 7.4). After injection of F-18-AZD9272 in two cynomolgus monkeys, the maximum whole brain radioactivity concentration was 4.9-6.7% of the injected dose (n = 2) and PET images showed a pattern of regional radioactivity consistent with that previously obtained for C-11-AZD9272. The percentage of parent radioligand in plasma was 59 and 64% (n = 2) at 120 min after injection of F-18-AZD9272, consistent with high metabolic stability. Two whole body PET scans were performed in nonhuman primates for a total of 231 min after injection of F-18-AZD9272. Highest uptakes were seen in liver and small intestine, followed by brain and kidney. The estimated effective dose was around 0.017 mSv/MBq. F-18-AZD9272 shows suitable properties as a PET radioligand for in vivo imaging of binding in the primate brain. F-18-labeled AZD9272 offers advantages over C-11-AZD9272 in terms of higher image resolution, combined with a longer half-life. Moreover, based on the distribution and the estimated radiation burden, imaging of F-18-AZD9272 could be used as an improved tool for quantitative assessment and characterization of AZD9272 binding sites in the human brain by using PET.
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