Showing papers by "Robert B. Innis published in 2020"

PET imaging of neuroinflammation in neurological disorders

Abstract: A growing need exists for reliable in-vivo measurement of neuroinflammation to better characterise the inflammatory processes underlying various diseases and to inform the development of novel therapeutics that target deleterious glial activity. PET is well suited to quantify neuroinflammation and has the potential to discriminate components of the neuroimmune response. However, there are several obstacles to the reliable quantification of neuroinflammation by PET imaging. Despite these challenges, PET studies have consistently identified associations between neuroimmune responses and pathophysiology in brain disorders such as Alzheimer's disease. Tissue studies have also begun to clarify the meaning of changes in PET signal in some diseases. Furthermore, although PET imaging of neuroinflammation does not have an established clinical application, novel targets are under investigation and a small but growing number of studies have suggested that this imaging modality could have a role in drug development. Future studies are needed to further improve our knowledge of the cellular mechanisms that underlie changes in PET signal, how immune response contributes to neurological disease, and how it might be therapeutically modified.

Guidelines for the content and format of PET brain data in publications and archives: A consensus paper:

Abstract: It is a growing concern that outcomes of neuroimaging studies often cannot be replicated. To counteract this, the magnetic resonance (MR) neuroimaging community has promoted acquisition standards and created data sharing platforms, based on a consensus on how to organize and share MR neuroimaging data. Here, we take a similar approach to positron emission tomography (PET) data. To facilitate comparison of findings across studies, we first recommend publication standards for tracer characteristics, image acquisition, image preprocessing, and outcome estimation for PET neuroimaging data. The co-authors of this paper, representing more than 25 PET centers worldwide, voted to classify information as mandatory, recommended, or optional. Second, we describe a framework to facilitate data archiving and data sharing within and across centers. Because of the high cost of PET neuroimaging studies, sample sizes tend to be small and relatively few sites worldwide have the required multidisciplinary expertise to properly conduct and analyze PET studies. Data sharing will make it easier to combine datasets from different centers to achieve larger sample sizes and stronger statistical power to test hypotheses. The combining of datasets from different centers may be enhanced by adoption of a common set of best practices in data acquisition and analysis.

PET measurement of cyclooxygenase-2 using a novel radioligand: upregulation in primate neuroinflammation and first-in-human study.

Abstract: Cyclooxygenase-2 (COX-2), which is rapidly upregulated by inflammation, is a key enzyme catalyzing the rate-limiting step in the synthesis of several inflammatory prostanoids. Successful positron emission tomography (PET) radioligand imaging of COX-2 in vivo could be a potentially powerful tool for assessing inflammatory response in the brain and periphery. To date, however, the development of PET radioligands for COX-2 has had limited success. The novel PET tracer [11C]MC1 was used to examine COX-2 expression [1] in the brains of four rhesus macaques at baseline and after injection of the inflammogen lipopolysaccharide (LPS) into the right putamen, and [2] in the joints of two human participants with rheumatoid arthritis and two healthy individuals. In the primate study, two monkeys had one LPS injection, and two monkeys had a second injection 33 and 44 days, respectively, after the first LPS injection. As a comparator, COX-1 expression was measured using [11C]PS13. COX-2 binding, expressed as the ratio of specific to nondisplaceable uptake (BPND) of [11C]MC1, increased on day 1 post-LPS injection; no such increase in COX-1 expression, measured using [11C]PS13, was observed. The day after the second LPS injection, a brain lesion (~ 0.5 cm in diameter) with high COX-2 density and high BPND (1.8) was observed. Postmortem brain analysis at the gene transcript or protein level confirmed in vivo PET results. An incidental finding in an unrelated monkey found a line of COX-2 positivity along an incision in skull muscle, demonstrating that [11C]MC1 can localize inflammation peripheral to the brain. In patients with rheumatoid arthritis, [11C]MC1 successfully imaged upregulated COX-2 in the arthritic hand and shoulder and apparently in the brain. Uptake was blocked by celecoxib, a COX-2 preferential inhibitor. Taken together, these results indicate that [11C]MC1 can image and quantify COX-2 upregulation in both monkey brain after LPS-induced neuroinflammation and in human peripheral tissue with inflammation. ClinicalTrials.gov NCT03912428. Registered April 11, 2019.

First-in-human evaluation of [11C]PS13, a novel PET radioligand, to quantify cyclooxygenase-1 in the brain.

Abstract: This study assessed whether the newly developed PET radioligand [11C]PS13, which has shown excellent in vivo selectivity in previous animal studies, could be used to quantify constitutive levels of cyclooxygenase-1 (COX-1) in healthy human brain. Brain test-retest scans with concurrent arterial blood samples were obtained in 10 healthy individuals. The one- and unconstrained two-tissue compartment models, as well as the Logan graphical analysis were compared, and test-retest reliability and time-stability of total distribution volume (VT) were assessed. Correlation analyses were conducted between brain regional VT and COX-1 transcript levels provided in the Allen Human Brain Atlas. In the brain, [11C]PS13 showed highest uptake in the hippocampus and occipital cortex. The pericentral cortex also showed relatively higher uptake compared with adjacent neocortices. The two-tissue compartment model showed the best fit in all the brain regions, and the results from the Logan graphical analysis were consistent with those from the two-tissue compartment model. VT values showed excellent test-retest variability (range 6.0–8.5%) and good reliability (intraclass correlation coefficient range 0.74–0.87). VT values also showed excellent time-stability in all brain regions, confirming that there was no radiometabolite accumulation and that shorter scans were still able to reliably measure VT. Significant correlation was observed between VT and COX-1 transcript levels (r = 0.82, P = 0.007), indicating that [11C]PS13 binding reflects actual COX-1 density in the human brain. These results from the first-in-human evaluation of the ability of [11C]PS13 to image COX-1 in the brain justifies extending the study to disease populations with neuroinflammation. NCT03324646 at https://clinicaltrials.gov/ . Registered October 30, 2017. Retrospectively registered.

PET ligands [18F]LSN3316612 and [11C]LSN3316612 quantify O-linked-β-N-acetyl-glucosamine hydrolase in the brain.

Abstract: We aimed to develop effective radioligands for quantifying brain O-linked-β-N-acetyl-glucosamine (O-GlcNAc) hydrolase (OGA) using positron emission tomography in living subjects as tools for evaluating drug target engagement. Posttranslational modifications of tau, a biomarker of Alzheimer's disease, by O-GlcNAc through the enzyme pair OGA and O-GlcNAc transferase (OGT) are inversely related to the amounts of its insoluble hyperphosphorylated form. Increase in tau O-GlcNAcylation by OGA inhibition is believed to reduce tau aggregation. LSN3316612, a highly selective and potent OGA ligand [half-maximal inhibitory concentration (IC50) = 1.9 nM], emerged as a lead ligand after in silico analysis and in vitro evaluations. [3H]LSN3316612 imaged and quantified OGA in postmortem brains of rat, monkey, and human. The presence of fluorine and carbonyl functionality in LSN3316612 enabled labeling with positron-emitting fluorine-18 or carbon-11. Both [18F]LSN3316612 and [11C]LSN3316612 bound reversibly to OGA in vivo, and such binding was blocked by pharmacological doses of thiamet G, an OGA inhibitor of different chemotype, in monkeys. [18F]LSN3316612 entered healthy human brain avidly (~4 SUV) without radiodefluorination or adverse effect from other radiometabolites, as evidenced by stable brain total volume of distribution (VT) values by 110 min of scanning. Overall, [18F]LSN3316612 is preferred over [11C]LSN3316612 for future human studies, whereas either may be an effective positron emission tomography radioligand for quantifying brain OGA in rodent and monkey.

PET quantification of brain O-GlcNAcase with [18F]LSN3316612 in healthy human volunteers.

Abstract: Previous studies found that [18F]LSN3316612 was a promising positron emission tomography (PET) radioligand for imaging O-GlcNAcase in nonhuman primates and human volunteers. This study sought to further evaluate the suitability of [18F]LSN3316612 for human clinical research. Kinetic evaluation of [18F]LSN3316612 was conducted in a combined set of baseline brain scans from 17 healthy human volunteers and test-retest imaging was conducted in 10 of these volunteers; another 6 volunteers had whole-body scans to measure radiation exposure to body organs. Total distribution volume (VT) estimates were compared for the one- and two-tissue compartment models with the arterial input function. Test-retest variability and reliability were evaluated via mean difference and intraclass correlation coefficient (ICC). The time stability of VT was assessed down to a 30-min scan time. An alternative quantification method for [18F]LSN3316612 binding without blood was also investigated to assess the possibility of eliminating arterial sampling. Brain uptake was generally high and could be quantified as VT with excellent identifiability using the two-tissue compartment model. [18F]LSN3316612 exhibited good absolute test-retest variability (12.5%), but the arithmetic test-retest variability was far from 0 (11.3%), reflecting a near-uniform increase of VT on the retest scan in nine of 10 volunteers. VT values were stable after 110 min in all brain regions, suggesting that no radiometabolites accumulated in the brain. Measurements obtained using only brain activity (i.e., area under the curve (AUC) from 150–180 min) correlated strongly with regional VT values during test-retest conditions (R2 = 0.84), exhibiting similar reliability to VT (ICC = 0.68 vs. 0.64). Estimated radiation exposure for [18F]LSN3316612 PET was 20.5 ± 2.1 μSv/MBq, comparable to other 18F-labeled radioligands for brain imaging. [18F]LSN3316612 is an excellent PET radioligand for imaging O-GlcNAcase in the human brain. Alternative quantification without blood is possible, at least for within-subject repeat studies. However, the unexplained increase of VT under retest conditions requires further investigation.

Evaluation of 11C-NR2B-SMe and its Enantiomers as PET Radioligands for Imaging the NR2B Subunit within the NMDA Receptor Complex in Rats

Abstract: 1212 Objectives: The pretargeting strategy, which has been proposed as an alternative to radioimmunotherapy, increases the absorbed dose to the tumor while reducing the side effects of normal tissues, resulting in a high therapeutic effect. From the early development stage, the combination of streptavidin and biotin for in vivo binding, which controls the pharmacokinetics and therapeutic effects, has been expected to have clinical application owing to its high affinity for the recognition pair. However, due to the strong immunogenicity of streptavidin in vivo, the streptavidin/biotin pair has not been studied extensively. Hence, we designed Cupid, which is a mutated streptavidin with low immunogenicity and can fuse to four single-chain Fv fragments (scFv), and synthesized Psyche, which is a corresponding biotin variant with a specific affinity to Cupid (Sugiyama et al., Prc. Jpn. Acad. Ser. B 95:602, 2019). In this study, in addition to using these combinations, we labeled Psyche with an alpha-emitting radionuclide 211At, which is expected to have high therapeutic effects in the tumor and fewer side effects on the surrounding tissue in vivo. The aim of this study was to evaluate the labeling, stability, and pharmacokinetics of 211At labeled Psyche and scFV-Cupid in mice. Methods: 211At was produced using 209Bi(α, 2n)211At reaction and isolated through dry distillation. 80 MBq of 211At was labeled to Psyche derivatives (Psyche-B) and purified as a no-carrier-added product using Reversed-Phase HPLC. For the stability evaluation, 211At-Psyche-B was incubated with PBS and serum for 24 h. In total, 1×107 human gastric cancer cells (MKN-45) with a high expression of CEA were xenografted subcutaneously in male, 9-week-old BALB/c nu/nu mice. Thirteen days after the transplantation, 100 pmol of anti-CEACAM5 scFv-Cupid was intravenously administered to mice. After an interval of 24 h, 250 kBq of 211At labeled Psyche-B was intravenously administered to mice. The mice were sacrificed 1 min, 1 h, 6 h, and 24 h after the 211At-Psyche-B injection (n = 3 in each group). Samples were collected from the blood, brain, thyroid, heart, lung, liver, spleen, stomach, small intestine, pancreas, kidney, adrenal gland, muscle, bone, urine, and feces. Subsequently, they were weighed and measured for radioactivity using a gamma counter. Results: The radiochemical yield of 211At-Psyche-B was 96%, as determined by a radio-HPLC analysis of the crude product. 211At-Psyche-B showed high stability in PBS after 24 h but dissociated in serum within 1 h. In the biodistribution study, the clearance of 211At-Psyche-B from blood was 46.4 ± 1.4%ID/g after 1 min, 40.6 ± 3.2 %ID/g after 1 h, 29.4 ± 0.9 %ID/g after 6 h, and 6.2 ± 1.3 %ID/g after 24 h. The accumulation of 211At-Psyche-B in blood decreased with time, and approximately 94% of the injected dose was excreted within 24 h. The accumulation of 211At-Psyche-B in the tumor was 0.6 ± 0.1 %ID/g in 1 min, 3.2 ± 0.4 %ID/g in 1 h, 11.8 ± 1.5 %ID/g in 6 h, and 16.3 ± 4.1 %ID/g in 24 h. The tumor-to-blood ratios were 0.01 ± 0.00 after 1 min, 0.08 ± 0.01 after 1 h, 0.40 ± 0.05 after 6 h, and 2.63 ± 0.10 after 24 h. 211At-Psyche-B showed high liver uptakes in the experiment period: 17.4 ± 2.3 %ID/g in 1 min, 13.6 ± 1.4 %ID/g in 1 h, 16.0 ± 1.4 %ID/g in 6 h, and 15.5 ± 0.1 %ID/g in 24 h. 211At-Psyche-B uptakes in the spleen were 4.9 ± 1.7 %ID/g in 1 min, 7.9 ± 1.1 %ID/g in 1 h, 10.1 ± 0.3 %ID/g in 6 h, and 9.6 ± 2.4 %ID/g in 24 h. 211At-Psyche-B uptakes in the kidney were 29.3 ± 1.5 %ID/g in 1 min, 14.2 ± 1.3 %ID/g in 1 h, 9.8 ± 0.7 %ID/g in 6 h, and 4.7 ± 0.8 %ID/g in 24 hours. Conclusion: Although 211At-Psyche-B showed low serum stability in vitro, tumor uptake was confirmed and accumulation increased over time in vivo. Thus, this study suggested that our pre-targeting strategy of low-immunogenic CEA scFv-Cupid and 211At labeled Psyche might work for advanced cancer treatment. Acknowledgments: This work was financially supported by AMED under Grant Number JP19ck0106414.

Discovery, Radiolabeling, and Evaluation of Subtype-Selective Inhibitors for Positron Emission Tomography Imaging of Brain Phosphodiesterase-4D.

Abstract: We aimed to develop radioligands for PET imaging of brain phosphodiesterase subtype 4D (PDE4D), a potential target for developing cognition enhancing or antidepressive drugs. Exploration of several chemical series gave four leads with high PDE4D inhibitory potency and selectivity, optimal lipophilicity, and good brain uptake. These leads featured alkoxypyridinyl cores. They were successfully labeled with carbon-11 (t1/2 = 20.4 min) for evaluation with PET in monkey. Whereas two of these radioligands did not provide PDE4D-specific signal in monkey brain, two others, [11C]T1660 and [11C]T1650, provided sizable specific signal, as judged by pharmacological challenge using rolipram or a selective PDE4D inhibitor (BPN14770) and subsequent biomathematical analysis. Specific binding was highest in prefrontal cortex, temporal cortex, and hippocampus, regions that are important for cognitive function. [11C]T1650 was progressed to evaluation in humans with PET, but the output measure of brain enzyme density (VT) increased with scan duration. This instability over time suggests that radiometabolite(s) were accumulating in the brain. BPN14770 blocked PDE4D uptake in human brain after a single dose, but the percentage occupancy was difficult to estimate because of the unreliability of measuring VT. Overall, these results show that imaging of PDE4D in primate brain is feasible but that further radioligand refinement is needed, most likely to avoid problematic radiometabolites.

PET imaging of phosphodiesterase-4 identifies affected dysplastic bone in McCune-Albright syndrome, a genetic mosaic disorder

Abstract: McCune-Albright syndrome (MAS) is a mosaic disorder arising from gain-of-function mutations in the GNAS gene, which encodes the 3′, 5′-cyclic adenosine monophosphate (cAMP) pathway-associated G-protein, Gsα. Clinical manifestations of MAS in a given individual, including fibrous dysplasia, are determined by the timing and location of the GNAS mutation during embryogenesis, the tissues involved, and the role of Gsα in the affected tissues. The Gsα mutation results in dysregulation of the cAMP signaling cascade, leading to upregulation of phosphodiesterase type 4 (PDE4), which catalyzes the hydrolysis of cAMP. Increased cAMP levels have been found in vitro in both animal models of fibrous dysplasia and in cultured cells from individuals with MAS, but not in humans with fibrous dysplasia. Positron emission tomography (PET) imaging of PDE4 with 11C-(R)-rolipram has been used successfully to study the in vivo activity of the cAMP cascade. To date, it remains unknown whether fibrous dysplasia and other symptoms of MAS, including neuropsychiatric impairments, are associated with increased PDE4 activity in humans. Methods:11C-(R)-rolipram whole-body and brain PET scans were performed in six individuals with MAS (three for brain scans and six for whole-body scans) and nine healthy controls (seven for brain scans and six for whole-body scans). Results:11C-(R)-rolipram binding correlated with known locations of fibrous dysplasia in the periphery of individuals with MAS; no uptake was observed in the bones of healthy controls. In peripheral organs and the brain, no difference in 11C-(R)-rolipram uptake was noted between participants with MAS and healthy controls. Conclusion: This study is the first to find evidence for increased cAMP activity in areas of fibrous dysplasia in vivo. No differences in brain uptake between MAS participants and controls were detected, which could be due to several reasons, including the limited anatomic resolution of PET. Nevertheless, the results confirm the usefulness of PET scans with 11C-(R)-rolipram to indirectly measure increased cAMP pathway activation in human disease.

Development of a non-radiometric method for measuring the arterial input function of a 11C-labeled PET radiotracer.

Abstract: Positron emission tomography (PET) uses radiotracers to quantify important biochemical parameters in human subjects. A radiotracer arterial input function (AIF) is often essential for converting brain PET data into robust output measures. For radiotracers labeled with carbon-11 (t1/2 = 20.4 min), AIF is routinely determined with radio-HPLC of blood sampled frequently during the PET experiment. There has been no alternative to this logistically demanding method, neither for regular use nor validation. A 11C-labeled tracer is always accompanied by a large excess of non-radioactive tracer known as carrier. In principle, AIF might be obtained by measuring the molar activity (Am; ratio of radioactivity to total mass; Bq/mol) of a radiotracer dose and the time-course of carrier concentration in plasma after radiotracer injection. Here, we implement this principle in a new method for determining AIF, as shown by using [11C]PBR28 as a representative tracer. The method uses liquid chromatography-tandem mass spectrometry for measuring radiotracer Am and then the carrier in plasma sampled regularly over the course of a PET experiment. Am and AIF were determined radiometrically for comparison. The new non-radiometric method is not constrained by the short half-life of carbon-11 and is an attractive alternative to conventional AIF measurement.

Syntheses and Evaluation of Phosphodiesterase Subtype 4D (PDE4D) PET Radioligand Candidates in Monkey

Abstract: 268 Objectives: Phosphodiesterase-4 (PDE4) has emerged as a therapeutic target for central nervous system, inflammatory, and respiratory diseases PDE4 has 4 isoforms (A-D) among which B and D are predominant in brain Allosteric modulators for PDE4D are sought as candidate cognition enhancers and antidepressants with low potential for emesis side-effect [1] [11C](R)-Rolipram readily images PDE4 in primate brain [2, 3] However, [11C](R)-Rolipram is a non-subtype selective inhibitor and has no utility for showing target engagement by experimental drugs at subtypes of PDE4 A radioligand selective for imaging PDE4D with positron emission tomography (PET) would be useful for drug development and for studying PDE4D regulation in neuropsychiatric disorders Here we describe the synthesis and evaluation of candidate radioligands ([11C]1−6 and [18F]7 for PET imaging of PDE4D in primate brain Methods: Ligands 1−7, des-methyl precursors for labeling with carbon-11 (8−13), and an iodonium salt for fluorine-18 labeling (14) were synthesized de novo 11C-Labeled tracers were prepared by treating a desmethyl precursor with [11C]iodomethane in the presence of a base at RT (eg, [11C]6 and 7 in Figure 1A] or at 80 °C for 5 min For 18F-labeling, iodonium salt precursor plus TEMPO in DMSO was added to dry[18F]fluoride ion (~ 8 GBq) and heated with microwaves (130 ⁰C for 25 min) [Figure 1B] Rhesus monkeys were administered with radioligand (015−03 GBq iv) and PET scans were acquired on a Focus 220 scanner The monkey was administered either rolipram (01, 02, 1 mg/kg, iv) or BPN14770, a selective PDE4D negative allosteric modulator (3 mg/kg, iv) Concurrent arterial blood sampling was performed to obtain the metabolite-corrected input function for quantification Total distribution volumes (VT) for different regions were calculated using a two-tissue compartmental model Results: In vitro enzyme inhibition assays revealed that 1igands 1-7 are potent (PDE4D IC50 values range subnanomolar to low nanomolar) and selective to PDE4D over PDE4B [11C]1-6 were obtained in 10-20% decay-corrected formulated yields in ~ 40 min synthesis time with a molar activity (Am) of > 200 GBq/µmol The yield of [18F]7 was 10% with Am of ~ 98 GBq/µmol at EOS (~ 90 min synthesis time) At baseline, each radioligand readily entered monkey brain with quick washout However, receptor pre-block revealed no appreciable specific signal for [11C]1-[11C]4 and[18F]7 Radioligands [11C]5 and [11C]6 performed best giving moderate brain uptake (SUV ~3) and appreciable specific signal [Figure 2] [11C]6 uptake was also appreciably blocked by BPN14770 [Figure 2C] VT time stability of [11C]5 and [11C]6 indicated possible accumulation of radiometabolites in the brain but was adequate for quantification as the normalized VT improves towards the end of the scan (120 min) Conclusions: [11C]5 and [11C]6 are the best among the radioligands evaluated with [11C]6 being the slightly superior PET radioligand and may be used for occupancy and target engagement by experimental drugs at PDE4D in monkeyAcknowledgements: The Intramural Research Program of NIH (NIMH)References [1] Burgin AB et al, 2010Nat Biotechnol 28, 63 [2] Fujita M et al, 2012Biol Psychiatry, 72, 548 [3] Ooms M et al, 2019J Cerebr Blood Flow Metab,39, 1306