The adenosine monophosphate-activated protein kinase (AMPK) induces glucose uptake by increasing the expression of glucose transporter 4 (GLUT4), and [18F]Fluorodeoxyglucose (FDG) is readily transported into tissues with high GLUT4 expression. Thus, positron emission tomography with FDG (FDG-PET) could serve as an important pharmacodynamic readout of AMPK activation. In this study, the impact of treatment with the pan-AMPK activator MK-8722 on FDG uptake was evaluated in rats and Rhesus monkeys. Rats were evaluated with FDG-PET following intravenous (IV) or oral (PO) administration of MK-8722. Rhesus monkeys were orally dosed and evaluated with FDG-PET. FDG uptake was measured in skeletal and cardiac muscle, and the incorporation rate was calculated using the Patlak graphical method. In rats, the highest IV dose of MK-8722 (5 mg/kg) and both PO doses (4 mg/kg and 10 mg/kg) given 4 h prior to FDG-PET resulted in a significant increase in forelimb skeletal muscle FDG uptake (p < 0.01). In Rhesus monkeys, chronic oral administration of 10 mg/kg MK-8722 QD resulted in significantly higher FDG uptake in bicep skeletal muscle than vehicle treatment after 2 and 4 weeks of treatment (p < 0.01), but no difference was observed after 5 weeks of drug washout (p > 0.05). FDG uptake in cardiac muscle was significantly reduced with MK-8722 treatment in rats, but no significant changes in cardiac muscle FDG uptake were measured in Rhesus monkey. FDG-PET can be used as a pharmacodynamic readout for systemic pharmacological activation of AMPK for preclinical studies and potentially be extended to study humans.

Many commercially available biologics, previously delivered only intravenously, are being re-formulated for subcutaneous delivery to improve patient access and compliance. However, due to inherent solubility limitations, large volume injections (more than 2 mL) are typically required. Different strategies are being explored to improve the tolerability of such injections, including the co-formulation with hyaluronidase and/or implementing different needle designs. While there have been separate reports of measuring injection forces and using imaging to track injection delivery and tissue response, there is no current set of methods to simultaneously characterize the injection delivery (bleb) and measure injection pressures. In this study we describe the development of Computed Tomography imaging methods in minipigs to characterize the morphology of the bleb following injection, along with inline pressure measurements to assess subcutaneous pressure during injection using two different injection volumes, 4.5 mL and 9 mL. We show that these parameters change with injection volume, and that inclusion of hyaluronidase in the injection increases bleb dispersion and reduces skin distention while also lowering the injection pressure. This method will likely be a valuable tool for assessing and comparing different injection delivery methods and formulations.

Deposition of misfolded α-synuclein (α-Syn) aggregates in the human brain is one of the major hallmarks of synucleinopathies. Positron Emission Tomography imaging (PET) of α-synuclein in Parkinson’s disease (PD) patients is highly desirable but remains elusive. An extensive PET radioligand discovery campaign was undertaken. Here, we present the in-vitro and preclinical in-vivo characterization of [ 3 H]- and [ 11 C]Compound S, representing an example of a promising PET radioligand for α-Syn. Competition binding studies and autoradiographic studies were performed in cortexes of PD patients, healthy control, and Alzheimer’s disease (AD) patients brain tissue, and mid-brain region of the aged A30P mouse using [ 3 H]S. Brain PET experiments using [ 11 C]S were carried out in a healthy non-human primate (NHP) and the aged homozygote A30P mice. Compound S is a sub-nanomolar affinity ligand to human and A30P mouse α-Syn (Kd = 0.15 nM and 0.5 nM resp.). Lower affinity was observed in AD and healthy control brain tissues. [ 3 H]S showed displaceable binding in PD brain cortex and A30P mid-brain and brainstem, and little binding in AD and healthy control. In the NHP, [ 11 C]S crosses the brain-blood barrier and the uptake phase of [ 11 C]S is followed by a washout. [ 11 C]S PET studies in A30P mice showed higher uptake in the mid brain and brainstem regions, with standard uptake value ratios in those regions higher than 2.0. Compound S is potent for α-Syn in vitro, and PET imaging in the A30P mice showed an elevated signal in regions known to accumulate α-Syn. This dataset suggests that the Compound S has promise towards imaging α-Syn in PD patients .

Positron emission tomography (PET) ligands play an important role in the development of therapeutics by serving as target engagement or pharmacodynamic biomarkers. Here, we describe the discovery and translation of the PET tracer [11C]MK-6884 from rhesus monkeys to patients with Alzheimer’s disease (AD). [3H]MK-6884/[11C]MK-6884 binds with high binding affinity and good selectivity to an allosteric site on M4 muscarinic cholinergic receptors (M4Rs) in vitro and shows a regional distribution in the brain consistent with M4R localization in vivo. The tracer demonstrates target engagement of positive allosteric modulators of the M4R (M4 PAMs) through competitive binding interactions. [11C]MK-6884 binding is enhanced in vitro by the orthosteric M4R agonist carbachol and indirectly in vivo by the acetylcholinesterase inhibitor donepezil in rhesus monkeys and healthy volunteers, consistent with its pharmacology as a highly cooperative M4 PAM. PET imaging of [11C]MK-6884 in patients with AD identified substantial regional differences quantified as nondisplaceable binding potential (BPND) of [11C]MK-6884. These results suggest that [11C]MK-6884 is a useful target engagement biomarker for M4 PAMs but may also act as a sensitive probe of neuropathological changes in the brains of patients with AD.

The cathepsin K (CatK) enzyme is abundantly expressed in osteoclasts, and CatK inhibitors have been developed for the treatment of osteoporosis. In our effort to support discovery and clinical evaluations of a CatK inhibitor, we sought to discover a radioligand to determine target engagement of the enzyme by therapeutic candidates using positron emission tomography (PET). L-235, a potent and selective CatK inhibitor, was labeled with carbon-11. PET imaging studies recording baseline distribution of [11 C]L-235, and chase and blocking studies using the selective CatK inhibitor MK-0674 were performed in juvenile and adult nonhuman primates (NHP) and ovariectomized rabbits. Retention of the PET tracer in regions expected to be osteoclast-rich compared with osteoclast-poor regions was examined. Increased retention of the radioligand was observed in osteoclast-rich regions of juvenile rabbits and NHP but not in the adult monkey or adult ovariectomized rabbit. Target engagement of CatK was observed in blocking studies with MK-0674, and the radioligand retention was shown to be sensitive to the level of MK-0674 exposure. [11 C]L-235 can assess target engagement of CatK in bone only in juvenile animals. [11 C]L-235 may be a useful tool for guiding the discovery of CatK inhibitors.

Purpose Programmed cell death-1 receptor (PD-1) and its ligand (PD-L1) are the targets for immunotherapy in many cancer types. Although PD-1 blockade has therapeutic effects, the efficacy differs between patients. Factors contributing to this variability are PD-L1 expression levels and immune cells present in tumors. However, it is not well understood how PD-1 expression in the tumor microenvironment impacts immunotherapy response. Thus, imaging of PD-1-expressing immune cells is of interest. This study aims to evaluate the biodistribution of Zirconium-89 ( 89 Zr)-labeled pembrolizumab, a humanized IgG4 kappa monoclonal antibody targeting PD-1, in healthy cynomolgus monkeys as a translational model of tracking PD-1-positive immune cells. Procedures Pembrolizumab was conjugated with the tetrafluorophenol-N-succinyl desferal-Fe(III) ester (TFP-N-sucDf) and subsequently radiolabeled with 89 Zr. Four cynomolgus monkeys with no previous exposure to humanized monoclonal antibodies received tracer only or tracer co-injected with pembrolizumab intravenously over 5 min. Thereafter, a static whole-body positron emission tomography (PET) scan was acquired with 10 min per bed position on days 0, 2, 5, and 7. Image-derived standardized uptake values (SUV mean ) were quantified by region of interest (ROI) analysis. Results 89 Zr-N-sucDf-pembrolizumab was synthesized with high radiochemical purity (> 99 %) and acceptable molar activity (> 7 MBq/nmol). In animals dosed with tracer only, 89 Zr-N-sucDf-pembrolizumab distribution in lymphoid tissues such as mesenteric lymph nodes, spleen, and tonsils increased over time. Except for the liver, low radiotracer distribution was observed in all non-lymphoid tissue including the lung, muscle, brain, heart, and kidney. When a large excess of pembrolizumab was co-administered with a radiotracer, accumulation in the lymph nodes, spleen, and tonsils was reduced, suggestive of target-mediated accumulation. Conclusions 89 Zr-N-sucDf-pembrolizumab shows preferential uptake in the lymphoid tissues including the lymph nodes, spleen, and tonsils. 89 Zr-N-sucDf-pembrolizumab may be useful in tracking the distribution of a subset of immune cells in non-human primates and humans. Trial Registration ClinicalTrials.gov Identifier: NCT02760225

PURPOSE:In vivo imaging of programmed death ligand 1 (PD-L1) during immunotherapy could potentially monitor changing PD-L1 expression and PD-L1 expression heterogeneity within and across tumors. Some protein constructs can be used for same-day positron emission tomography (PET) imaging. Previously, we evaluated the PD-L1-targeting Affibody molecule [18F]AlF-NOTA-ZPD-L1_1 as a PET tracer in a mouse tumor model of human PD-L1 expression. In this study, we evaluated the affinity-matured Affibody molecule ZPD-L1_4, to determine if improved affinity for PD-L1 resulted in increased in vivo targeting of PD-L1. PROCEDURES:ZPD-L1_4 was conjugated with NOTA and radiolabeled with either [18F]AlF or 68Ga. [18F]AlF-NOTA-ZPD-L1_4 and [68Ga]NOTA-ZPD-L1_4 were evaluated in immunocompromised mice with LOX (PD-L1+) and SUDHL6 (PD-L1-) tumors with PET and ex vivo biodistribution measurements. In addition, whole-body PET studies were performed in rhesus monkeys to predict human biodistribution in a model with tracer binding to endogenous PD-L1, and to calculate absorbed radiation doses. RESULTS:Ex vivo biodistribution measurements showed that both tracers had > 25 fold higher accumulation in LOX tumors than SUDHL6 ([18F]AlF-NOTA-ZPD-L1_4: LOX: 8.7 ± 0.7 %ID/g (N = 4) SUDHL6: 0.2 ± 0.01 %ID/g (N = 6), [68Ga]NOTA-ZPD-L1_4: LOX: 15.8 ± 1.0 %ID/g (N = 6) SUDHL6: 0.6 ± 0.1 %ID/g (N = 6)), considerably higher than ZPD-L1_1. In rhesus monkeys, both PET tracers showed fast clearance through kidneys and low background signal in the liver ([18F]AlF-NOTA-ZPD-L1_4: 1.26 ± 0.13 SUV, [68Ga]NOTA-ZPD-L1_4: 1.11 ± 0.06 SUV). PD-L1-expressing lymph nodes were visible in PET images, indicating in vivo PD-L1 targeting. Dosimetry estimates suggest that both PET tracers can be used for repeated clinical studies, although high kidney accumulation may limit allowable radioactive doses. CONCLUSIONS:[18F]AlF-NOTA-ZPD-L1_4 and [68Ga]NOTA-ZPD-L1_4 are promising candidates for same-day clinical PD-L1 PET imaging, warranting clinical evaluation. The ability to use either [18F] or [68Ga] may expand access to clinical sites.

The measurement of receptor occupancy (RO) using positron emission tomography (PET) has been instrumental in guiding discovery and development of CNS directed therapeutics. We and others have investigated muscarinic acetylcholine receptor 4 (M4) positive allosteric modulators (PAMs) for the treatment of symptoms associated with neuropsychiatric disorders. In this article, we describe the synthesis, in vitro, and in vivo characterization of a series of central pyridine-related M4 PAMs that can be conveniently radiolabeled with carbon-11 as PET tracers for the in vivo imaging of an allosteric binding site of the M4 receptor. We first demonstrated its feasibility by mapping the receptor distribution in mouse brain and confirming that a lead molecule 1 binds selectively to the receptor only in the presence of the orthosteric agonist carbachol. Through a competitive binding affinity assay and a number of physiochemical properties filters, several related compounds were identified as candidates for in vivo evaluation. These candidates were then radiolabeled with 11C and studied in vivo in rhesus monkeys. This research eventually led to the discovery of the clinical radiotracer candidate [11C]MK-6884.

In the past, diagnosis of Alzheimer’s disease (AD) could only be performed post-mortem by staining of amyloid-beta (Aß) plaque and neurofibrillary tangles (NFTs). Non-invasive clinical imaging Aß can now be routinely performed using Positron Emission Tomography (PET). However, dementia correlates more strongly with the extent of NFT than with Aß. Hence, efforts are currently directed towards the identification of a PET radiotracer suitable for measuring the NFT load clinically, which will be complementary to the Aß probes. Our effort was directed towards identifying a PET radioligand that is specific and sensitive. Here, we present the preclinical characterization of [18F]MK-6240 as well as its first-in-man evaluation. Immunohistochemistry, autoradiography (ARG) studies and tissue homogenates binding studies were carried out in AD and healthy control (HC) brains. Non-human primate (NHP) brain PET imaging was carried out to determine the pharmacokinetic (PK) profile, brain uptake and non-specific binding as well as lack of off-target binding. A dosimetry study was performed for the first-in-human evaluation of [18F]MK-6240. Study design for the evaluation of [18F]MK-6240 included HC and probable AD patient brain PET scans. In vitro experiments using AD tissues showed that MK-6240 is a potent (KD = 0.28 nM) and selective (Ki Aß >10 μM) NFT ligand. Using HC tissues, no saturable MK-6240 binding was observed. [18F]MK-6240 displayed a high initial uptake followed by a fast washout in NHP in vivo. Non-specific and white matter binding were low. Self-block studies showed no off-target binding. Dosimetry evaluation in human resulted in an effective dose of ∼29.4 μSv/MBq. A Similar PK profile as in NHP were observed for HCs. Extensive in-vitro characterization showed that MK-6240 is a potent and selective tau tracer with great binding potential. In NHP, [18F]MK-6240 displayed a favorable PK profile and no potential liabilities were found. In vivo in human, we observed similar results in HC. Further PET evaluation is currently ongoing to evaluate the sensitivity of the radioligand in healthy elderly and AD subjects.

Neurofibrillary tangles (NFTs) made up of aggregated tau protein have been identified as the pathologic hallmark of several neurodegenerative diseases including Alzheimer's disease. In vivo detection of NFTs using PET imaging represents a unique opportunity to develop a pharmacodynamic tool to accelerate the discovery of new disease modifying therapeutics targeting tau pathology. Herein, we present the discovery of 6-(fluoro-18F)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine, 6 ([18F]-MK-6240), as a novel PET tracer for detecting NFTs. 6 exhibits high specificity and selectivity for binding to NFTs, with suitable physicochemical properties and in vivo pharmacokinetics.

In the past, diagnosis of Alzheimer’s disease (AD) could only be performed post-mortem by staining of amyloid-beta (Aß) plaque and neurofibrillary tangles (NFTs). Non-invasive clinical imaging of Aß can now be routinely performed using Positron Emission Tomography (PET). However, dementia correlates more strongly with the extent of NFT than with Aß. Hence, efforts are currently directed towards the identification of a PET radiotracer suitable for measuring the NFT load clinically, which will be complementary to the Aß probes. Our effort was directed towards identifying a PET radioligand that is specific and sensitive. Here, we present the preclinical characterization of [18F]MK-6240 as well as its first-in-man evaluation. Immunohistochemistry, autoradiography (ARG) studies and tissue homogenates binding studies were carried out in AD and healthy control (HC) brains. Non-human primate (NHP) brain PET imaging was carried out to determine the pharmacokinetic (PK) profile, brain uptake and non-specific binding as well as lack of off-target binding. A dosimetry study was performed for the first-in-human evaluation of [18F]MK-6240. Study design for the evaluation of [18F]MK-6240 included HC and probable AD patient brain PET scans. In vitro experiments using AD tissues showed that MK-6240 is a potent (KD = 0.28 nM) and selective (Ki Aß >10 μM) NFT ligand. Using HC tissues, no saturable MK-6240 binding was observed. [18F]MK-6240 displayed a high initial uptake followed by a fast washout in NHP in vivo. Non-specific and white matter binding were low. Self-block studies showed no off-target binding. Dosimetry evaluation in human resulted in an effective dose of ∼29.4 μSv/MBq. A Similar PK profile as in NHP were observed for HCs. Extensive in-vitro characterization showed that MK-6240 is a potent and selective tau tracer with great binding potential. In NHP, [18F]MK-6240 displayed a favorable PK profile and no potential liabilities were found. In vivo in human, we observed similar results in HC. Further PET evaluation is currently ongoing to evaluate the sensitivity of the radioligand in healthy elderly and AD subjects.

PURPOSE:A positron emission tomography (PET) tracer for the enzyme phosphodiesterase 10A (PDE10A) is desirable to guide the discovery and development of PDE10A inhibitors as potential therapeutics. The preclinical characterization of the PDE10A PET tracer [(11)C]MK-8193 is described.PROCEDURES:In vitro binding studies with [(3)H]MK-8193 were conducted in rat, monkey, and human brain tissue. PET studies with [(11)C]MK-8193 were conducted in rats and rhesus monkeys at baseline and following administration of a PDE10A inhibitor.RESULTS:[(3)H]MK-8193 is a high-affinity, selective PDE10A radioligand in rat, monkey, and human brain tissue. In vivo, [(11)C]MK-8193 displays rapid kinetics, low test-retest variability, and a large specific signal that is displaced by a structurally diverse PDE10A inhibitor, enabling the determination of pharmacokinetic/enzyme occupancy relationships.CONCLUSIONS:[(11)C]MK-8193 is a useful PET tracer for the preclinical characterization of PDE10A therapeutic candidates in rat and monkey. Further evaluation of [(11)C]MK-8193 in humans is warranted.

1362 Objectives Imaging of the folate receptor (FR) is important for assessing treatment options of patients with FR-expressing cancers, and has been primarily accomplished with the FR-targeting SPECT tracer [Tc-99m]-EC20 (Fisher, et al., 2008). However, clinical PET provides superior spatial resolution, shorter scan times, and greater dynamic imaging capabilities than SPECT. The PET tracer Folate-NOTA-Al-[F-18] has been shown to successfully target FR in vivo (Meng, et al., 2016). In this study, the in vivo biodistribution of Folate-NOTA-Al-[F-18] was compared to [Tc-99m]-EC20, and radiation dosimetry estimates for humans were obtained. Methods Rhesus monkeys (N=3) were scanned with both radiotracers. Monkeys were fasted and maintained under anesthesia with propofol. Following a CT scan, PET data were acquired for 180 minutes following IV administration of Folate-NOTA-Al-[F18] (137-178 MBq, 1.9-2.5 μg), while SPECT data were acquired for 150 min, starting 60 min after administration of [Tc-99m]-EC20 (390-399 MBq, 39-48 μg). Venous blood samples were taken throughout the study and metabolite-corrected blood levels of radiotracer were calculated. Regions of interest (ROI) were drawn for liver, kidney cortex, lumbar spine, lung, small intestine, spleen, bladder, and heart muscle, and average radiotracer uptake from ~60-150 minutes for each ROI was calculated. Absorbed radiation doses were calculated using OLINDA/EXM v1 (Organ Level Internal Dose Assessment) with adult human model inputs. Results PET and SPECT images revealed similar biodistribution for the two radiotracers (Table 1). Based on a mixed-effect model analysis, significant differences in accumulation between radiotracers were detected for spleen, (p= 0.02), heart muscle (p = 0.03), and lumbar spine (p = 0.05). Dosimetry analysis of Folate-NOTA-Al-[F-18] found that the critical organ for males was the testes, while for females the urinary bladder wall was the critical organ. A conservative estimate of the recommended maximum allowable dose of folate-NOTA-Al-[F-18] for clinical research subjects (U.S) is 197 MBq per dose and 328 MBq per year for males, and 235 MBq per dose and 704 MBq per year for females. Conclusions Folate-NOTA-Al-[F-18] showed similar biodistribution to [Tc-99m]-EC20 in Rhesus monkey, and an analysis of radiation dosimetry indicated that human studies will be feasible. These results add support for the PET radiotracer Folate-NOTA-Al-[F-18] as a viable alternative [Tc-99m]-EC20 for FR imaging.