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PET imaging of synaptic vesicle glycoprotein 2A allows for noninvasive quantification of synapses. This first-in-human study aimed to evaluate the kinetics, test-retest reproducibility, and extent of specific binding of a recently developed synaptic vesicle glycoprotein 2A PET ligand, ( R )-4-(3-( 18 F-fluoro)phenyl)-1-((3-methylpyridin-4-yl)methyl)pyrrolidine-2-one ( 18 F-SynVesT-2), with fast brain kinetics. Methods: Nine healthy volunteers participated in this study and were scanned on a High Resolution Research Tomograph scanner with 18 F-SynVesT-2. Five volunteers were scanned twice on 2 different days. Five volunteers were rescanned with preinjected levetiracetam (20 mg/kg, intravenously). Arterial blood was collected to calculate the plasma free fraction and generate the arterial input function. Individual MR images were coregistered to a brain atlas to define regions of interest for generating time-activity curves, which were fitted with 1- and 2-tissue-compartment (1TC and 2TC) models to derive the regional distribution volume ( V T ). The regional nondisplaceable binding potential ( BP ND ) was calculated from 1TC V T , using the centrum semiovale (CS) as the reference region. Results: 18 F-SynVesT-2 was synthesized with high molar activity (187 ± 69 MBq/nmol, n = 19). The parent fraction of 18 F-SynVesT-2 in plasma was 28% ± 8% at 30 min after injection, and the plasma free fraction was high (0.29 ± 0.04). 18 F-SynVesT-2 entered the brain quickly, with an SUV peak of 8 within 10 min after injection. Regional time-activity curves fitted well with both the 1TC and the 2TC models; however, V T was estimated more reliably using the 1TC model. The 1TC V T ranged from 1.9 ± 0.2 mL/cm 3 in CS to 7.6 ± 0.8 mL/cm 3 in the putamen, with low absolute test-retest variability (6.0% ± 3.6%). Regional BP ND ranged from 1.76 ± 0.21 in the hippocampus to 3.06 ± 0.29 in the putamen. A 20-min scan was sufficient to provide reliable V T and BP ND Conclusion: 18 F-SynVesT-2 has fast kinetics, high specific uptake, and low nonspecific uptake in the brain. Consistent with the nonhuman primate results, the kinetics of 18 F-SynVesT-2 is faster than the kinetics of 11 C-UCB-J and 18 F-SynVesT-1 in the human brain and enables a shorter dynamic scan to derive physiologic information on cerebral blood flow and synapse density., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Arginine vasopressin is a hormone that is synthesized mainly in the hypothalamus and stored in the posterior pituitary. Receptors for vasopressin are categorized into at least 3 subtypes (V 1A , V 1B , and V 2 ). Among these subtypes, the V 1B receptor (V 1B R), highly expressed in the pituitary, is a primary regulator of hypothalamic-pituitary-adrenal axis activity and thus a potential target for treatment of neuropsychiatric disorders such as depression and anxiety. N - tert -butyl-2-[2-(6-methoxypyridine-2-yl)-6-[3-(morpholin-4-yl)propoxy]-4-oxopyrido[2,3-d]pyrimidin-3(4 H )-yl]acetamide (TASP699) is a novel PET radiotracer with high affinity and selectivity for V 1B R. The purpose of this study was to characterize the pharmacokinetic and binding profiles of 11 C-TASP699 in humans and determine its utility in an occupancy study of a novel V 1B R antagonist, TS-121. Methods: Six healthy subjects were scanned twice with 11 C-TASP699 to determine the most appropriate kinetic model for analysis of imaging data and test-retest reproducibility of outcome measures. Nine healthy subjects were scanned before and after administration of TS-121 (active component: THY1773) to assess V 1B R occupancy. Metabolite-corrected arterial input functions were obtained. Pituitary time-activity curves were analyzed with 1- and 2-tissue-compartment (1TC and 2TC, respectively) models and multilinear analysis 1 (MA1) to calculate distribution volume ( V T ). Relative test-retest variability (TRV) and absolute TRV were calculated. Since no brain region could be used as a reference region, percentage change in V T after TS-121 administration was computed to assess its receptor occupancy and correlate with plasma concentrations of the drug. Results: 11 C-TASP699 showed high uptake in the pituitary and no uptake in any brain region. The 2TC model provided better fits than the 1TC model. Because the MA1 V T estimates were similar to the 2TC V T estimates, MA1 was the model of choice. The TRV of V T was good (TRV, -2% ± 14%; absolute TRV, 11%). THY1773 reduced V T in a dose-dependent fashion, with a half-maximal inhibitory concentration of 177 ± 52 ng/mL in plasma concentration. There were no adverse events resulting in discontinuation from the study. Conclusion: 11 C-TASP699 was shown to display appropriate kinetics in humans, with substantial specific binding and good reproducibility of V T Therefore, this tracer is suitable for measurement of V 1B R in the human pituitary and the V 1B R occupancy of TS-121, a novel V 1B R antagonist., (© 2022 by the Society of Nuclear Medicine and Molecular Imaging.)

The positron emission tomography radioligand [ 11 C]UCB-J binds to synaptic vesicle glycoprotein 2 A (SV2A), a regulator of vesicle release. Increased neuronal firing could potentially affect tracer concentrations if binding site availability is altered during vesicle exocytosis. This study assessed whether physiological brain activation induces changes in [ 11 C]UCB-J tissue influx ( K 1 ), volume of distribution ( V T ), or binding potential ( BP ND ). Healthy volunteers ( n  = 7) underwent 60-min [ 11 C]UCB-J PET scans at baseline and during intermittent presentation of 8-Hz checkerboard visual stimulation. Sensitivity to intermittent changes in kinetic parameters was assessed in simulations, and visual stimulation was repeated using functional magnetic resonance imaging to characterize neural responses. V T  and K 1 were determined using the one-tissue compartment model and BP ND using the simplified reference tissue model. In primary visual cortex, K 1 increased 34.3 ± 15.5% ( p  = 0.001) during stimulation, with no change in other regions ( p s   >   0.12). K 1 change was correlated with fMRI BOLD response (r = 0.77, p  = 0.043). There was no change in V T (-3.9 ± 8.8%, p  =   0.33) or BP ND (-0.2 ± 9.6%, p  =   0.94) in visual cortex nor other regions ( p s   >   0.19). Therefore, despite robust increases in regional tracer influx due to blood flow increases, binding measures were unchanged during stimulation. [ 11 C]UCB-J V T and BP ND are likely to be stable in vivo measures of synaptic density.

The use of synaptic vesicle glycoprotein 2A radiotracers with PET imaging could provide a way to measure synaptic density quantitatively in living humans. 11 C-UCB-J (( R )-1-((3-( 11 C-methyl- 11 C)pyridin-4-yl)methyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-2-one), previously developed and assessed in nonhuman primates and humans, showed excellent kinetic properties as a PET radioligand. However, it is labeled with the short half-life isotope 11 C. We developed a new tracer, an 18 F-labeled difluoro-analog of UCB-J ( 18 F-SynVesT-1, also known as 18 F-SDM-8), which displayed favorable properties in monkeys. The purpose of this first-in-human study was to assess the kinetic and binding properties of 18 F-SynVesT-1 and compare with 11 C-UCB-J. Methods: Eight healthy volunteers participated in a baseline study of 18 F-SynVesT-1. Four of these subjects were also scanned after a blocking dose of the antiepileptic drug levetiracetam (20 mg/kg). Metabolite-corrected arterial input functions were measured. Regional time-activity curves were analyzed using 1-tissue-compartment (1TC) and 2-tissue-compartment (2TC) models and multilinear analysis 1 to compute total distribution volume ( V T ) and binding potential ( BP ND ). The centrum semiovale was used as a reference region. The Lassen plot was applied to compute levetiracetam occupancy and nondisplaceable distribution volume. SUV ratio-1 (SUVR-1) over several time windows was compared with BP ND Results: Regional time-activity curves were fitted better with the 2TC model than the 1TC model, but 2TC V T estimates were unstable. The 1TC V T values matched well with those from the 2TC model (excluding the unstable values). Thus, 1TC was judged as the most useful model for quantitative analysis of 18 F-SynVesT-1 imaging data. The minimum scan time for stable V T measurement was 60 min. The rank order of V T and BP ND was similar between 18 F-SynVesT-1 and 11 C-UCB-J. Regional V T was slightly higher for 11 C-UCB-J, but BP ND was higher for 18 F-SynVesT-1, though these differences were not significant. Levetiracetam reduced the uptake of 18 F-SynVesT-1 in all regions and produced occupancy of 85.7%. The SUVR-1 of 18 F-SynVesT-1 from 60 to 90 min matched best with 1TC BP ND Conclusion: The novel synaptic vesicle glycoprotein 2A tracer, 18 F-SynVesT-1, displays excellent kinetic and in vivo binding properties in humans and holds great potential for the imaging and quantification of synaptic density in neuropsychiatric disorders., (© 2021 by the Society of Nuclear Medicine and Molecular Imaging.)

This was a first-in-human study of the PET radiotracer 11 C-LSN3172176 for the muscarinic acetylcholine receptor subtype M1. The objectives of the study were to determine the appropriate kinetic model to quantify binding of the tracer to M1 receptors, and the reliability of the chosen quantification method. Methods: Six healthy subjects completed the test-retest protocol, and 5 healthy subjects completed the baseline-scopolamine blocking protocol. Multiple modeling methods were applied to calculate total distribution volume ( V T ) and nondisplaceable binding potential ( BP ND ) in various brain regions. The reference region was selected from the blocking study. The occupancy plot was applied to compute receptor occupancy by scopolamine and nondisplaceable distribution volume. Results: Tracer uptake was highest in the striatum, followed by neocortical regions and white matter, and lowest in the cerebellum. Regional time-activity curves were fitted well by all models. The 2-tissue-compartment (2TC) model fits were good, but the 2TC parameters often could not be reliably estimated. Because V T correlated well between the 2TC and 1-tissue-compartment (1TC) models after exclusion of unreliable estimates, the 1TC model was chosen as the most appropriate. The cerebellum showed the lowest V T , consistent with preclinical studies showing little to no specific binding in the region. Further, cerebellar V T did not change between baseline and blocking scans, indicating that the cerebellum is a suitable reference region. The simplified reference tissue model (SRTM) slightly underestimated 1TC BP ND , and the simplified reference tissue model 2 (SRTM2) improved BP ND estimation. An 80-min scan was sufficient to quantify V T and BP ND The test-retest study showed excellent absolute test-retest variability for 1TC V T (≤5%) and BP ND (≤10%). In the baseline and blocking studies, occupancy values were lower in the striatum than in nonstriatal regions, as may be attributed to differences in regional acetylcholine concentrations. Conclusion: The 1TC and SRTM2 models are appropriate for quantitative analysis of 11 C-LSN3172176 imaging data. 11 C-LSN3172176 displayed excellent test-retest reproducibility and is a highly promising ligand to quantify M1 receptors in the human brain., (© 2021 by the Society of Nuclear Medicine and Molecular Imaging.)

11 C-UCB-J (( R )-1-((3-( 11 C-methyl- 11 C)pyridin-4-yl)methyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-2-one) is a PET tracer for synaptic vesicle glycoprotein 2A, which may be a marker of synaptic density. To simplify the scan protocol, SUV ratios (SUVRs) were compared with model-based nondisplaceable binding potential ( BP ND ) to select the optimal time window in healthy and neuropsychiatric subjects. Methods: In total, 141 scans were acquired for 90 min. Arterial blood sampling and metabolite analysis were conducted. SUVR-1 (centrum semiovale reference region) was computed for six 30-min windows and compared with 1-tissue-compartment model BP ND Simulations were performed to assess the time dependency of SUVR-1. Results: Greater correlation and less bias were observed for SUVR-1 at later time windows for all subjects. Simulations showed that the agreement between SUVR-1 and BP ND is time-dependent. Conclusion: The 60- to 90-min period provided the best match between SUVR-1 and BP ND (-1% ± 7%); thus, a short scan is sufficient for accurate quantification of 11 C-UCB-J-specific binding., (© 2021 by the Society of Nuclear Medicine and Molecular Imaging.)

The κ-opioid receptor (KOR) is implicated in various neuropsychiatric disorders. We previously evaluated an agonist tracer, 11 C-GR103545, for PET imaging of KOR in humans. Although 11 C-GR103545 showed high brain uptake, good binding specificity, and selectivity for KOR, it displayed slow kinetics and relatively large test-retest variability of total distribution volume ( V T ) estimates (15%). Therefore, we set out to develop 2 novel KOR agonist radiotracers, 11 C-EKAP and 11 C-FEKAP. In nonhuman primates, both tracers exhibited faster kinetics than 11 C-GR103545 and comparable binding parameters to 11 C-GR103545. The aim of this study was to assess their kinetic and binding properties in humans. Methods: Six healthy subjects underwent 120-min test-retest PET scans with both 11 C-EKAP and 11 C-FEKAP. Metabolite-corrected arterial input functions were measured. Regional time-activity curves were generated for 14 regions of interest. One-tissue-compartment and 2-tissue-compartment (2TC) models and the multilinear analysis-1 (MA1) method were applied to the regional time-activity curves to calculate V T The time stability of V T and test-retest reproducibility were evaluated. Levels of specific binding, as measured by the nondisplaceable binding potential ( BP ND ) for the 3 tracers ( 11 C-EKAP, 11 C-FEKAP, and 11 C-GR103545), were compared using a graphical method. Results: For both tracers, regional time-activity curves were fitted well with the 2TC model and MA1 method ( t * = 20 min) but not with the 1-tissue-compartment model. Given the unreliably estimated parameters in several fits with the 2TC model and a good V T match between MA1 and 2TC, MA1 was chosen as the appropriate model for both tracers. Mean MA1 V T was highest for 11 C-GR103545, followed by 11 C-EKAP and then 11 C-FEKAP. The minimum scan time for stable V T measurement was 90 and 110 min for 11 C-EKAP and 11 C-FEKAP, respectively, compared with 140 min for 11 C-GR103545. The mean absolute test-retest variability in MA1 V T estimates was 7% and 18% for 11 C-EKAP and 11 C-FEKAP, respectively. BP ND levels were similar for 11 C-FEKAP and 11 C-GR103545 but were about 25% lower for 11 C-EKAP. Conclusion: The 2 novel KOR agonist tracers showed faster tissue kinetics than 11 C-GR103545. Even with a slightly lower BP ND , 11 C-EKAP is judged to be a better tracer for imaging and quantification of KOR in humans, on the basis of the shorter minimum scan time and the excellent test-retest reproducibility of regional V T ., (© 2020 by the Society of Nuclear Medicine and Molecular Imaging.)

Development of medications selective for dopamine D 2 or D 3 receptors is an active area of research in numerous neuropsychiatric disorders including addiction and Parkinson's disease. The positron emission tomography (PET) radiotracer [ 11 C]-(+)-PHNO, an agonist that binds with high affinity to both D 2 and D 3 receptors, has been used to estimate relative receptor subtype occupancy by drugs based on a priori knowledge of regional variation in the expression of D 2 and D 3 receptors. The objective of this work was to use a data-driven independent component analysis (ICA) of receptor blocking scans to separate D 2 -and D 3 -related signal in [ 11 C]-(+)-PHNO binding data in order to improve the precision of subtype specific measurements of binding and occupancy. Eight healthy volunteers underwent [ 11 C]-(+)-PHNO PET scans at baseline and at two time points following administration of the D 3 -preferring antagonist ABT-728 (150-1000 ​mg). Parametric binding potential (BP ND ) images were analyzed as four-dimensional image series using ICA to extract two independent sources of variation in [ 11 C]-(+)-PHNO BP ND . Spatial source maps for each component were consistent with respective regional patterns of D 2 -and D 3 -related binding. ICA-derived occupancy estimates from each component were similar to D 2 -and D 3 -specific occupancy estimated from a region-based approach (intraclass correlation coefficients ​> ​0.95). ICA-derived estimates of D 3 receptor occupancy improved quality of fit to a single site binding model. Furthermore, ICA-derived estimates of the regional fraction of [ 11 C]-(+)-PHNO binding related to D 3 receptors was generated for each subject and values showed good agreement with region-based model estimates and prior literature values. In summary, ICA successfully separated D 2 -and D 3 -related components of the [ 11 C]-(+)-PHNO binding signal, establishing this approach as a powerful data-driven method to quantify distinct biological features from PET data composed of mixed data sources., Competing Interests: Declaration of competing interest None., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme converts cortisone to cortisol and participates in the regulation of glucocorticoid levels in tissues. 11β-HSD1 is expressed in the liver, kidney, adipose tissue, placenta, and brain. 11β-HSD1 is a target for treatment of depression, anxiety, posttraumatic stress disorder, and also against age-related cognitive function and memory loss. In this study, we evaluated the radiotracer 11 C-AS2471907 (3-(2-chlorophenyl)-4-(methyl- 11 C )-5-[2-[2,4,6-trifluorophenoxy]propan-2-yl]-4 H -1,2,4-triazole) to image 11β-HSD1 availability in the human brain with PET. Methods: Fifteen subjects were included in the study. All subjects underwent one 2-h scan after a bolus administration of 11 C-AS2471907. Two subjects underwent an additional scan after blockade with the selective and high-affinity 11β-HSD1 inhibitor ASP3662 to evaluate 11 C-AS2471907 nondisplaceable distribution volume. Five subjects also underwent an additional scan to evaluate the within-day test-retest variability of 11 C-AS2471907 volumes of distribution ( V T ). Results: 11 C-AS2471907 time-activity curves were best fitted by the 2-tissue-compartment (2TC) model. 11 C-AS2471907 exhibited a regionally varying pattern of uptake throughout the brain. The V T of 11 C-AS2471907 ranged from 3.7 ± 1.5 mL/cm 3 in the caudate nucleus to 14.5 ± 5.3 mL/cm 3 in the occipital cortex, with intermediate values in the amygdala, white matter, cingulum, insula, frontal cortex, putamen, temporal and parietal cortices, cerebellum, and thalamus (from lowest to highest V T ). From the blocking scans, nondisplaceable distribution volume was determined to be 0.16 ± 0.04 mL/cm 3 for 11 C-AS2471907. Thus, nearly all uptake was specific and the binding potential ranged from 22 in the caudate to 90 in the occipital cortex. Test-retest variability of 2TC V T values was less than 10% in most large cortical regions (14% in parietal cortex) and ranged from 14% (cerebellum) to 51% (amygdala) in other regions. The intraclass correlation coefficient of 2TC V T values ranged from 0.55 in the white matter to 0.98 in the cerebellum. Conclusion: 11 C-AS2471907 has a high fraction of specific binding in vivo in humans and reasonable within-day reproducibility of binding parameters., (© 2019 by the Society of Nuclear Medicine and Molecular Imaging.)

The M 1 muscarinic acetylcholine receptor (mAChR) plays an important role in learning and memory, and therefore is a target for development of drugs for treatment of cognitive impairments in Alzheimer disease and schizophrenia. The availability of M 1 -selective radiotracers for PET will help in developing therapeutic agents by providing an imaging tool for assessment of drug dose-receptor occupancy relationship. Here we report the synthesis and evaluation of 11 C-LSN3172176 (ethyl 4-(6-(methyl- 11 C )-2-oxoindolin-1-yl)-[1,4'-bipiperidine]-1'-carboxylate) in nonhuman primates. Methods: 11 C-LSN3172176 was radiolabeled via the Suzuki-Miyaura cross-coupling method. PET scans in rhesus macaques were acquired for 2 h with arterial blood sampling and metabolite analysis to measure the input function. Blocking scans with scopolamine (50 μg/kg) and the M 1 -selective agent AZD6088 (0.67 and 2 mg/kg) were obtained to assess tracer binding specificity and selectivity. Regional brain time-activity curves were analyzed with the 1-tissue-compartment model and the multilinear analysis method (MA1) to calculate regional distribution volume. Nondisplaceable binding potential values were calculated using the cerebellum as a reference region. Results: 11 C-LSN3172176 was synthesized with greater than 99% radiochemical purity and high molar activity. In rhesus monkeys, 11 C-LSN3172176 metabolized rapidly (29% ± 6% parent remaining at 15 min) and displayed fast kinetics and extremely high uptake in the brain. Imaging data were modeled well with the 1-tissue-compartment model and MA1 methods. MA1-derived distribution volume values were high (range, 10-81 mL/cm 3 ) in all known M 1 mAChR-rich brain regions. Pretreatment with scopolamine and AZD6088 significantly reduced the brain uptake of 11 C-LSN3172176, thus demonstrating its binding specificity and selectivity in vivo. The cerebellum appeared to be a suitable reference region for derivation of nondisplaceable binding potential, which ranged from 2.42 in the globus pallidus to 8.48 in the nucleus accumbens. Conclusion: 11 C-LSN3172176 exhibits excellent in vivo binding and imaging characteristics in nonhuman primates and appears to be the first appropriate radiotracer for PET imaging of human M 1 AChR., (© 2019 by the Society of Nuclear Medicine and Molecular Imaging.)

Studies have shown κ-opioid receptor (KOR) abnormalities in addictive disorders, other central nervous system diseases, and Alzheimer's disease. We have developed the first set of agonist 11 C-GR103545 and antagonist 11 C-LY2795050 radiotracers for PET imaging of KOR in humans. Nonetheless, 11 C-GR103545 displays protracted uptake kinetics and is not an optimal radiotracer. Here, we report the development and evaluation of 11 C - methyl-( R )-4-(2-(3,4-dichlorophenyl)acetyl)-3-((diethylamino)methyl)piperazine-1-carboxylate ( 11 C-EKAP) and its comparison with 11 C-GR103545. Methods: EKAP was synthesized and assayed for in vitro binding affinities and then radiolabeled. PET studies were performed on rhesus monkeys. Blocking studies were performed with naloxone and the selective KOR antagonists LY2795050 and LY2456302. Arterial input functions were generated for use in kinetic modeling. Brain TACs were analyzed with multilinear analysis 1 to derive binding parameters. Results: EKAP has high KOR affinity (inhibition constant, 0.28 nM) and good selectivity in vitro. 11 C-EKAP was prepared in good radiochemical purity. 11 C-EKAP rapidly metabolized in plasma and displayed fast and reversible kinetics in brain, with peak uptake at less than 20 min after injection. Preblocking with naloxone (1 mg/kg) or LY2795050 (0.2 mg/kg) produced 84%-89% receptor occupancy, whereas LY2456302 (0.05 and 0.3 mg/kg) dose-dependently reduced 11 C-EKAP-specific binding, thus demonstrating its binding specificity and selectivity in vivo. Mean multilinear analysis 1-derived nondisplaceable binding potential values were 1.74, 1.79, 1.46, 0.80, and 0.77 for cingulate cortex, globus pallidus, insula, striatum, and frontal cortex, respectively, consistent with the known KOR distribution in primate brains. Conclusion: We have successfully developed 11 C-EKAP as a KOR agonist tracer with dual attractive imaging properties of fast uptake kinetics and high specific binding in vivo., (© 2019 by the Society of Nuclear Medicine and Molecular Imaging.)

The lysophosphatidic acid receptor type 1 (LPA1) is 1 of 6 known receptors of the extracellular signaling molecule lysophosphatidic acid. It mediates effects such as cell proliferation, migration, and differentiation. In the lung, LPA1 is involved in pathways leading, after lung tissue injury, to pulmonary fibrosis instead of normal healing, by mediating fibroblast recruitment and vascular leakage. Thus, a LPA1 PET radiotracer may be useful for studying lung fibrosis or for developing LPA1-targeting drugs. We developed and evaluated the radiotracer 11 C-BMT-136088 (1-(4'-(3-methyl-4-(((1( R )-(3- 11 C-methylphenyl)ethoxy)carbonyl)amino)isoxazol-5-yl)-[1,1'-biphenyl]-4-yl)cyclopropane-1-carboxylic acid) in rhesus monkeys to image LPA1 in the lung in vivo with PET. Methods: The study consisted of 3 parts: test-retest scans; self-saturation to estimate the tracer's in vivo dissociation constant, nondisplaceable volume of distribution ( V ND ), and nondisplaceable binding potential ( BP ND ); and dosimetry. In the first 2 parts, the radiotracer was administered using a bolus-plus-infusion protocol, the arterial input function was measured, and the animals underwent 2 scans per day separated by about 4 h. Lung regions of interest were segmented, and the tissue density estimated, from CT images. A fixed blood volume correction was applied. The tracer volume of distribution ( V T ) was estimated using multilinear analysis 1 (MA1) or equilibrium analysis (EA). Results: 11 C-BMT-136088 baseline V T was 1.83 ± 0.16 (MA1, n = 5) or 2.1 ± 0.55 (EA, n = 7) mL of plasma per gram of tissue in the left and right lung regions of interest, with a test-retest variability of -6% (MA1, n = 1) or -1% ± 14% (EA, n = 2). For the self-saturation study, 11 C-BMT-136088 V ND and BP ND were estimated to be 0.9 ± 0.08 mL of plasma per gram of tissue and 1.1 ± 0.14, respectively. The unlabeled drug dose and plasma concentration leading to a 50% reduction of 11 C-BMT-136088 specific binding were 73 ± 30 nmol/kg and 28 ± 12 nM, respectively. The average plasma free fraction was 0.2%; thus, the tracer's in vivo dissociation constant was estimated to be 55 pM. For the dosimetry study, the highest organ dose was in the liver (43.1 ± 4.9 and 68.9 ± 9.4 μSv/MBq in reference human male and female phantoms, respectively), and the effective dose equivalent was 6.9 ± 0.6 and 8.7 ± 0.6 μSv/MBq, respectively. Conclusion: Specific binding of 11 C-BMT-136088 can be reliably measured to quantify LPA1 in the lungs of rhesus monkeys in vivo., (© 2018 by the Society of Nuclear Medicine and Molecular Imaging.)

The κ-opioid receptor (KOR) has been implicated in depression, addictions, and other central nervous system disorders and, thus, is an important target for drug development. We previously developed several 11 C-labeled PET radiotracers for KOR imaging in humans. Here we report the synthesis and evaluation of 18 F-LY2459989 as the first 18 F-labeled KOR antagonist radiotracer in nonhuman primates and its comparison with 11 C-LY2459989. Methods: The novel radioligand 18 F-LY2459989 was synthesized by 18 F displacement of a nitro group or an iodonium ylide. PET scans in rhesus monkeys were obtained on a small-animal scanner to assess the pharmacokinetic and in vivo binding properties of the ligand. Metabolite-corrected arterial activity curves were measured and used as input functions in the analysis of brain time-activity curves and the calculation of binding parameters. Results: With the iodonium ylide precursor, 18 F-LY2459989 was prepared at high radiochemical yield (36% ± 7% [mean ± SD]), radiochemical purity (>99%), and mean molar activity (1,175 GBq/μmol; n = 6). In monkeys, 18 F-LY2459989 was metabolized at a moderate rate, with a parent fraction of approximately 35% at 30 min after injection. Fast and reversible kinetics were observed, with a regional peak uptake time of less than 20 min. Pretreatment with the selective KOR antagonist LY2456302 (0.1 mg/kg) decreased the activity level in regions with high levels of binding to that in the cerebellum, thus demonstrating the binding specificity and selectivity of 18 F-LY2459989 in vivo. Regional time-activity curves were well fitted by the multilinear analysis 1 kinetic model to derive reliable estimates of regional distribution volumes. With the cerebellum as the reference region, regional binding potentials were calculated and ranked as follows: cingulate cortex > insula > caudate/putamen > frontal cortex > temporal cortex > thalamus, consistent with the reported KOR distribution in the monkey brain. Conclusion: The evaluation of 18 F-LY2459989 in nonhuman primates demonstrated many attractive imaging properties: fast tissue kinetics, specific and selective binding to the KOR, and high specific binding signals. A side-by-side comparison of 18 F-LY2459989 and 11 C-LY2459989 indicated similar kinetic and binding profiles for the 2 radiotracers. Taken together, the results indicated that 18 F-LY2459989 appears to be an excellent PET radiotracer for the imaging and quantification of the KOR in vivo., (© 2018 by the Society of Nuclear Medicine and Molecular Imaging.)

Measurements of drug occupancies using positron emission tomography (PET) can be biased if the radioligand concentration exceeds "tracer" levels. Negative bias would also arise in successive PET scans if clearance of the radioligand is slow, resulting in a carryover effect. We developed a method to (1) estimate the in vivo dissociation constant K d of a radioligand from PET studies displaying a non-tracer carryover (NTCO) effect and (2) correct the NTCO bias in occupancy studies taking into account the plasma concentration of the radioligand and its in vivo K d . This method was applied in a study of healthy human subjects with the histamine H 3 receptor radioligand [ 11 C]GSK189254 to measure the PK-occupancy relationship of the H 3 antagonist PF-03654746. From three test/retest studies, [ 11 C]GSK189254 K d was estimated to be 9.5 ± 5.9 pM. Oral administration of 0.1 to 4 mg of PF-03654746 resulted in occupancy estimates of 71%-97% and 30%-93% at 3 and 24 h post-drug, respectively. NTCO correction adjusted the occupancy estimates by 0%-15%. Analysis of the relationship between corrected occupancies and PF-03654746 plasma levels indicated that PF-03654746 can fully occupy H 3 binding sites ( RO max  = 100%), and its IC 50 was estimated to be 0.144 ± 0.010 ng/mL. The uncorrected IC 50 was 26% higher.

Unlabelled: This was a first-in-human study of the novel phosphodiesterase-2A (PDE2A) PET ligand (18)F-PF-05270430. The primary goals were to determine the appropriate tracer kinetic model to quantify brain uptake and to examine the within-subject test-retest variability., Methods: In advance of human studies, radiation dosimetry was determined in nonhuman primates. Six healthy male subjects participated in a test-retest protocol with dynamic scans and metabolite-corrected input functions. Nine brain regions of interest were studied, including the striatum, white matter, neocortical regions, and cerebellum. Multiple modeling methods were applied to calculate volume of distribution (VT) and binding potentials relative to the nondisplaceable tracer in tissue (BPND), concentration of tracer in plasma (BPP), and free tracer in tissue (BPF). The cerebellum was selected as a reference region to calculate binding potentials., Results: The dosimetry study provided an effective dose of less than 0.30 mSv/MBq, with the gallbladder as the critical organ; the human target dose was 185 MBq. There were no adverse events or clinically detectable pharmacologic effects reported. Tracer uptake was highest in the striatum, followed by neocortical regions and white matter, and lowest in the cerebellum. Regional time-activity curves were well fit by multilinear analysis-1, and a 70-min scan duration was sufficient to quantify VT and the binding potentials. BPND, with mean values ranging from 0.3 to 0.8, showed the best intrasubject and intersubject variability and reliability. Test-retest variability in the whole brain (excluding the cerebellum) of VT, BPND, and BPP were 8%, 16%, and 17%, respectively., Conclusion: (18)F-PF-05270430 shows promise as a PDE2A PET ligand, albeit with low binding potential values., (© 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

The Radiotracer [(11)C]OMAR was developed for positron emission tomography (PET) imaging of cannabinoid type-1 receptors (CB1R). The objectives of the present study were to evaluate kinetic analysis methods, determine test-retest reliability, and assess gender differences in receptor availability. Dynamic PET data were acquired in 10 human subjects, and analyzed with one-tissue (1T) and two-tissue (2T) compartment models and by the Logan and multilinear analysis (MA1) methods to estimate regional volume of distribution (VT). The 2T model inclusive of a vascular component (2TV) and MA1 were the preferred techniques. Test-retest reliability of VT was good (mean absolute deviation ~9%; intraclass correlation coefficient ~0.7). Tracer parent fraction in plasma was lower in women (P<0.0001). Cerebral uptake normalized by body weight and injected dose was higher in men by 17% (P<0.0001), but VT was significantly greater in women by 23% (P<0.0001). These findings show that [(11)C]OMAR binding can be reliably quantified by the 2T model or MA1 method and demonstrate the utility of this tracer for in vivo imaging of CB1R. In addition, results from the present study indicate that gender difference in receptor binding should be taken into consideration when [(11)C]OMAR is used to quantify CB1R availability in neuropsychiatric disorders.

Unlabelled: (11)C-LY2795050 is a new antagonist PET radioligand for the κ opioid receptor (KOR). In this study, we assessed the reproducibility of the binding parameters of (11)C-LY2795050 in healthy human subjects., Methods: Sixteen healthy subjects (11 men and 5 women) underwent 2 separate 90-min PET scans with arterial input function and plasma free fraction (fP) measurements. The 2-tissue-compartment model and multilinear analysis-1 were applied to calculate 5 outcome measures in 14 brain regions: distribution volume (VT), VT normalized by fP (VT/fP), and 3 binding potentials (nondisplaceable binding potential, binding potential relative to total plasma concentration, and binding potential relative to free plasma concentration: BPND, BPP, BPF, respectively). Since KOR is distributed ubiquitously throughout the brain, there are no suitable reference regions. We used a fixed fraction of individual cerebellar VT value (VT,CER) as the nondisplaceable VT (VND) (VND = VT,CER/1.17). The relative and absolute test-retest variability and intraclass correlation coefficient were evaluated for the outcome measures of (11)C-LY2795050., Results: The test-retest variability of (11)C-LY2795050 for VT was no more than 10% in any region and was 12% in the amygdala. For binding potential (BPND and BPP), the test-retest variability was good in regions of moderate and high KOR density (BPND > 0.4) and poor in regions of low density. Correction by fP (VT/fP or BPF) did not improve the test-retest performance., Conclusion: Our results suggest that quantification of (11)C-LY2795050 imaging is reproducible and reliable in regions with moderate and high KOR density. Therefore, we conclude that this first antagonist radiotracer is highly useful for PET studies of KOR., (© 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

(11)C-LY2795050 is a novel kappa opioid receptor (KOR) antagonist tracer for positron emission tomography (PET) imaging. The purpose of this first-in-human study was to determine the optimal kinetic model for analysis of (11)C-LY2795050 imaging data. Sixteen subjects underwent baseline scans and blocking scans after oral naltrexone. Compartmental modeling and multilinear analysis-1 (MA1) were applied using the arterial input functions. Two-tissue compartment model and MA1 were found to be the best models to provide reliable measures of binding parameters. The rank order of (11)C-LY2795050 distribution volume (VT) matched the known regional KOR densities in the human brain. Blocking scans with naltrexone indicated no ideal reference region for (11)C-LY2795050. Three methods for calculation of the nondisplaceable distribution volume (VND) were assessed: (1) individual VND estimated from naltrexone occupancy plots, (2) mean VND across subjects, and (3) a fixed fraction of cerebellum VT. Approach (3) produced the lowest intersubject variability in the calculation of binding potentials (BPND, BPF, and BPP). Therefore, binding potentials of (11)C-LY2795050 can be determined if the specific binding fraction in the cerebellum is presumed to be unchanged by diseases and experimental conditions. In conclusion, results from the present study show the suitability of (11)C-LY2795050 to image and quantify KOR in humans.

Unlabelled: (11)C-(+)-4-propyl-9-hydroxynaphthoxazine ((11)C-(+)-PHNO) is an agonist radioligand for imaging dopamine D2 and D3 receptors in the human brain with PET. In this study we evaluated the reproducibility of (11)C-(+)-PHNO binding parameters using a within-day design and assessed parametric imaging methods., Methods: Repeated studies were performed in 8 subjects, with simultaneous measurement of the arterial input function and plasma free fraction. Two (11)C-(+)-PHNO scans for the same subject were separated by 5.4 ± 0.7 h. After compartment models were evaluated, (11)C-(+)-PHNO volumes of distribution (V(T)) and binding potentials relative to the concentration of tracer in plasma (BP(P)), nondisplaceable tracer in tissue (BP(ND)), and free tracer in tissue (BP(F)) were quantified using the multilinear analysis MA1 method, with the cerebellum as the reference region. Parametric images of BP(ND) were also computed using the simplified reference tissue model (SRTM) and SRTM2., Results: The test-retest variability of (11)C-(+)-PHNO BP(ND) was 9% in D2-rich regions (caudate and putamen). Among D3-rich regions, variability was low in the pallidum (6%) but higher in substantia nigra (19%), thalamus (14%), and hypothalamus (21%). No significant mass carry-over effect was observed in D3-rich regions, although a trend in BP(ND) was present in the substantia nigra (-14% ± 15%). Because of the relatively fast kinetics, low-noise BP(ND) parametric images were obtained with both SRTM and SRTM2 without spatial smoothing., Conclusion: (11)C-(+)-PHNO can be used to compute low-noise parametric images in both D2- and D3-rich regions in humans., (© 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Unlabelled: Four novel phosphodiesterase 10A (PDE10A) PET tracers have been synthesized, characterized in preclinical studies, and compared with the previously reported (11)C-MP-10., Methods: On the basis of in vitro data, IMA102, IMA104, IMA107, and IMA106 were identified as potential PDE10A radioligand candidates and labeled with either (11)C via N-methylation or with (18)F through an SN2 reaction, in the case of IMA102. These candidates were compared with (11)C-MP-10 in pilot in vivo studies in the pig brain. On the basis of these data, (11)C-IMA106 and (11)C-IMA107 were taken into further evaluation and comparison with (11)C-MP-10 in the primate brain. Finally, the most promising radioligand candidate was progressed into human evaluation., Results: All 5 tracers were produced with good radiochemical yield and specific activity. All candidates readily entered the brain and demonstrated a heterogeneous distribution consistent with the known expression of PDE10A. Baseline PET studies in the pig and baboon showed that (11)C-IMA107 and (11)C-MP-10 displayed the most favorable tissue kinetics and imaging properties. The administration of selective PDE10A inhibitors reduced the binding of (11)C-IMA107 and (11)C-MP-10 in the PDE10A-rich brain regions, in a dose-dependent manner. In the nonhuman primate brain, the tissue kinetics of (11)C-IMA107 and (11)C-MP-10 were well described by a 2-tissue-compartment model, allowing robust estimates of the regional total volume of distribution. Blockade with unlabeled MP-10 confirmed the suitability of the cerebellum as a reference tissue and enabled the estimation of regional binding potential as the outcome measure of specific binding., Conclusion: (11)C-IMA107 was identified as the ligand with the highest binding potential while still possessing reversible kinetics. The first human administration of (11)C-IMA107 has demonstrated the expected regional distribution and suitably fast kinetics, indicating that (11)C-IMA107 will be a useful tool for the investigation of PDE10A status in the living human brain.

The radiotracer [(11)C]PHNO may have advantages over other dopamine (DA) D2/D3 receptor ligands because, as an agonist, it measures high-affinity, functionally active D2/D3 receptors, whereas the traditionally used radiotracer [(11)C]raclopride measures both high- and low-affinity receptors. Our aim was to take advantage of the strength of [(11)C]PHNO for measuring the small DA signal induced by nicotine, which has been difficult to measure in preclinical and clinical neuroimaging studies. Nicotine- and amphetamine-induced DA release in non-human primates was measured with [(11)C]PHNO and [(11)C]raclopride positron emission tomography (PET) imaging. Seven adult rhesus monkeys were imaged on a FOCUS 220 PET scanner after injection of a bolus of [(11)C]PHNO or [(11)C]raclopride in three conditions: baseline; preinjection of nicotine (0.1 mg/kg bolus+0.08 mg/kg infusion over 30 min); preinjection of amphetamine (0.4 mg/kg, 5 min before radiotracer injection). DA release was measured as change in binding potential (BPND). Nicotine significantly decreased BPND in the caudate (7 ± 8%), the nucleus accumbens (10 ± 7%), and in the globus pallidus (13 ± 15%) measured with [(11)C]PHNO, but did not significantly decrease BPND in the putamen or the substantia nigra or in any region when measured with [(11)C]raclopride. Amphetamine significantly reduced BPND in all regions with both radiotracers. In the striatum, larger amphetamine-induced changes were detected with [(11)C]PHNO compared with [(11)C]raclopride (52-64% vs 33-35%, respectively). We confirmed that [(11)C]PHNO is more sensitive than [(11)C]raclopride to nicotine- and amphetamine-induced DA release. [(11)C]PHNO PET may be more sensitive to measuring tobacco smoking-induced DA release in human tobacco smokers.

[(11)C]AFM, or [(11)C]2-[2-(dimethylaminomethyl)phenylthio]-5-fluoromethylphenylamine, is a new positron emission tomography (PET) radioligand with high affinity and selectivity for the serotonin transporter (SERT). The purpose of this study was to determine the most appropriate kinetic model to quantify [(11)C]AFM binding in the healthy human brain. Positron emission tomography data and arterial input functions were acquired from 10 subjects. Compartmental modeling and the multilinear analysis-1(MA1) method were tested using the arterial input functions. The one-tissue model showed a lack of fit in low-binding regions, and the two-tissue model failed to estimate parameters reliably. Regional time-activity curves were well described by MA1. The rank order of [(11)C]AFM binding potential (BPND) matched well with the known regional SERT densities. For routine use of [(11)C]AFM, several noninvasive methods for quantification of regional binding were evaluated, including simplified reference tissue models (SRTM and SRTM2), and multilinear reference tissue models (MRTM and MRTM2). The best methods for region of interest (ROI) analysis were MA1, MRTM2, and SRTM2, with fixed population kinetic values ( or b') for the reference methods. The MA1 and MRTM2 methods were best for parametric imaging. These results showed that [(11)C]AFM is a suitable PET radioligand to image and quantify SERT in humans.

Unlabelled: Neuroreceptor imaging in the nonhuman primate (NHP) is valuable for translational research approaches in humans. However, most NHP studies are conducted under anesthesia, which affects the interpretability of receptor binding measures. The aims of this study were to develop awake NHP imaging with minimal head restraint and to compare in vivo binding of the γ-aminobutyric acid type A (GABAA)-benzodiazepine radiotracer (11)C-flumazenil under anesthetized and awake conditions. We hypothesized that (11)C-flumazenil binding potential (BPND) would be higher in isoflurane-anesthetized monkeys., Methods: The small animal PET scanner was fitted to a mechanical device that raised and tilted the scanner 45° while the awake NHP was tilted back 35° in a custom chair for optimal brain positioning, which required acclimation of the animals to the chair, touch-screen tasks, intravenous catheter insertion, and tilting. For PET studies, the bolus-plus-constant infusion method was used for (11)C-flumazenil administration. Two rhesus monkeys were scanned under the awake (n = 6 scans) and isoflurane-anesthetized (n = 4 scans) conditions. An infrared camera was used to track head motion during PET scans. Under the awake condition, emission and head motion-tracking data were acquired for 40-75 min after injection. Anesthetized monkeys were scanned for 90 min. Cortisol measurements were acquired during awake and anesthetized scans. Equilibrium analysis was used for both the anesthetized (n = 4) and the awake (n = 5) datasets to compute mean BPND images in NHP template space, using the pons as a reference region. The percentage change per minute in radioactivity concentration was calculated in high- and low-binding regions to assess the quality of equilibrium., Results: The monkeys acclimated to procedures in the NHP chair necessary to perform awake PET imaging. Image quality was comparable between awake and anesthetized conditions. The relationship between awake and anesthetized values was BPND (awake) = 0.94 BPND (anesthetized) + 0.36 (r(2) = 0.95). Cortisol levels were significantly higher under the awake condition (P < 0.05)., Conclusion: We successfully performed awake NHP imaging with minimal head restraint. There was close agreement in (11)C-flumazenil BPND values between awake and anesthetized conditions.

Unlabelled: (11)C-LY2795050 is a novel κ-selective antagonist PET tracer. The in vitro binding affinities (Ki) of LY2795050 at the κ-opioid (KOR) and μ-opioid (MOR) receptors are 0.72 and 25.8 nM, respectively. Thus, the in vitro KOR/MOR binding selectivity is about 36:1. Our goal in this study was to determine the in vivo selectivity of this new KOR antagonist tracer in the monkey., Methods: To estimate the ED50 value (dose of a compound [or drug] that gives 50% occupancy of the target receptor) of LY2795050 at the MOR and KOR sites, 2 series of blocking experiments were performed in 3 rhesus monkeys using (11)C-LY2795050 and (11)C-carfentanil with coinjections of various doses of unlabeled LY2795050. Kinetic modeling was applied to calculate regional binding potential (BP(ND)), and 1- and 2-site binding curves were fitted to these data to measure (11)C-LY2795050 binding selectivity., Results: The LY2795050 ED50 at MOR was 119 μg/kg based on a 1-site model for (11)C-carfentanil. The 1-site binding model was also deemed sufficient to describe the specific binding of (11)C-LY2795050 at KOR. The ED50 at KOR estimated from the 1-site model was 15.6 μg/kg. Thus, the ED50 ratio for MOR:KOR was 7.6., Conclusion: The in vivo selectivity of (11)C-LY2795050 for KOR over MOR is 7.6. (11)C-LY2795050 has 4.7-fold-lower selectivity at KOR over MOR in vivo as compared with in vitro. Nevertheless, on the basis of our finding in vivo, 88% of the PET-observed specific binding of (11)C-LY2795050 under baseline conditions will be due to binding of the tracer at the KOR site in a region with similar prevalence of KOR and MOR. (11)C-LY2795050 is sufficiently selective for KOR over MOR in vivo to be considered an appropriate probe for studying the KOR with PET.

In vivo characterization of the brain pharmacokinetics of novel compounds provides important information for drug development decisions involving dose selection and the determination of administration regimes. In this context, the compound-target affinity is the key parameter to be estimated. However, if compounds exhibit a dynamic lag between plasma and target bound concentrations leading to pharmacological hysteresis, care needs to be taken to ensure the appropriate modeling approach is used so that the system is characterized correctly and that the resultant estimates of affinity are correct. This work focuses on characterizing different pharmacokinetic models that relate the plasma concentration to positron emission tomography outcomes measurements (e.g., volume of distribution and target occupancy) and their performance in estimating the true in vivo affinity. Measured (histamine H3 receptor antagonist--GSK189254) and simulated data sets enabled the investigation of different modeling approaches. An indirect pharmacokinetic-receptor occupancy model was identified as a suitable model for the calculation of affinity when a compound exhibits pharmacological hysteresis.

[(18)F]FPEB is a positron emission tomography tracer which, in preclinical studies, has shown high specificity and selectivity toward the metabotropic glutamate receptor 5 (mGluR5). It possesses the potential to be used in human studies to evaluate mGluR5 function in a range of neuropsychiatric disorders, such as anxiety and Fragile X syndrome. To define optimal scan methodology, healthy human subjects were scanned for 6 hours following either a bolus injection (n=5) or bolus-plus-constant-infusion (n=5) of [(18)F]FPEB. Arterial blood samples were collected and parent fraction measured by high-performance liquid chromatography (HPLC) to determine the metabolite-corrected plasma input function. Time activity curves were extracted from 13 regions and fitted by various models to estimate V(T) and BPND. [(18)F]FPEB was well fitted by the two-tissue compartment model, MA1 (t*=30), and MRTM (using cerebellum white matter as a reference). Highest V(T) values were observed in the anterior cingulate and caudate, and lowest V(T) values were observed in the cerebellum and pallidum. For kinetic modeling studies, VT and BPND were estimated from bolus or bolus-plus-constant-infusion scans as short as 90 minutes. Bolus-plus-constant-infusion of [(18)F]FPEB reduced intersubject variability in V(T) and allowed equilibrium analysis to be completed with a 30-minute scan, acquired 90-120 minutes after the start of injection.

Unlabelled: We describe the preparation of 7 alpha-[125I]iodo-5 alpha-dihydrotestosterone (7 alpha-[125I]IDHT) and its characterization as a ligand for the androgen receptor., Methods: We designed a route to prepare the radioiodine-labeled androgen on microscale through treatment of the 7 beta-tosylate of 7 beta-hydroxy-5 alpha-dihydrotestosterone-17 beta-p-nitro-benzoate with Na125I, followed by alkaline hydrolysis. The radiolabeled steroid was tested as a ligand for the androgen receptor in cytosol from MCF-7 cells, and for its in vivo tissue distribution in the rat. In addition, we tested 7 alpha-[125I]IDHT as a ligand in a novel assay for the detection and quantification of the ligand activated androgen receptor by in vitro autoradiography., Results: The above synthetic route produced the 17 beta-p-nitrobenzoate of 7 alpha-[125I]IDHT in carrier-free form and in good yield. The 17 beta-ester was removed with alkali and the resulting 7 alpha-[125I]IDHT was purified by HPLC. 7 alpha-[125I]IDHT bound with high affinity, Kd = 0.26 nM, to the androgen receptor and showed low nonspecific binding. Since the ligand was carrier free and thus of very high specific activity, approximately 2,200 Ci/mmole, the sensitivity of the assay was much greater than with [3H]R1881, the classical androgen receptor ligand with which it was compared. When tested as a ligand for in vitro autoradiography, 7 alpha-[125I]IDHT produced excellent autoradiograms of the activated receptor with very low nonspecific binding and with only overnight exposure of the film., Conclusion: These studies demonstrate that 7 alpha-[125I]IDHT is an excellent ligand for the androgen receptor.

Three bleomycin (BLM)-resistant sublines were isolated from a human head and neck squamous cell carcinoma cell line (A-253); these sublines (C-10, D-10, and G-11) were 4-, 9-, and 21-fold resistant to BLM A2, respectively. These sublines were selectively resistant to other members of the BLM class, namely BLM B2, peplomycin, talisomycin S10b, and bleomycinic acid; none of the sublines displayed cross-resistance to vincristine, doxorubicin, cis-diamminedichloroplatinum or melphalan; only one subline (G-11) was cross-resistant to X-irradiation. None of the BLM-resistant cell lines demonstrated resistance to the novel BLM analogue liblomycin, which contains a lipophilic terminal amine. The cell cycle distributions of the clonally derived BLM-resistant cell populations were similar to the distribution of the parental cell population. In vitro BLM hydrolase activity in homogenates of D-10 and G-11 BLM-resistant cell lines was two- to threefold higher than that in homogenates of A-253 or C-10 cells. Nonetheless, no deamido BLM A2 was found associated with any cell type or in the culture medium and more than 80% of the radioactivity in all cells appeared as unmetabolized BLM A2 by high pressure liquid chromatography. Thus, the appearance of large quantities of the deamido BLM metabolite was not a prominent feature of acquired resistance to BLM in these human tumor cells. The cellular accumulation of radiolabeled BLM A2 by C-10 and G-11 cells during a 1-h incubation with [3H]BLM A2 was 1/2 that seen with A-253 and D-10 cells. C-10 cells maintained a lower nuclear content of radioactivity than A-253, G-11, or D-10 cells. Initial single strand DNA damage, based upon alkaline elution analysis, also was lower in C-10 cells compared to A-253 cells. D-10 cells, in contrast, exhibited high initial genomic DNA damage but demonstrated a greater repair rate than either A-253 or C-10 cells. Thus, multiple BLM-resistant phenotypes can be obtained from a population of human squamous carcinoma cells, and modification of the terminal amine in the BLM molecule can produce compounds capable of circumventing all of these BLM-resistant phenotypes. Liblomycin, which appears to be a nonclassical BLM, may be a useful therapeutic agent with a spectrum of activity distinct from other members of the BLM class.

The cytosolic estrogen receptor (ERc) and progestin receptor (PRc) levels were measured in 56 human epithelial ovarian tumors. The maximum ERc content in a tumor sample was 163 fmol/mg cytosolic protein. Forty-six of the tumor samples were evaluable for clonogenic growth in soft agar, and 19 samples produced 15 or more colonies per 10(5) cells plated. Four of the samples that grew had ERc levels of greater than 30 fmol/mg cytosolic protein. No correlation, however, between growth in soft agar and ERc or PRc content was observed. The antiproliferative properties of the antiestrogen, tamoxifen, were studied. Although no decrease in colony formation was observed after a 1-hr exposure to 0.2 or 2 microM tamoxifen, continuous exposure of cells to 2 microM tamoxifen reduced clonogenicity in 8 of 18 solid ovarian carcinomas examined. The maximum diminution in colony formation was approximately 50% of that of the control and was seen in 2 tumor samples. Both tumors that displayed the maximum response to continuous tamoxifen treatment had ERc and PRc levels greater than 30 fmol/mg cytosolic protein. None of the 14 tumors with ERc levels less than or equal to 30 fmol/mg cytosolic protein exhibited a decrease in colony formation of more than 50%. Exposure of cells for 1 hr to the combination of doxorubicin and tamoxifen produced a significant antagonism of the individual doxorubicin or tamoxifen antiproliferative effects in 7 of 9 samples examined. These data suggest that in a subset of human ovarian epithelial carcinomas tamoxifen alone can have some direct antiproliferative action on the clonogenic cells. The maximum antiproliferative effect of tamoxifen observed was related to ERc content in ovarian tumors.