Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)-mediated ER stress crosstalk with autophagy is involved in tris(2-chloroethyl) phosphate stress-induced cardiac fibrosis.

Excessive organophosphate flame retardant (OPFR) use in consumer products has been reported to increase human disease susceptibility. However, the adverse effects of tris(2-chloroethyl) phosphate (TCEP) (a chlorinated alkyl OPFR) on the heart remain unknown. In this study, we tested whether cardiac fibrosis occurred in animal models of TCEP (10 mg/kg b.w./day) administered continuously by gavage for 30 days and evaluated the specific role of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). First, we confirmed that TCEP could trigger cardiac fibrosis by histopathological observation and cardiac fibrosis markers. We further verified that cardiac fibrosis occurred in animal models of TCEP exposure accompanied by SERCA2a, SERCA2b and SERCA2c downregulation. Notably, inductively coupled plasma-mass spectrometry (ICP-MS) analysis revealed that the cardiac concentrations of Ca2+ increased by 45.3% after TCEP exposure. Using 4-Isopropoxy-N-(2-methylquinolin-8-yl)benzamide (CDN1163, a small molecule SERCA activator), we observed that Ca2+ overload and subsequent cardiac fibrosis caused by TCEP were both alleviated. Simultaneously, the protein levels of endoplasmic reticulum (ER) markers (protein kinase R-like endoplasmic reticulum kinase (PERK), inositol requiring protein 1α (IRE1α), eukaryotic initiation factor 2 α (eIF2α)) were upregulated by TCEP, which could be abrogated by CDN1163 pretreatment. Furthermore, we observed that CDN1163 supplementation prevented overactive autophagy induced by TCEP in the heart. Mechanistically, TCEP could lead to Ca2+ overload by inhibiting the expression of SERCA, thereby triggering ER stress and overactive autophagy, eventually resulting in cardiac fibrosis. Together, our results suggest that the Ca2+ overload/ER stress/autophagy axis can act as a driver of cardiotoxicity induced by TCEP.

Design and Evaluation of a Novel Peptide-Drug Conjugate Covalently Targeting SARS-CoV-2 Papain-like Protease.

Coronavirus disease 2019 (COVID-19) pandemic, a global health threat, was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 papain-like cysteine protease (PLpro) was recognized as a promising drug target because of multiple functions in virus maturation and antiviral immune responses. Inhibitor GRL0617 occupied the interferon-stimulated gene 15 (ISG15) C-terminus-binding pocket and showed an effective antiviral inhibition. Here, we described a novel peptide-drug conjugate (PDC), in which GRL0617 was linked to a sulfonium-tethered peptide derived from PLpro-specific substrate LRGG. The EM-C and EC-M PDCs showed a promising in vitro IC50 of 7.40 ± 0.37 and 8.63 ± 0.55 µM, respectively. EC-M could covalently label PLpro active site C111 and display anti-ISGylation activities in cellular assays. The results represent the first attempt to design PDCs composed of stabilized peptide inhibitors and GRL0617 to inhibit PLpro. These novel PDCs provide promising opportunities for antiviral drug design.

Entrectinib-A SARS-CoV-2 Inhibitor in Human Lung Tissue (HLT) Cells.

Since the start of the COVID-19 outbreak, pharmaceutical companies and research groups have focused on the development of vaccines and antiviral drugs against SARS-CoV-2. Here, we apply a drug repurposing strategy to identify drug candidates that are able to block the entrance of the virus into human cells. By combining virtual screening with in vitro pseudovirus assays and antiviral assays in Human Lung Tissue (HLT) cells, we identify entrectinib as a potential antiviral drug.

Revefenacin for the treatment of chronic obstructive pulmonary disease.

Introduction: Chronic obstructive pulmonary disease (COPD) is a progressive condition characterized by irreversible or incompletely reversible airflow limitation. Long-acting bronchodilators, including ß2 agonists (LABA) and muscarinic antagonists (LAMA), serve as the standard of care for maintenance therapy in COPD. Individualizing therapy to optimize selection of delivery device has the potential to improve medication adherence and clinical outcomes among COPD patients.Areas covered: Revefenacin (Yupelri) is the only LAMA approved for once-daily administration via standard jet nebulizer for the maintenance therapy in patients with COPD. Revefenacin has a unique biphenyl carbamate tertiary amine structure, differing from the quaternary amine structure of previously approved LAMAs. Here we summarize the available clinical data for this new agent and discuss its potential place in the treatment of COPD.Expert opinion: Based on available clinical trial data, revefenacin appears to be an effective and safe option for long-term maintenance therapy of COPD. Revefenacin offers a once-daily option for LAMA therapy for patients who prefer or require nebulized drug delivery. The availability of this agent can allow patients to combine nebulized therapies that could improve clinical outcomes in appropriately selected patients with COPD.

Tomato Root Growth Inhibition by Salinity and Cadmium Is Mediated By S-Nitrosative Modifications of ROS Metabolic Enzymes Controlled by S-Nitrosoglutathione Reductase.

S-nitrosoglutathione reductase (GSNOR) exerts crucial roles in the homeostasis of nitric oxide (NO) and reactive nitrogen species (RNS) in plant cells through indirect control of S-nitrosation, an important protein post-translational modification in signaling pathways of NO. Using cultivated and wild tomato species, we studied GSNOR function in interactions of key enzymes of reactive oxygen species (ROS) metabolism with RNS mediated by protein S-nitrosation during tomato root growth and responses to salinity and cadmium. Application of a GSNOR inhibitor N6022 increased both NO and S-nitrosothiol levels and stimulated root growth in both genotypes. Moreover, N6022 treatment, as well as S-nitrosoglutathione (GSNO) application, caused intensive S-nitrosation of important enzymes of ROS metabolism, NADPH oxidase (NADPHox) and ascorbate peroxidase (APX). Under abiotic stress, activities of APX and NADPHox were modulated by S-nitrosation. Increased production of H2O2 and subsequent oxidative stress were observed in wild Solanumhabrochaites, together with increased GSNOR activity and reduced S-nitrosothiols. An opposite effect occurred in cultivated S. lycopersicum, where reduced GSNOR activity and intensive S-nitrosation resulted in reduced ROS levels by abiotic stress. These data suggest stress-triggered disruption of ROS homeostasis, mediated by modulation of RNS and S-nitrosation of NADPHox and APX, underlies tomato root growth inhibition by salinity and cadmium stress.

Selective targeting of tumor cells and tumor associated macrophages separately by twin-like core-shell nanoparticles for enhanced tumor-localized chemoimmunotherapy.

Tumor associated macrophage (TAM)-based immunotherapy has been presented as a promising strategy in cancer therapy. The combination of TAM-based immunotherapy with sorafenib (SF) could be conceivably quite more effective in hepatocellular carcinoma (HCC) treatment. A co-delivery system was superior in improving the co-accumulation of two drugs in tumor tissues for chemoimmunotherapy, while in the case of selective targeting of separated cells such as tumor cells and immune cells, a novel targeted co-delivery strategy was badly required. In this study, twin-like core-shell nanoparticles (TCN) were developed for synchronous biodistribution and separated cell targeting delivery of SF and TAM re-polarization agents IMD-0354 to cancer cells and TAM to enhance tumor-localized chemoimmunotherapy, respectively. First of all, SF loaded cationic lipid-based nanoparticles (SF-CLN) and mannose-modified IMD-0354 loaded cationic lipid-based nanoparticles (M-IMD-CLN) were prepared, respectively. SF on the surface of SF-CLN and mannose on the M-IMD-CLN were regarded as targeting ligands for selective targeting delivery of SF-CLN and M-IMD-CLN to cancer cells and TAM separately. Then, pH-responsive charge reversal polymer O-carboxymethyl-chitosan (CMCS) was coated on the SF-CLN and M-IMD-CLN to obtain twin-like CMCS/SF-CLN and CMCS/M-IMD-CLN, respectively. The results of cellular uptake assay on Hepa1-6 cells and RAW 264.7 cells in vitro, respectively, as well as the results of tumor tissue distribution of SF and IMD-0354 in vivo suggested that CMCS/SF-CLN and CMCS/M-IMD-CLN exhibited similar properties in vitro and synchronous biodistribution in vivo, and were efficient at separated cell targeting delivery. What's more, the results of antitumor efficiency in vivo and phenotype analysis of TAM in tumor tissues proved that CMCS/SF-CLN and CMCS/M-IMD-CLN exhibited superior synergistic antitumor efficacy and M2-type TAM polarization ability compared with SF treatment in Hepa1-6 tumor bearing mice. Consequently, TCN which was the combination of co-administration and nano-drug delivery systems has great potential to be used in tumor-localized chemoimmunotherapy in clinics.

Selective Inhibition of Histone Deacetylase 10: Hydrogen Bonding to the Gatekeeper Residue is Implicated.

The discovery of isozyme-selective histone deacetylase (HDAC) inhibitors is critical for understanding the biological functions of individual HDACs and for validating HDACs as drug targets. The isozyme HDAC10 contributes to chemotherapy resistance and has recently been described to be a polyamine deacetylase, but no studies toward selective HDAC10 inhibitors have been published. Using two complementary assays, we found Tubastatin A, an HDAC6 inhibitor, to potently bind HDAC10. We synthesized Tubastatin A derivatives and found that a basic amine in the cap group was required for strong HDAC10 binding. HDAC10 inhibitors mimicked knockdown by causing dose-dependent accumulation of acidic vesicles in a neuroblastoma cell line. Furthermore, docking into human HDAC10 homology models indicated that a hydrogen bond between a cap group nitrogen and the gatekeeper residue Glu272 was responsible for potent HDAC10 binding. Taken together, our data provide an optimal platform for the development of HDAC10-selective inhibitors, as exemplified with the Tubastatin A scaffold.

Hit discovery of 4-amino-N-(4-(3-(trifluoromethyl)phenoxy)pyrimidin-5-yl)benzamide: A novel EGFR inhibitor from a designed small library.

Searching for hit compounds within the huge chemical space resembles the attempt to find a needle in a haystack. Cheminformatics-guided selection of few representative molecules of a rationally designed virtual combinatorial library is a powerful tool to confront this challenge, speed up hit identification and cut off costs. Herein, this approach has been applied to identify hit compounds with novel scaffolds able to inhibit EGFR kinase. From a generated virtual library, six 4-aryloxy-5-aminopyrimidine scaffold-derived compounds were selected, synthesized and evaluated as hit EGFR inhibitors. 4-Aryloxy-5-benzamidopyrimidines inhibited EGFR with IC50 1.05-5.37 µM. Cell-based assay of the most potent EGFR inhibitor hit (10ac) confirmed its cytotoxicity against different cancerous cells. In spite of no EGFR, HER2 or VEGFR1 inhibition was elicited by 4-aryloxy-5-(thio)ureidopyrimidine derivatives, cell-based evaluation suggested them as antiproliferative hits acting by other mechanism(s). Molecular docking study provided a plausible explanation of incapability of 4-aryloxy-5-(thio)ureidopyrimidines to inhibit EGFR and suggested a reasonable binding mode of 4-aryloxy-5-benzamidopyrimidines which provides a basis to develop more optimized ligands.

Histone deacetylase inhibitors containing a benzamide functional group and a pyridyl cap are preferentially effective human immunodeficiency virus-1 latency-reversing agents in primary resting CD4+ T cells.

Antiretroviral therapy (ART) can control human immunodeficiency virus-1 (HIV-1) replication in infected individuals. Unfortunately, patients remain persistently infected owing to the establishment of latent infection requiring that ART be maintained indefinitely. One strategy being pursued involves the development of latency-reversing agents (LRAs) to eliminate the latent arm of the infection. One class of molecules that has been tested for LRA activity is the epigenetic modulating compounds histone deacetylases inhibitors (HDACis). Previously, initial screening of these molecules typically commenced using established cell models of viral latency, and although certain drugs such as the HDACi suberoylanilide hydroxamic acid demonstrated strong activity in these models, it did not translate to comparable activity with patient samples. Here we developed a primary cell model of viral latency using primary resting CD4+ T cells infected with Vpx-complemented HIV-1 and found that the activation profile using previously described LRAs mimicked that obtained with patient samples. This primary cell model was used to evaluate 94 epigenetic compounds. Not surprisingly, HDACis were found to be the strongest activators. However, within the HDACi class, the most active LRAs with the least pronounced toxicity contained a benzamide functional moiety with a pyridyl cap group, as exemplified by the HDACi chidamide. The results indicate that HDACis with a benzamide moiety and pyridyl cap group should be considered for further drug development in the pursuit of a successful viral clearance strategy.

Discovery, structure-activity relationship studies, and anti-nociceptive effects of N-(1,2,3,4-tetrahydro-1-isoquinolinylmethyl)benzamides as novel opioid receptor agonists.

µ-Opioid receptor (MOR) agonists are analgesics used clinically for the treatment of moderate to severe pain, but their use is associated with severe adverse effects such as respiratory depression, constipation, tolerance, dependence, and rewarding effects. In this study, we identified N-({2-[(4-bromo-2-trifluoromethoxyphenyl)sulfonyl]-1,2,3,4-tetrahydro-1-isoquinolinyl}methyl)cyclohexanecarboxamide (1) as a novel opioid receptor agonist by high-throughput screening. Structural modifications made to 1 to improve potency and blood-brain-barrier (BBB) penetration resulted in compounds 45 and 46. Compound 45 was a potent MOR/KOR (κ-opioid receptor) agonist, and compound 46 was a potent MOR and medium KOR agonist. Both 45 and 46 demonstrated a significant anti-nociceptive effect in a tail-flick test performed in wild type (WT) B6 mice. The ED50 value of 46 was 1.059 mg/kg, and the brain concentrations of 45 and 46 were 7424 and 11696 ng/g, respectively. Accordingly, compounds 45 and 46 are proposed for lead optimization and in vivo disease-related pain studies.

Discovery and SAR study of 3-(tert-butyl)-4-hydroxyphenyl benzoate and benzamide derivatives as novel farnesoid X receptor (FXR) antagonists.

3-(tert-Butyl)-4-hydroxyphenyl 2,4-dichlorobenzoate (1) was discovered in our in-house high throughput screening as a moderate FXR antagonist. To improve the potency and the stability of the hit 1, forty derivatives were synthesized and SAR was systematically explored. The results turn out that replacing the 2,4-dichlorophenyl with 2,6-dichloro-4-amidophenyl shows great improvement in potency, replacing the benzoate with benzamide shows improvement in stability and slight declining of potency and 3-(tert-butyl)-4-hydroxyphenyl unit is essential in obtaining the FXR antagonistic activity.

Discovery of novel oxazepine and diazepine carboxamides as two new classes of heat shock protein 90 inhibitors.

Two novel series of oxazepine and diazepine based HSP90 inhibitors are reported. This effort relied on structure based design and isothermal calorimetry to identify small drug like macrocycles. Computational modelling was used to build into a solvent exposed pocket near the opening of the ATP binding site, which led to potent inhibitors of HSP90 (25-30).

Discovery and characterization of 2-(cyclopropanesulfonamido)-N-(2-ethoxyphenyl)benzamide, ML382: a potent and selective positive allosteric modulator of MrgX1.

Previous studies have shown that the activation of mouse MrgC11, a G-protein-coupled receptor, by its peptide ligand BAM8-22 can inhibit chronic pain. A large-scale screen has been carried out to isolate small-molecule allosteric agonists of MrgX1, the human homologue of MrgC11. The goal of this study is to improve the efficacy and potency of positive allosteric modulators (PAMs) with therapeutic implications in combating chronic pain. Herein we report an iterative parallel synthesis effort and a structure-activity relationship study of a series of arylsulfonamides which led to the discovery of the first PAM of MrgX1, ML382.

Mortality and deformities in European lobster (Homarus gammarus) juveniles exposed to the anti-parasitic drug teflubenzuron.

This study describes experiments carried out to examine effects of the antiparasitic drug teflubenzuron, used in delousing farmed salmon, on a non-target species, the European lobster (Homarus gammarus). Juvenile lobsters were fed two doses of teflubenzuron, 10 and 20mg/kg successively for 7 days corresponding to a standard medication of the fish (10mg/kg day) and twice the standard dose (20mg/kg day). Monitoring lasted 3 months to include at least one moulting period for all individuals. Cumulative mortality was higher in all replicates given medicated feed compared with the control group. Mean cumulative mortality for each dosing was 41 ± 13% for 10mg/kg and 38 ± 8% for 20mg/kg, i.e. no difference. Drug residue was analysed in all juveniles that died, in addition to 12 juveniles at day 8 and the first 12 surviving lobsters. A decline in concentration of teflubenzuron from over 8,000 ng/g (day 5) to 14 ng/g (day 70) was observed in the juveniles that died during the experiment. Twelve individuals that died contained 82 ng/g or less whereas the mean concentration in the first 12 lobsters that survived moulting was 152 ng/g. Following a single oral administration, the half-life of teflubenzuron in lobster was estimated to 3.4 days and the initial concentration (C0) to 515 ng/g at time t0. At the end of the study a considerable number of juvenile lobsters were observed with deformities in various organs; carapace, walking legs, cheliped, tail fan, abdomen and antenna. The occurrence of observed deformities varied from 0 to 15% in treated replicates and will most likely affect ability to locate and consume food (antenna, claw and walking legs), respiration (carapace) and ability to move/swim (walking legs, tail fan and abdomen). In total, the mortality and senescent damages were close to 50% in all replicates. Juveniles that survived medication without deformities however, moulted and increased in size at each moult equally well as the unmedicated controls.

Schizophrenia thalamus imaging: low benzamide binding to dopamine D2 receptors suggests fewer D2Short receptors and fewer presynaptic terminals.

The dopamine D2 receptor continues to be the major target for the treatment of schizophrenia and is one of many genes genetically associated with this disease. Recent data show that fewer short forms of the D2 receptor protein are synthesized if there is a genetic variant in the D2 receptor (with a T in rs 1076560 in intron 6). At the same time, at least six publications report that the binding of radioactive benzamides is reduced in the schizophrenia thalamus. A review of the benzamide pharmacology of the short and long forms of the D2 receptor shows that benzamides have a 2.4-fold higher affinity for the D2Short receptor relative to the D2Long form. Hence, the reduced amount of benzamide binding to the D2 receptors in the schizophrenia thalamus suggests that there is a reduced amount of D2Short receptors in this diseased region, and may possibly also mean fewer presynaptic terminals because that is where D2Short receptors mostly reside. If so, fewer presynaptic dopamine terminals in various brain regions may be the basis of the known behavioural dopamine supersensitivity in schizophrenia.

Discovery of aryl ureas and aryl amides as potent and selective histamine H3 receptor antagonists for the treatment of obesity (part II).

A novel series of histamine H3 receptor (H3R) antagonists was derived from an arylurea lead series (1) via bioisosteric replacement of the urea functionality by an amide linkage. The arylamide series was optimized through SAR studies by a broad variation of substituents in the left-hand side benzoyl residue (analogs 2a-2ag) or replacement of the benzoyl moiety by heteroarylcarbonyl residues (analogs 5a-5n). Compounds 2p and 2q were identified within the series as potent and selective H3R antagonists/inverse agonists with acceptable overall profile. Compound 2q was orally active in food intake inhibition in diet-induced obese (DIO) mice. Compound 2q represents a novel H3R antagonist template with improved in vitro potency and oral efficacy and has its merits as a new lead for further optimization.

The development of a high-content screening binding assay for the smoothened receptor.

In this study, the development of an image-based high-content screening (HCS) binding assay for the seven-transmembrane (7TM) receptor Smoothened (Smo) is described. Using BacMam-based gene delivery of Smo, BODIPY-cyclopamine as a fluorescent probe, and a confocal imaging system, a robust 384-well assay that could be used for high-throughput compound profiling activities was developed. The statistically robust HCS binding assay was developed through optimization of multiple parameters, including cell transduction conditions, Smo expression levels, the image analysis algorithm, and staining procedures. Evaluation of structurally diverse compounds, including functional Smo activators, inhibitors, and related analogs, demonstrated good compound potency correlations between high-content imaging binding, membrane fluorescence polarization binding, and gene reporter assays. Statistical analysis of data from a screening test set of compounds at a single 10-µM concentration suggested that the high-content imaging Smo binding assay is amenable for use in hit identification. The 384-well HCS assay was rapidly developed and met statistical assay performance targets, thus demonstrating its utility as a fluorescent whole-cell binding assay suitable for compound screening and profiling.

Thermodynamics of nucleotide and inhibitor binding to wild-type and ispinesib-resistant forms of human kinesin spindle protein.

Current antimitotic cancer chemotherapy based on vinca alkaloids and taxanes target tubulin, a protein required not only for mitotic spindle formation but also for the overall structural integrity of terminally differentiated cells. Among many innovations targeting specific mitotic events, inhibition of motor enzymes including KSP (or Eg5) has been validated as a highly productive approach. Many reported KSP inhibitors bind to an induced allosteric site near the site of ATP hydrolysis, and some have been tested in clinical trials with varying degrees of success. This allosteric site was defined in detail by X-ray crystallography of inhibitor complexes, yet complementary information on binding thermodynamics is still lacking. Using two model ATP-uncompetitive inhibitors, monastrol and ispinesib, we report here the results of thermal denaturation and isothermal titration calorimetric studies. These binding studies were conducted with the wild-type KSP motor domain as well as two ispinesib mutants (D130V and A133D) identified to confer resistance to ispinesib treatment. The thermodynamic parameters obtained were placed in the context of the available structural information and corresponding models of the two ispinesib-resistant mutants. The resulting overall information formed a strong basis for future structure-based design of inhibitors of KSP and related motor enzymes.

Cutaneous biotransformation of N-(4-bromobenzoyl)-S,S-dimethyliminosulfurane and its product, 4-bromobenzamide, leading to percutaneous penetration enhancement of drugs: initial evidence using hydrocortisone.

The role of the skin's metabolism of N-(4-bromobenzoyl)-S,S-dimethyliminosulfurane (DMBIS), an effective penetration enhancer, on its enhancement activity was investigated. It has been found that DMBIS hydrolyzes very fast in physiological buffer to 4-bromobenzamide (BBA), and even faster and almost completely in the presence of skin tissue. It was further shown that in the presence of skin from different species incubated at physiological conditions, the concentration of BBA (DMBIS' immediate product) dropped sharply to 70-80% in 10 min followed by a slower decrease of 0.35-0.50 microg/h. This metabolism was partially inhibited by a continuous application of iodine, and more profoundly, by iodoacetic acid (IAA) and dithiothreitol (DTT) combination treatment. This indicates that at least a part of the metabolism of BBA involves enzymes that are sensitive to reactions with their sulfhydryl groups. In an in vitro permeation study using human epidermis and conventional diffusion cells, we compared between the permeabilities of untreated epidermis and IAA/DTT-treated epidermis to hydrocortisone in the presence of BBA. Due to its metabolic inhibition, we noted a higher penetration of BBA through IAA/DTT-treated epidermis than through the untreated epidermis. Contrary to these results, the extent of the penetration of hydrocortisone was higher through the untreated epidermis with only 1.6 h lag time relative to its penetration through IAA/DTT-treated epidermis, which exhibited a lag time of 12.4 h. It is evident, therefore, that the skin enhancement activity of DMBIS/BBA depends on BBA metabolism in the skin, presumably through its in situ biotransformation into an active enhancer.

Evaluation of [11C]PipISB and [18F]PipISB in monkey as candidate radioligands for imaging brain cannabinoid type-1 receptors in vivo.

N-(4-Fluorobenzyl)-4-[3-(piperidin-1-yl)indole-1-sulfonyl]benzamide] (PipISB, 3) is a selective and high-potency cannabinoid subtype-1 (CB1) receptor inverse agonist. We have previously reported radiosyntheses of [11C]3 and [18F]3. Here, we aimed to evaluate the uptake and CB(1) receptor-specific binding of each radioligand in monkey brain in vivo with positron emission tomography (PET). [11C]3 or [18F]3 was injected intravenously into rhesus or cynomolgus monkey, respectively, and examined with PET at baseline or after pretreatment with a receptor-saturating dose of CB1 receptor-selective ligand (3 for [11C]3 or 8 for [18F]3). In one PET experiment, the dose of 3 was administered at 100 min after [11C]3. Relative plasma concentrations of radioligand and radiometabolites were concurrently measured in baseline experiments with high-performance liquid chromatography. Brain radioactivity uptake was highest in striatum and cerebellum, and it reached 170-270% standardized uptake value (SUV) at 120 min after injection of [11C]3 and 180% SUV at 240 min after injection of [18F]3. Radioactivity was well retained in all CB1 receptor-rich regions. No reference region could be identified for nonspecifically bound radioligand. Under CB1 receptor pretreatment and displacement conditions, initial brain uptakes of radioactivity were similar to those at baseline. Regional brain radioactivity concentrations then became homogeneous and diminished to between 70 and 80% SUV at 120 min after injection of [11C]3 and to 25% SUV at 240 min after injection of [18F]3. [18F]3 was not defluorinated but was metabolized to less lipophilic radiometabolites, as was [11C]3. Hence, [11C]3 and [18F]3 showed high CB1 receptor-specific binding in monkey brain in vivo and merit further investigation as prospective PET radioligands in humans.