Radioligand Binding Research Articles

Synthesis of Fentanyl Triazole Derivatives and their Affinity for Mu-Opioid and Sigma-1 Receptors

The search for compounds with affinity for both mu-opioid receptor (MOR) and sigma-1 receptor (σ1R) is one of the innovative directions to develop painkillers with reduced side effects. Additionally, triazole scaffolds have been extensively explored in the last two decades in medicinal chemistry. In this context, we synthesized a series of new triazole fentanyl derivatives and evaluated their affinity for both MOR and σ1R. The binding affinity of the compounds for human MOR was determined in competitive radioligand binding assays, using fentanyl as standard. For the assays with σ1R, a σ1R agonist (SKF10047) was employed. The most active analogue was 6d which moderately binds to MOR with half-maximal inhibitory concentrations (IC50) = 1.9 μM and to σ1R with IC50 = 6.9 μM. Molecular docking calculations were carried out, providing a structural elucidation for the observed values of affinity. Absorption, distribution, metabolism, and excretion toxicity (ADMET) parameters for the new compounds were simulated with the SwissADME tool

Nuclear Receptor Imaging In Vivo-Clinical and Research Advances.

Nuclear receptors are transcription factors that function in normal physiology and play important roles in diseases such as cancer, inflammation, and diabetes. Noninvasive imaging of nuclear receptors can be achieved using radiolabeled ligands and positron emission tomography (PET). This quantitative imaging approach can be viewed as an in vivo equivalent of the classic radioligand binding assay. A main clinical application of nuclear receptor imaging in oncology is to identify metastatic sites expressing nuclear receptors that are targets for approved drug therapies and are capable of binding ligands to improve treatment decision-making. Research applications of nuclear receptor imaging include novel synthetic ligand and drug development by quantifying target drug engagement with the receptor for optimal therapeutic drug dosing and for fundamental research into nuclear receptor function in cells and animal models. This mini-review provides an overview of PET imaging of nuclear receptors with a focus on radioligands for estrogen receptor, progesterone receptor, and androgen receptor and their use in breast and prostate cancer.

A new serotonin 2A receptor antagonist with potential benefits in Non-Alcoholic Fatty Liver Disease

Peripheral 5-hydroxytryptamine 2A receptor (5-HT2AR) could be a new pharmacological target for NASH, an evolution of NAFLD characterized by hepatic steatosis, cytoskeletal alterations, and hepatic inflammation that can arise with or without fibrosis. SJT4a is a synthetic β-carboline antagonist for 5-HT2AR developed by SJT molecular research to treat NASH. We performed a combined in silico/in vivo study on this potential drug to elucidate its activity and possible mechanism of action.The in silico protocol compares SJT4a with four known 5-HT2AR ligands with different activities (LSD, methiothepin, zotepine, risperidone). We performed molecular docking calculations, evaluation of binding energy by AI-based methods and Molecular Dynamics simulations of the five ligand-target complexes. Moreover, we used a pseudo-semantic analysis to evaluate the potential mechanism of action of SJT4a. In silico predictions and pseudo-semantic analysis suggested antagonistic activity for SJT4a. The in silico prediction was confirmed by [3H]-5HT radioligand binding together with SJT4a competition analysis in CHO-K1 cell cultures expressing 5-HT2AR.SJT4a was then tested in vivo. We investigated the effect of 8 weeks of treatment with SJT4A on metabolic parameters, liver pathology, NAFLD activity score, and fibrosis stage in male DIO-NASH C57BL/6 J mice diet-induced obesity fed with an obesogenic diet compared with DIO-NASH and LEAN-CHOW vehicles. In our tests, SJT4a showed intense activity in diminishing the most relevant hallmarks of NASH in the DIO-NASH mice model. We proposed a possible mode of action for SJT4a based on its 5-HT2AR antagonist activity.

Novel Arylpiperazine Derivatives of Salicylamide with α1-Adrenolytic Properties Showed Antiarrhythmic and Hypotensive Properties in Rats.

Cardiovascular diseases remain one of the leading causes of death worldwide. Unfortunately, the available pharmacotherapeutic options have limited effectiveness. Therefore, developing new drug candidates remains very important. We selected six novel arylpiperazine alkyl derivatives of salicylamide to investigate their cardiovascular effects. Having in mind the beneficial role of α1-adrenergic receptors in restoring sinus rhythm and regulating blood pressure, first, using radioligand binding assays, we evaluated the affinity of the tested compounds for α-adrenergic receptors. Our experiments revealed their high to moderate affinity for α1- but not α2-adrenoceptors. Next, we aimed to determine the antiarrhythmic potential of novel derivatives in rat models of arrhythmia induced by adrenaline, calcium chloride, or aconitine. All compounds showed potent prophylactic antiarrhythmic activity in the adrenaline-induced arrhythmia model and no effects in calcium chloride- or aconitine-induced arrhythmias. Moreover, the tested compounds demonstrated therapeutic antiarrhythmic activity, restoring a normal sinus rhythm immediately after the administration of the arrhythmogen adrenaline. Notably, none of the tested derivatives affected the normal electrocardiogram (ECG) parameters in rodents, which excludes their proarrhythmic potential. Finally, all tested compounds decreased blood pressure in normotensive rats and reversed the pressor response to methoxamine, suggesting that their hypotensive mechanism of action is connected with the blockade of α1-adrenoceptors. Our results confirm the antiarrhythmic and hypotensive activities of novel arylpiperazine derivatives and encourage their further investigation as model structures for potential drugs.

Ketamine preservative benzethonium chloride potentiates hippocampal synaptic transmission and binds neurotransmitter receptors and transporters

Benzethonium chloride (BZT) is an excipient used in numerous products including (R,S)-ketamine (ketamine) drug formulations for human and veterinary use. Emerging evidence indicates BZT is pharmacologically active. BZT may therefore contribute to some of the clinical or preclinical effects observed with ketamine. In the present study, we evaluated: (i) the affinity of BZT for neurotransmitter receptors and transporters, (ii) the effects of BZT on hippocampal synaptic transmission in vitro, and (iii) plasma and brain concentrations of BZT following its intraperitoneal administration to male CD1 mice. Radioligand binding assays determined the affinity of BZT for neurotransmitter targets. Effects of BZT on field excitatory postsynaptic potentials (fEPSPs) were established via electrophysiological recordings from slices collected from male C57BL/6J mice. The binding assays revealed that BZT binds to numerous receptors (e.g., σ2 Ki = 7 nM) and transporters (e.g., dopamine transporter Ki = 545 nM). Bath application of BZT potentiated hippocampal fEPSPs in mouse hippocampal slices with an EC50 of 2.03 nM. Following intraperitoneal administration, BZT was detected in the plasma, but not in the brain of mice. These data highlight that studies measuring peripheral endpoints or directly exposing systems, in vitro, intracerebroventricularly, or intracortically, to BZT-containing formulations should account for the direct effects of BZT. Our findings also suggest that earlier data attributing pharmacological effects to ketamine may be confounded by BZT and that additional investigation into the functional impact of BZT is warranted.This article is part of the Special Issue on ‘Ketamine and its Metabolites’.

Pharmacokinetic and Pharmacologic Characterization of the Dihydrotetrabenazine Isomers of Deutetrabenazine and Valbenazine.

Valbenazine and deutetrabenazine are vesicular monoamine transporter 2 (VMAT2) inhibitors approved for tardive dyskinesia. The clinical activity of valbenazine is primarily attributed to its only dihydrotetrabenazine (HTBZ) metabolite, [+]-α-HTBZ. Deutetrabenazine is a deuterated form of tetrabenazine and is metabolized to four deuterated HTBZ metabolites: [+]-α-deuHTBZ, [+]-β-deuHTBZ, [-]-α-deuHTBZ, and [-]-β-deuHTBZ. An open-label, crossover study characterized the pharmacokinetic profiles of the individual deuHBTZ metabolites, which have not been previously reported. VMAT2 inhibition and off-target interactions of the deuHTBZ metabolites were evaluated using radioligand binding. The only valbenazine HTBZ metabolite, [+]-α-HTBZ, was a potent VMAT2 inhibitor, with negligible affinity for off-target dopamine, serotonin, and adrenergic receptors. Following deutetrabenazine administration, [-]-α-deuHTBZ represented 66% of circulating deuHTBZ metabolites and was a relatively weak VMAT2 inhibitor with appreciable affinity for dopamine (D2S , D3 ) and serotonin (5-HT1A , 5-HT2B , 5-HT7 ) receptors. [+]-β-deuHTBZ was the most abundant deuHTBZ metabolite that potently inhibited VMAT2, but it represented only 29% of total circulating deuHTBZ metabolites. The mean half-life of [+]-α-HTBZ (22.2hours) was ∼3× longer than that of [+]-β-deuHTBZ (7.7hours). These findings are similar to studies with tetrabenazine, in that deutetrabenazine is metabolized to four deuHTBZ stereoisomers, the most abundant of which has negligible interaction with VMAT2 in vitro and appreciable affinity for several off-target receptors. In contrast, valbenazine's single HTBZ metabolite is a potent VMAT2 inhibitor in vitro with no discernible off-target activity. Determination of the effects of intrinsic/extrinsic variables on deutetrabenazine's safety/efficacy profile should incorporate assessment of the effects on all deuHTBZ metabolites.

Plasma pharmacokinetics and pharmacodynamic effects of the 2-benzylbenzimidazole synthetic opioid, isotonitazene, in male rats

Isotonitazene is an illicit synthetic opioid associated with many intoxications and fatalities. Recent studies show that isotonitazene is a potent µ-opioid receptor (MOR) agonist in vitro, but little information is available about its in vivo effects. The aims of the present study were to investigate the pharmacokinetics of isotonitazene in rats, and relate pharmacokinetic parameters to pharmacodynamic effects. Isotonitazene and its metabolites were identified and quantified by liquid chromatography tandem quadrupole mass spectrometry (LC-QQQ-MS). Male Sprague-Dawley rats with jugular catheters and subcutaneous (s.c.) temperature transponders received isotonitazene (3, 10, 30µg/kg, s.c.) or its vehicle. Blood samples were drawn at 15, 30, 60, 120, and 240min post-injection, and plasma was assayed using LC-QQQ-MS. At each blood draw, body temperature, catalepsy scores, and hot plate latencies were recorded. Maximum plasma concentrations of isotonitazene rose in parallel with increasing dose (range 0.2-9.8ng/mL) and half-life ranged from 23.4 to 63.3min. The metabolites 4'-hydroxy nitazene and N-desethyl isotonitazene were detected, and plasma concentrations were below the limit of quantitation (0.5ng/mL) but above the limit of detection (0.1ng/mL). Isotonitazene produced antinociception (ED50 = 4.22µg/kg), catalepsy-like symptoms (ED50 = 8.68µg/kg), and hypothermia (only at 30µg/kg) that were significantly correlated with concentrations of isotonitazene. Radioligand binding in rat brain tissue revealed that isotonitazene displays nM affinity for MOR (Ki = 15.8nM), while the N-desethyl metabolite shows even greater affinity (Ki = 2.2nM). In summary, isotonitazene is a potent MOR agonist whose pharmacodynamic effects are related to circulating concentrations of the parent drug. The high potency of isotonitazene portends substantial risk to users who are exposed to the drug.

In vitro assessment of the binding and functional responses of ozanimod and its plasma metabolites across human sphingosine 1-phosphate receptors

Ozanimod is approved in multiple countries for the treatment of adults with either relapsing multiple sclerosis or moderately to severely active ulcerative colitis. Ozanimod is metabolized in humans to form seven active plasma metabolites, including two major active metabolites CC112273 and CC1084037, and an inactive metabolite. Here, the binding and activity of ozanimod and its metabolites across human sphingosine 1-phosphate receptors were determined. Binding affinity was assessed in Chinese hamster ovary cell membranes expressing recombinant human sphingosine 1-phosphate receptors 1 and 5 via competitive radioligand binding using tritium-labeled ozanimod; selectivity via functional potency assessment was performed using [35S]-guanosine-5'-(γ-thio)-triphosphate binding assays. Receptor internalization was assessed in human embryonic kidney 293 cells overexpressing sphingosine 1-phosphate receptor 1–green fluorescent protein and Chinese hamster ovary cells overexpressing sphingosine 1-phosphate receptor 5–hemagglutinin via fluorescence activated cell sorting. Functional activity was assessed in primary cultures of human astrocytes via phosphorylation assays. Ozanimod and its functionally active metabolites bound to the same sites within sphingosine 1-phosphate receptors 1 and 5, with metabolites displaying the same selectivity profile as ozanimod. Agonism at sphingosine 1-phosphate receptor 1 induced receptor internalization, whereas sphingosine 1-phosphate receptor 5 did not. Ozanimod, CC112273, and CC1084037 elicited functional intracellular signaling in human astrocytes, pharmacologically characterized to be mediated by sphingosine 1-phosphate receptor 1. The active plasma metabolites of ozanimod bound to sphingosine 1-phosphate receptors 1 and 5 and displayed similar pharmacologic profiles as their parent compound, likely contributing to clinical efficacy in patients with relapsing multiple sclerosis or moderately to severely active ulcerative colitis.

Assessing synaptic integrity in Alzheimer’s brain with UCB‐J

AbstractBackgroundSynaptic loss is an eminent feature of Alzheimer’s disease (AD). Synapses critical for neuronal transmission are regulated by synaptic vesicles (SV), which expresses an integral transmembrane protein called SV protein 2A (SV2A) throughout the brain. Notably, reduced SV2A expression has been implicated in AD and other proteinopathies. UCB‐J is a recently developed PET‐tracer, which target SV2A, to study synaptic integrity in different neurological disorders. Though several UCB‐J in vivo PET studies have been published in AD, there have been some discrepancies between the findings. Many of these studies indicated potential correlations between tau deposition, atrophy, cognitive‐impairment, and loss of UCB‐J binding. Based on these in vivo observations, we found it of utmost relevance to perform extensive pre‐clinical validation of UCB‐J in AD brains with postmortem brain imaging techniques.MethodWe performed radioligand binding assays (saturation and competition) alongside large brain section autoradiography in AD and control brains.Result 3H‐UCB‐J competition studies with unlabelled‐UCB‐J showed one‐binding site (IC50: ∼ 10 nM) in the frontal cortex (FC) of AD and control brains. Saturation studies in FC region of AD and control brains showed much higher specific binding in synaptosomal membrane P2‐fraction (Bmax: 5.8 ‐ 8.7 pmol/mg, Kd ∼ 5.0 nM) as compared to brain homogenates (Bmax: 0.09 ‐ 0.18 pmol/mg, Kd ∼ 4.0 nM). P2‐fraction saturation binding study clearly highlighted the loss of UCB‐J binding in FC of AD brain as compared to control brain. In contrast, large brain hemisphere section autoradiography incubated with 3H‐UCB‐J showed unexpectedly higher regional binding in AD brain sections as compared to control, motivating further mechanistic pre‐clinical validation with other synaptic markers in AD and other proteinopathies.ConclusionOur findings explicitly demonstrated high specificity of UCB‐J for SV2A in AD and control brains. UCB‐J specific binding was much higher in P2‐fraction than brain homogenate (P2‐fraction > brain homogenate) and highlighted the expected loss of UCB‐J binding in AD brain. Competition and saturation binding studies complemented each other and showed only one‐binding site with nM‐affinity in AD and control brains. Unanticipated high UCB‐J binding in AD brain in autoradiography studies indicate potential interaction with other brain tissue component, warrant further investigation.

Understanding reactive astrogliosis in degenerative proteinopathies through the lens of postmortem brain imaging techniques

AbstractBackgroundThe discovery and validation of biomarkers beyond amyloid‐β and tau is now considered as a research priority in the Alzheimer’s disease (AD) field. In this context, astrocytes ‘key homeostatic cells’ in the central nervous system (CNS) are potential targets thanks to their swift response in AD pathology. Astrocytes tend to changes in brain physiology after CNS injury/insults via a specific defence response called reactive astrogliosis. The role of astrocytes in AD pathology is still under examination, however recent studies have openly suggested that reactive astrogliosis is an early event which may precede other hallmarks of the disease. In our recent work, we have speculated, based on clinical/translational in vivo and in vitro brain imaging studies, that there are two waves, ‘first (early) and second (late)’, of reactive astrogliosis in AD pathology with a distinct, close‐knit relationship with other pathological markers. Reactive astrogliosis also seems to play a critical role in other proteinopathies, such as progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). The PET imaging field is advancing very fast and different astrocytic‐tracers are now available, such as 11C‐Deprenyl (DED) and SMBT‐1 (both targeting MAO‐B) as well as 11C‐BU99008 (detect imidazoline2 binding sites), which we believe is of utmost relevance to shed more light on the role of reactive astrogliosis, including astrocytic heterogeneity and improve our understanding of the underlying mechanism in these proteinopathies.MethodWe used a mixed battery of specialized in vitro radioligand binding assays (saturation and competition studies in brain tissues) and large frozen human brain section autoradiography in AD, PSP, CBD and control brains.ResultIn our studies, we demonstrated that BU99008 could visualize reactive astrogliosis and target multiple binding sites in AD brain. We observed that BU99008 and DED varied in their binding behavior in AD and control brains, suggesting that they might be targeting a specific state/or a different sub‐population of astrocytes.ConclusionPostmortem brain imaging techniques are valuable tools for validation (pre‐clinical) of novel PET‐tracers. Our ongoing studies in PSP and CBD brains as well as with SMBT‐1 will provide deeper insight into the role of reactive astrogliosis in these deadly diseases.

Multi‐tracer comparison in Alzheimer’s disease, Corticobasal Degeneration and Progressive Supranuclear Palsy brains

AbstractBackgroundRecent CryoEM studies in tauopathies clearly bought forward one more time the complexity of these pathologies, and showed that it goes beyond the tau isoforms. Tau PET tracers play an important role in the understanding of this aspect. We have observed in post‐mortem brain tissue from Alzheimer’s disease (AD) patients that the second‐generation of tau PET tracers 3H‐PI2620, 3H‐MK6240, 3H‐RO948, all show similar regional distribution, but also comparable binding affinities to multiple binding sites in AD brain large hemispherical tissue section autoradiography and homogenates. In vitro autoradiography can mirror the in vivo binding pattern of PET tracers and be useful in pre‐clinical validations. The aim of this study was to perform a comparative multi‐tracer study in AD, PSP, CBD and control brains with several second generation tau‐tracers to obtain a deeper insight into tau spatial distribution and discriminative power of tau tracers in degenerative proteinopathies. We also want to study how the binding of the tracers correlate with other pathological hallmarks such as amyloid‐β and reactive astrogliosis.Methodswe performed radioligand binding assays in brain homogenates, in small tissue sections and large frozen hemispherical brain sections autoradiography using tau PET tracers (3H‐PI2620, 3H‐MK6240, 3H‐RO948, and 3H‐JNJ067) in comparison to reactive astrogliosis PET tracers (3H‐BU99008 and 3H‐Deprenyl) and amyloid tracer (3H‐PIB).ResultsIn ongoing studies, 3H‐PI2620, 3H‐MK6240 and 3H‐RO948 clearly followed a two binding sites model in AD brains. 3H‐PI2620 bound to two binding sites in the frontal cortex of both AD and CBD but to one site in PSP brains, but in similar high affinity range in the three pathologies. 3H‐RO948 and 3H‐MK6240 showed low binding in comparison to 3H‐PI2620 in CBD and PSP.ConclusionTau PET tracers showed similar affinities to multiple binding sites and similar regional distribution in AD brains. 3H‐MK6240 and 3H‐RO948 displayed selectivity for AD brains, while 3H‐PI2620 also showed similar binding affinity for CBD and PSP brains. Ongoing studies with several PET tracers targeting different pathological hallmarks will increase possible discriminative properties and provide new knowledge on the spatial distribution of tau and its correlation with other biomarkers in different tauopathies.

Discriminative binding of tau PET tracers PI2620, MK6240 and RO948 in Alzheimer’s disease, corticobasal degeneration and progressive supranuclear palsy brains

Recent mechanistic and structural studies have challenged the classical tauopathy classification approach and revealed the complexity and heterogeneity of tau pathology in Alzheimer’s disease (AD) and primary tauopathies such as corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP), progressing beyond distinct tau isoforms. In this multi-tau tracer study, we focused on the new second-generation tau PET tracers PI2620, MK6240 and RO948 to investigate this tau complexity in AD, CBD, and PSP brains using post-mortem radioligand binding studies and autoradiography of large and small frozen brain sections. Saturation binding studies indicated multiple binding sites for 3H-PI2620 in AD, CBD and PSP brains with different binding affinities (Kd ranging from 0.2 to 0.7 nM) and binding site densities (following the order: BmaxAD > BmaxCBD > BmaxPSP). Competitive binding studies complemented these findings, demonstrating the presence of two binding sites [super-high affinity (SHA): IC50(1) = 8.1 pM; and high affinity (HA): IC50(2) = 4.9 nM] in AD brains. Regional binding distribution studies showed that 3H-PI2620 could discriminate between AD (n = 6) and control cases (n = 9), especially in frontal cortex and temporal cortex tissue (p < 0.001) as well as in the hippocampal region (p = 0.02). 3H-PI2620, 3H-MK6240 and 3H-RO948 displayed similar binding behaviour in AD brains (in both homogenate competitive studies and one large frozen hemispherical brain section autoradiography studies) in terms of binding affinities, number of sites and regional patterns. Our small section autoradiography studies in the frontal cortex of CBD (n = 3) and PSP brains (n = 2) showed high specificity for 3H-PI2620 but not for 3H-MK6240 or 3H-RO948. Our findings clearly demonstrate different binding properties among the second-generation tau PET tracers, which may assist in further understanding of tau heterogeneity in AD versus non-AD tauopathies and suggests potential for development of pure selective 4R tau PET tracers.

Pharmacological Characterization of Purified Full-Length Dopamine Transporter from Drosophila melanogaster

The dopamine transporter (DAT) is a member of the neurotransmitter:sodium symporter (NSS) family, mediating the sodium-driven reuptake of dopamine from the extracellular space thereby terminating dopaminergic neurotransmission. Our current structural understanding of DAT is derived from the resolutions of DAT from Drosophila melanogaster (dDAT). Despite extensive structural studies of purified dDAT in complex with a variety of antidepressants, psychostimulants and its endogenous substrate, dopamine, the molecular pharmacology of purified, full length dDAT is yet to be elucidated. In this study, we functionally characterized purified, full length dDAT in detergent micelles using radioligand binding with the scintillation proximity assay. We elucidate the consequences of Na+ and Cl- binding on [3H]nisoxetine affinity and use this to evaluate the binding profiles of substrates and inhibitors to the transporter. Additionally, the technique allowed us to directly determine a equilibrium binding affinity (Kd) for [3H]dopamine to dDAT. To compare with a more native system, the affinities of specified monoamines and inhibitors was determined on dDAT, human DAT and human norepinephrine transporter expressed in COS-7 cells. With our gathered data, we established a pharmacological profile for purified, full length dDAT that will be useful for subsequent biophysical studies using dDAT as model protein for the mammalian NSS family of proteins.

Design, synthesis, and structure-activity relationships of diindolylmethane derivatives as cannabinoid CB2 receptor agonists.

3,3'-Diindolylmethane (DIM), a natural product-derived compound formed upon ingestion of cruciferous vegetables, was recently described to act as a partial agonist of the anti-inflammatory cannabinoid (CB) receptor subtype CB2 . In the present study, we synthesized and evaluated a series of DIM derivatives and determined their affinities for human CB receptor subtypes in radioligand binding studies. Potent compounds were additionally evaluated in functional cAMP accumulation and β-arrestin recruitment assays. Small substituents in the 4-position of both indole rings of DIM were beneficial for high CB2 receptor affinity and efficacy. Di-(4-cyano-1H-indol-3-yl)methane (46, PSB-19837, EC50 : cAMP, 0.0144 µM, 95% efficacy compared to the full standard agonist CP55,940; β-arrestin, 0.0149 µM, 67% efficacy) was the most potent CB2 receptor agonist of the present series. Di-(4-bromo-1H-indol-3-yl)methane (44, PSB-19571) showed higher potency in β-arrestin (EC50 0.0450 µM, 61% efficacy) than in cAMP accumulation assays (EC50 0.509 µM, 85% efficacy) while 3-((1H-indol-3-yl)methyl)-4-methyl-1H-indole (149, PSB-18691) displayed a 19-fold bias for the G protein pathway (EC50 : cAMP, 0.0652 µM; β-arrestin, 1.08 µM). DIM and its analogs act as allosteric CB2 receptor agonists. These potent CB2 receptor agonists have potential as novel drugs for the treatment of inflammatory diseases.

An optimized reference tissue method for quantification of tau protein depositions in diverse neurodegenerative disorders by PET with 18F-PM-PBB3 (18F-APN-1607)

Positron emission tomography (PET) with 18F-PM-PBB3 (18F-APN-1607, 18F-Florzolotau) enables high-contrast detection of tau depositions in various neurodegenerative dementias, including Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD). A simplified method for quantifying radioligand binding in target regions is to employ the cerebellum as a reference (CB-ref) on the assumption that the cerebellum has minimal tau pathologies. This procedure is typically valid in AD, while FTLD disorders exemplified by progressive supranuclear palsy (PSP) are characterized by occasional tau accumulations in the cerebellum, hampering the application of CB-ref. The present study aimed to establish an optimal method for defining reference tissues on 18F-PM-PBB3-PET images of AD and non-AD tauopathy brains. We developed a new algorithm to extract reference voxels with a low likelihood of containing tau deposits from gray matter (GM-ref) or white matter (WM-ref) by a bimodal fit to an individual, voxel-wise histogram of the radioligand retentions and applied it to 18F-PM-PBB3-PET data obtained from age-matched 40 healthy controls (HCs) and 23 CE, 40 PSP, and five other tau-positive FTLD patients. PET images acquired at 90–110 min after injection were averaged and co-registered to corresponding magnetic resonance imaging space. Subsequently, we generated standardized uptake value ratio (SUVR) images estimated by CB-ref, GM-ref and WM-ref, respectively, and then compared the diagnostic performances. GM-ref and WM-ref covered a broad area in HCs and were free of voxels located in regions known to bear high tau burdens in AD and PSP patients. However, radioligand retentions in WM-ref exhibited age-related declines. GM-ref was unaffected by aging and provided SUVR images with higher contrast than CB-ref in FTLD patients with suspected and confirmed corticobasal degeneration. The methodology for determining reference tissues as optimized here improves the accuracy of 18F-PM-PBB3-PET measurements of tau burdens in a wide range of neurodegenerative illnesses.

Pharmacological Optimization of PSMA-Based Radioligand Therapy.

Prostate cancer (PCa) is the most common malignancy in men of middle and older age. The standard treatment strategy for PCa ranges from active surveillance in low-grade, localized PCa to radical prostatectomy, external beam radiation therapy, hormonal treatment and chemotherapy. Recently, the use of prostate-specific membrane antigen (PSMA)-targeted radioligand therapy (RLT) for metastatic castration-resistant PCa has been approved. PSMA is predominantly, but not exclusively, expressed on PCa cells. Because of its high expression in PCa, PSMA is a promising target for diagnostics and therapy. To understand the currently used RLT, knowledge about pharmacokinetics (PK) and pharmacodynamics (PD) of the PSMA ligand and the PSMA protein itself is crucial. PK and PD properties of the ligand and its target determine the duration and extent of the effect. Knowledge on the concentration-time profile, the target affinity and target abundance may help to predict the effect of RLT. Increased specific binding of radioligands to PSMA on PCa cells may be associated with better treatment response, where nonspecific binding may increase the risk of toxicity in healthy organs. Optimization of the radioligand, as well as synergistic effects of concomitant agents and an improved dosing strategy, may lead to more individualized treatment and better overall survival.

Anti-inflammatory effects of new human histamine H3 receptor ligands with flavonoid structure on BV-2 neuroinflammation

ObjectiveMicroglia play an important role in the neuroinflammation developed in response to various pathologies. In this study, we examined the anti-inflammatory effect of the new human histamine H3 receptor (H3R) ligands with flavonoid structure in murine microglial BV-2 cells.Material and methodsThe affinity of flavonoids (E243 -flavone and IIIa–IIIc—chalcones) for human H3R was evaluated in the radioligand binding assay. The cytotoxicity on BV-2 cell viability was investigated with the MTS assay. Preliminary evaluation of anti-inflammatory properties was screened by the Griess assay in an in vitro neuroinflammation model of LPS-treated BV-2 cells. The expression and secretion of pro-inflammatory cytokines were evaluated by real-time qPCR and ELISA, respectively. The expression of microglial cell markers were determined by immunocytochemistry.ResultsChalcone derivatives showed high affinity at human H3R with Ki values < 25 nM. At the highest nontoxic concentration (6.25 μM) compound IIIc was the most active in reducing the level of nitrite in Griess assay. Additionally, IIIc treatment attenuated inflammatory process in murine microglia cells by down-regulating pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) at both the level of mRNA and protein level. Our immunocytochemistry studies revealed expression of microglial markers (Iba1, CD68, CD206) in BV-2 cell line.ConclusionsThese results emphasize the importance of further research to accurately identify the anti-inflammatory mechanism of action of chalcones.

HBK-15, a Multimodal Compound, Showed an Anxiolytic-Like Effect in Rats

Anxiety is a common mental disorder, and its prevalence has lately increased because of the COVID-19 pandemic. Unfortunately, the available anxiolytics are often ineffective, and most possess addictive potential. Thus, searching for novel compounds is essential. In our previous studies, we selected a multimodal compound, HBK-15, which showed a fast antidepressant-like effect in animal models of depression. HBK-15 demonstrated a high affinity for serotonin 5-HT1A receptors and moderate for 5-HT7, dopamine D2, and α1-adrenoceptors. Based on the receptor profile and preliminary studies, we aimed to investigate the anxiolytic potential of HBK-15 using the conditioned-response rat model of anxiety, i.e., the Vogel drinking test. We performed hot plate and free-drinking tests to exclude false positive results in the Vogel test. Using radioligand binding studies, we also investigated the affinity of the compound for the selected biological targets, which play a role in anxiety. Our experiments revealed that HBK-15 showed an anxiolytic-like effect in rats (5 mg/kg) without influencing the pain threshold or the amount of water consumed in the free-drinking test. Furthermore, the tested compound did not show a significant affinity for the selected biological targets, which suggests that its anxiolytic-like mechanism of action could be connected with the interaction with other receptors. This study indicates that multimodal compounds with a receptor profile similar to HBK-15 could be an attractive therapeutic option for patients with a generalized anxiety disorder. However, more studies are required to determine the exact mechanism of action of HBK-15 and its safety profile.

A PDE10A inhibitor CPL500036 is a novel agent modulating striatal function devoid of most neuroleptic side-effects.

Background: Phosphodiesterase 10A (PDE10A) is expressed almost exclusively in the striatum and its inhibition is suggested to offer potential treatment in disorders associated with basal ganglia. We evaluated the selectivity, cytotoxicity, genotoxicity, pharmacokinetics and potential adverse effects of a novel PDE10A inhibitor, CPL500036, in vivo. Methods: The potency of CPL500036 was demonstrated by microfluidic technology, and selectivity was investigated in a radioligand binding assay against 44 targets. Cardiotoxicity in vitro was evaluated in human ether-a-go-go related gene (hERG)-potassium channel-overexpressing cells by the patch-clamp method and by assessing key parameters in 3D cardiac spheroids. Cytotoxicity was determined in H1299, HepG2 and SH-SY5Y cell lines. The Ames test was used for genotoxicity analyses. During in vivo studies, CPL500036 was administered by oral gavage. CPL500036 exposure were determined by liquid chromatography-tandem mass spectrometry and plasma protein binding was assessed. The bar test was employed to assess catalepsy. Prolactin and glucose levels in rat blood were measured by ELISAs and glucometers, respectively. Cardiovascular safety in vivo was investigated in dogs using a telemetry method. Results: CPL500036 inhibited PDE10A at an IC50 of 1nM, and interacted only with the muscarinic M2 receptor as a negative allosteric modulator with an IC50 of 9.2µM. Despite inhibiting hERG tail current at an IC25 of 3.2μM, cardiovascular adverse effects were not observed in human cardiac 3D spheroids or in vivo. Cytotoxicity in vitro was observed only at > 60μM and genotoxicity was not recorded during the Ames test. CPL500036 presented good bioavailability and penetration into the brain. CPL500036 elicited catalepsy at 0.6mg/kg, but hyperprolactinemia or hyperglycemic effects were not observed in doses up to 3mg/kg. Conclusion: CPL500036 is a potent, selective and orally bioavailable PDE10A inhibitor with a good safety profile distinct from marketed antipsychotics. CPL500036 may be a compelling drug candidate.

Structure-Activity Relationships for Psilocybin, Baeocystin, Aeruginascin, and Related Analogues to Produce Pharmacological Effects in Mice.

4-Phosphoryloxy-N,N-dimethyltryptamine (psilocybin) is a naturally occurring tertiary amine found in many mushroom species. Psilocybin is a prodrug for 4-hydroxy-N,N-dimethyltryptamine (psilocin), which induces psychedelic effects via agonist activity at the serotonin (5-HT) 2A receptor (5-HT2A). Several other 4-position ring-substituted tryptamines are present in psilocybin-containing mushrooms, including the secondary amine 4-phosphoryloxy-N-methyltryptamine (baeocystin) and the quaternary ammonium 4-phosphoryloxy-N,N,N-trimethyltryptamine (aeruginascin), but these compounds are not well studied. Here, we investigated the structure-activity relationships for psilocybin, baeocystin, and aeruginascin, as compared to their 4-acetoxy and 4-hydroxy analogues, using in vitro and in vivo methods. Broad receptor screening using radioligand binding assays in transfected cells revealed that secondary and tertiary tryptamines with either 4-acetoxy or 4-hydroxy substitutions display nanomolar affinity for most human 5-HT receptor subtypes tested, including the 5-HT2A and the serotonin 1A receptor (5-HT1A). The same compounds displayed affinity for 5-HT2A and 5-HT1A in mouse brain tissue in vitro and exhibited agonist efficacy in assays examining 5-HT2A-mediated calcium mobilization and β-arrestin 2 recruitment. In mouse experiments, only the tertiary amines psilocin, psilocybin, and 4-acetoxy-N,N-dimethyltryptamine (psilacetin) induced head twitch responses (ED50 0.11-0.29 mg/kg) indicative of psychedelic-like activity. Head twitches were blocked by 5-HT2A antagonist pretreatment, supporting 5-HT2A involvement. Both secondary and tertiary amines decreased body temperature and locomotor activity at higher doses, the effects of which were blocked by 5-HT1A antagonist pretreatment. Across all assays, the pharmacological effects of 4-acetoxy and 4-hydroxy compounds were similar, and these compounds were more potent than their 4-phosphoryloxy counterparts. Importantly, psilacetin appears to be a prodrug for psilocin that displays substantial serotonin receptor activities of its own.