High Nonspecific Binding Research Articles

[18F]RS-127445 radiosynthesis and evaluation as a 5-HT2B receptor PET radiotracer in rat brain

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in many physiological and pathological mechanisms through its numerous receptors. Among these, the 5-HT2B receptor is known to play a key role in multiple brain disorders but remains poorly understood. Positron emission tomography (PET) can contribute to a better understanding of pathophysiological mechanisms regulated by the 5-HT2B receptor. To develop the first PET radiotracer for the 5-HT2B receptor, RS-127445, a well-known 5-HT2B receptor antagonist, was labeled with fluorine-18. [18F]RS-127445 was synthesized in a high radiochemical purity and with a good molar activity and radiochemical yield. Preliminary PET scans in rats showed good brain penetration of [18F]RS-127445. However, competition experiments and in vitro autoradiography showed high non-specific binding, especially to brain white matter.

High pH stability and detection of α-synuclein using an EGFET biosensor with an HfO2 gate deposited by high-power pulsed magnetron sputtering

In this research, hafnium oxide (HfO2) was first fabricated in an in-house-developed high-power pulsed magnetron sputtering system (HiPIMS) functionalized for application as a high-performance extended gate field-effect transistor (EGFET) for the detection of pH and proteins associated with Parkinson's disease (PD). PD is the second most common neurodegenerative disease in elderly people after Alzheimer's disease. The α-synuclein protein in patients’ blood may be a potential biomarker in the early stage of PD. Detection of α-synuclein with high sensitivity and nonspecific binding through HfO2-EGFET biosensors has the advantages of low cost and simple fabrication from disposable EGs. Among the duty cycle conditions tested, HfO2 deposited at a 25% duty cycle in HiPIMS exhibited superior pH sensitivity and linearity, with values of 56.4 mV/pH and 99.7%, respectively. The hysteresis width and drift coefficient are −3 mV and 0.3 mV/h, respectively. To overcome the Debye length limitation, 100-fold diluted phosphate buffer solution and the PMOS configuration were used to determine the α-synuclein for which the isoelectric point was 4.67. The negative gate bias in the PMOS configuration successfully causes α-synuclein to approach the surface of HfO2 EG to achieve a sensitivity of 12.1 mV/dec and a linearity of 99.5% in the range of 0.1–1000 pg/mL. The limit of detection is 0.198 pg/mL, and the immobilized surface ultimately binds specifically, resulting in low interference signals from other related biomarkers. This promising study shows that detecting various proteins involved in different diseases and performing clinical tests can be accomplished in the future.

Evaluating the chaos game representation of proteins for applications in machine learning models: prediction of antibody affinity and specificity as a case study.

Machine learning techniques are becoming increasingly important in the selection and optimization of therapeutic molecules, as well as for the selection of formulation components and the prediction of long-term stability. Compared to first-principle models, machine learning techniques are easier to implement, and can identify correlations that would be hard to describe at a mechanistic level, but strongly rely on high-quality input training data. Here, we evaluate the potential of the "chaos game" representation to provide input data for machine learning models. The chaos game is an algorithm originally developed for the production of fractal structures, and later on applied also to the representation of biological sequences, such as genes and proteins. Our results show that the combination of the chaos game representation with convolutional neural networks results in comparable accuracy to other machine learning approaches, thus indicating that chaos game representations could be a valid alternative to existing featurization strategies for machine learning models of biological sequences. We implement the chaos game in Python 3.8.10, and use it to produce fractal as well as novel expanding representations of protein sequences. We then feed the resulting images to a convolutional neural network, built in Python 3.8.10, using TensorFlow 2.9.1, Keras 2.9.0, and the scikit-learn 1.1.1 packages. We select as case study a recently published dataset for the antibody emibetuzumab, with the objective of co-optimizing antibodies variants with both high affinity and low non-specific binding.

Preparation of lysozyme-imprinted mesoporous Zr-based metal-organic frameworks with remarkable specific recognition

The development of high-performance protein-imprinted materials remains challenging due to defects concerning high mass transfer resistance and non-specific binding, which are crucial for protein purification and enrichment. In this paper, lysozyme-imprinted mesoporous Zr-based MOF (mesoUiO-66-NH2@MIPs) with specific and selective recognition of lysozyme (Lyz) were prepared by surface imprinting technology. In particular, the excellent hydrophilicity mesoporous MOFs (mesoUiO-66-NH2) with a pore size of 10 nm was prepared as a carrier for Lyz immobilization by an auxiliary modulation strategy to regulate the microporous structure of UiO-66-NH2 with the propionic acid solution, enabling massive loading of the macromolecular protein Lyz. The mesoUiO-66-NH2@MIPs reached a maximum saturation adsorption of 206.54 mg g−1 on Lyz in 20 min at 25 °C with an imprinting factor of 2.57 and selection factors of 2.02, 2.34, and 2.45 for cytochrome c (Cyt c), bovine serum albumin (BSA) and bovine hemoglobin (BHb), respectively. More importantly, the mesoUiO-66-NH2@MIPs could specifically recognize Lyz from the mixed protein system. The adsorption capacity of Lyz could still reach 78.55% after 5 cycles with good cyclic regeneration performance. This provides a new research option for developing and applying novel porous MOF in biomolecule imprinting technology and the specific separation of biomolecules.

GATS tag system is compatible with biotin labelling methods for protein analysis

Polypeptide tags and biotin labelling technologies are widely used for protein analyses in biochemistry and cell biology. However, many peptide tag epitopes contain lysine residues (or amino acids) that are masked after biotinylation. Here, we propose the GATS tag system without a lysine residue and with high sensitivity and low non-specific binding using a rabbit monoclonal antibody against Plasmodium falciparum glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (PfGAMA). From 14 monoclonal clones, an Ra3 clone was selected as it recognized an epitope—TLSVGVQNTF—without a lysine residue; this antibody and epitope tag set was called the GATS tag system. Surface plasmon resonance analysis showed that the tag system had a high affinity of 8.71 × 10–9 M. GATS tag indicated a very low background with remarkably high sensitivity and specificity in immunoblotting using the lysates of mammalian cells. It also showed a high sensitivity for immunoprecipitation and immunostaining of cultured human cells. The tag system was highly sensitive in both biotin labelling methods for proteins using NHS-Sulfo-biotin and BioID (proximity-dependent biotin identification) in the human cells, as opposed to a commercially available tag system having lysine residues, which showed reduced sensitivity. These results showed that the GATS tag system is suitable for methods such as BioID involving labelling lysine residues.

Identification of a Putative α-synuclein Radioligand Using an in silico Similarity Search.

Previous studies from our lab utilized an ultra-high throughput screening method to identify compound 1 as a small molecule that binds to alpha-synuclein (α-synuclein) fibrils. The goal of the current study was to conduct a similarity search of 1 to identify structural analogs having improved in vitro binding properties for this target that could be labeled with radionuclides for both in vitro and in vivo studies for measuring α-synuclein aggregates. Using 1 as a lead compound in a similarity search, isoxazole derivative 15 was identified to bind to α-synuclein fibrils with high affinity in competition binding assays. A photocrosslinkable version was used to confirm binding site preference. Derivative 21, the iodo-analog of 15, was synthesized, and subsequently radiolabeled isotopologs [125I]21 and [11C]21 were successfully synthesized for use in in vitro and in vivo studies, respectively. [125I]21 was used in radioligand binding studies in post-mortem Parkinson's disease (PD) and Alzheimer's disease (AD) brain homogenates. In vivo imaging of an α-synuclein mouse model and non-human primates was performed with [11C]21. In silico molecular docking and molecular dynamic simulation studies for a panel of compounds identified through a similarity search, were shown to correlate with Ki values obtained from in vitro binding studies. Improved affinity of isoxazole derivative 15 for α-synuclein binding site 9 was indicated by photocrosslinking studies with CLX10. Design and successful (radio)synthesis of iodo-analog 21 of isoxazole derivative 15 enabled further in vitro and in vivo evaluation. Kd values obtained in vitro with [125I]21 for α-synuclein and Aβ42 fibrils were 0.48 ± 0.08 nM and 2.47 ± 1.30 nM, respectively. [125I]21 showed higher binding in human postmortem PD brain tissue compared with AD tissue, and low binding in control brain tissue. Lastly, in vivo preclinical PET imaging showed elevated retention of [11C]21 in PFF-injected mouse brain. However, in PBS-injected control mouse brain, slow washout of the tracer indicates high non-specific binding. [11C]21 showed high initial brain uptake in a healthy non-human primate, followed by fast washout that may be caused by rapid metabolic rate (21% intact [11C]21 in blood at 5 min p.i.). Through a relatively simple ligand-based similarity search, we identified a new radioligand that binds with high affinity (<10 nM) to α-synuclein fibrils and PD tissue. Although the radioligand has suboptimal selectivity for α-synuclein towards Aβ and high non-specific binding, we show here that a simple in silico approach is a promising strategy to identify novel ligands for target proteins in the CNS with the potential to be radiolabeled for PET neuroimaging studies.

Can We Predict Clinical Pharmacokinetics of Highly Lipophilic Compounds by Integration of Machine Learning or In Vitro Data into Physiologically Based Models? A Feasibility Study Based on 12 Development Compounds.

While high lipophilicity tends to improve potency, its effects on pharmacokinetics (PK) are complex and often unfavorable. To predict clinical PK in early drug discovery, we built human physiologically based PK (PBPK) models integrating either (i) machine learning (ML)-predicted properties or (ii) discovery stage in vitro data. Our test set was composed of 12 challenging development compounds with high lipophilicity (mean calculated log P 4.2), low plasma-free fraction (50% of compounds with fu,p < 1%), and low aqueous solubility. Predictions focused on key human PK parameters, including plasma clearance (CL), volume of distribution at steady state (Vss), and oral bioavailability (%F). For predictions of CL, the ML inputs showed acceptable accuracy and slight underprediction bias [an average absolute fold error (AAFE) of 3.55; an average fold error (AFE) of 0.95]. Surprisingly, use of measured data only slightly improved accuracy but introduced an overprediction bias (AAFE = 3.35; AFE = 2.63). Predictions of Vss were more successful, with both ML (AAFE = 2.21; AFE = 0.90) and in vitro (AAFE = 2.24; AFE = 1.72) inputs showing good accuracy and moderate bias. The %F was poorly predicted using ML inputs [average absolute prediction error (AAPE) of 45%], and use of measured data for solubility and permeability improved this to 34%. Sensitivity analysis showed that predictions of CL limited the overall accuracy of human PK predictions, partly due to high nonspecific binding of lipophilic compounds, leading to uncertainty of unbound clearance. For accurate predictions of %F, solubility was the key factor. Despite current limitations, this work encourages further development of ML models and integration of their results within PBPK models to enable human PK prediction at the drug design stage, even before compounds are synthesized. Further evaluation of this approach with more diverse chemical types is warranted.

Imaging of Transmembrane AMPA Receptor Regulatory Proteins by Positron Emission Tomography.

The transmembrane α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) receptor regulatory protein γ-8 (TARP γ-8) constitutes an auxiliary subunit of AMPA receptors, which mediates various brain functions including learning and memory. TARP γ-8 has emerged as a promising therapeutic target for central nervous system disorders. Despite considerable efforts, previously reported TARP γ-8 PET radioligands, such as [11C]TARP-1903 and [11C]TARP-1811 series, were plagued by limited brain uptake and/or high nonspecific binding in vivo. Herein, we developed two novel 11C-labeled probes, [11C]8 and [11C]15 (also named as [11C]TARP-2105), of which the latter exhibited a reasonable brain uptake as well as specific binding toward TARP γ-8 both in vitro and in vivo, as confirmed by blocking experiments with the commercially available TARP γ-8 inhibitor, JNJ-55511118 in the TARP γ-8-rich hippocampus. Overall, [11C]15 exhibited promising tracer characteristics and proved to be a lead positron-emission tomography ligand for the non-invasive quantification of TARP γ-8 in the mammalian brain.

Physisorption of Affinity Ligands Facilitates Extracellular Vesicle Detection with Low Non-Specific Binding to Plasmonic Gold Substrates.

Plasmonic biosensors are increasingly being used for the analysis of extracellular vesicles (EVs) originating from disease areas. However, the high non-specific binding of EVs to a gold-sensing surface has been a critical problem and hindered the true translational potential. Here, we report that direct antibody immobilization on the plasmonic gold surface via physisorption shows excellent capture of cancer-derived EVs with ultralow non-specific binding even at very high concentrations. Contrary to commonly used methods that involve thiol-based linker attachment and an EDC/sulfo-NHS reaction, we show a higher specific capture rate and >50-fold lower non-specific on citrate-capped plain and nanopatterned gold surfaces. The method provides a simple, fast, and reproducible means to functionalize plasmonic gold surfaces with antibodies for robust EV biosensing.

Anion Exchange Affinity-Based Controllable Surface Imprinting Synthesis of Ultrathin Imprinted Films for Protein Recognition.

Anion exchange affinity-based controllable surface imprinting is an effective approach to overcome low imprinting efficiency and high non-specific binding capacity. The template proteins were first immobilized on the anchored tetraalkylammonium groups of the nanoparticles via anion exchange affinity-based interactions, enabling monolayer sorption using a low template concentration. The combined use of surface-initiated photoiniferter-mediated polymerization to precisely control the imprinted film thickness, allowing the formation of homogeneous binding cavities, and the construction of effective binding sites resulted in a low non-specific binding capacity and high imprinting efficiency. The obtained imprinted films benefited from the anion exchange mechanism, exhibiting a higher imprinting factor and faster binding rate than the reference material. Binding tests revealed that the binding strength and selective recognition properties could be tuned to a certain extent by adjusting the NaCl concentration. Additionally, in contrast to the harsh template elution conditions of the covalent immobilization approach, over 80% of the template molecules were readily removed from the imprinted films using supersonic elution with an aqueous mixture of NaCl and HAc. Introducing template immobilization by anion exchange interactions to the synthesis of imprinted materials may provide a new approach for effective biomacromolecular imprinting.

Magnetic solid phase extraction followed by in-situ derivatization with core–shell structured magnetic graphene oxide nanocomposite for the accurate quantification of free testosterone and free androstenedione in human serum

Circulating free androgens are important indicators for a variety of diseases, so accurate determination of these hormones in serum is of great clinical significance. However, there are still many challenges for the accurate quantification of free androgens using mass methods because of their very low levels and complex interferences in serum, as well as the high serum protein binding rate and high nonspecific binding (NSB) rate. Here, an HPLC–MS/MS method coupled with magnetic solid phase extraction (MSPE) and in-situ derivatization was developed to quantify two free androgens—free testosterone (FT) and free androstenedione (FA4)—in human serum simultaneously and accurately. Ultrafiltration was used to obtain free androgens in serum. To minimize the NSB rate and obtain accurate results, the ultrafiltration membrane was doubly modified with surfactant followed by a silane-coupling agent. Multiple pre-saturation with the tested samples was also used. With these strategies, the ultrafiltration recoveries were up to 95.4% for FT and 94.0% for FA4, so the NSB was negligible. After that, the extremely low levels of free androgens in ultrafiltrates were extracted and enriched using MSPE with core–shell structured ferroferric oxide coated with graphene oxide. Hydroxylamine hydrochloride was used to derivatize the analytes and the reaction took place on the surface of the adsorbent. All the extraction and derivatization conditions were optimized. Under such conditions, the assays were linear for FT within the range of 2–100 pg mL−1 and for FA4 within the range of 5–500 pg mL−1. The intra- and inter-run CV was less than 12.3% and 10.9% for FT and less than 7.2% and 8.3% for FA4, respectively. For the intra- and inter-run accuracies, the relative error of the mean was 9.9% and 8.4% for FT, and 11.5% and 7.3% for FA4, respectively. The total extraction recoveries with MSPE in-situ derivatization were 93.2% for FT, 93.8% for FA4 and 95.7% for the internal standard. The method was validated and was used to quantify the trace level of these two free androgens in serum samples from female patients suspected of having polycystic ovary syndrome (PCOS) accurately. It is expected to improve the diagnosis accuracy of PCOS when combined with other clinical indicators.

Chemoselective Bioconjugation of Amyloidogenic Protein Antigens to PEGylated Microspheres Enables Detection of α-Synuclein Autoantibodies in Human Plasma.

The misfolding and subsequent aggregation of amyloidogenic proteins is a classic pathological hallmark of neurodegenerative diseases. Aggregates of the α-synuclein protein (αS) are implicated in Parkinson's disease (PD) pathogenesis, and naturally occurring autoantibodies to these aggregates are proposed to be potential early-stage biomarkers to facilitate the diagnosis of PD. However, upon misfolding, αS forms a multitude of quaternary structures of varying functions that are unstable ex vivo. Thus, when used as a capture agent in enzyme-linked immunosorbent assays (ELISAs), significant variance among laboratories has prevented the development of these valuable diagnostic tests. We reasoned that those conflicting results arise due to the high nonspecific binding and amyloid nucleation that are typical of ELISA platforms. In this work, we describe a multiplexed, easy-to-operate immunoassay that is generally applicable to quantify the levels of amyloid proteins and their binding partners, named Oxaziridine-Assisted Solid-phase Immunosorbent (OASIS) assay. The assay is built on a hydrophilic poly(ethylene glycol) scaffold that inhibits aggregate nucleation, which we show reduces assay variance when compared to similar ELISA measurements. To validate our OASIS assay in patient-derived samples, we measured the levels of naturally occurring antibodies against the αS monomer and oligomers in a cohort of donor plasma from patients diagnosed with PD. Using OASIS assays, we observed significantly higher titers of immunoglobulin G antibody recognizing αS oligomers in PD patients compared to those in healthy controls, while there was no significant difference in naturally occurring antibodies against the αS monomer. In addition to its development into a blood test to potentially predict or monitor PD, we anticipate that the OASIS assay will be of high utility for studies aimed at understanding protein misfolding, its pathology and symptomology in PD, and other neurodegenerative diseases.

A microdosing framework for absolute bioavailability assessment of poorly soluble drugs: A case study on cold-labeled venetoclax, from chemistry to the clinic.

This work presents an end‐to‐end approach for assessing the absolute bioavailability of highly hydrophobic, poorly water‐soluble compounds that exhibit high nonspecific binding using venetoclax as a model drug. The approach utilizes a stable labeled i.v. microdose and requires fewer resources compared with traditional approaches that use radioactive 14C‐labeled compounds. The stable labeled venetoclax and internal standard were synthesized, then an i.v. formulation was developed. In the clinical study, female subjects received a single oral dose of venetoclax 100 mg followed by a 100‐µg i.v. dose of cold‐labeled 13C‐venetoclax at the oral time of maximum concentration (Tmax). The i.v. microdose was prepared as an extemporaneous, sterile compounded solution on the dosing day by pharmacists at the clinical site. Several measures were taken to ensure the sterility and safety of the i.v. preparation. A sensitive liquid chromatography‐tandem mass spectrometry method was developed to allow the detection of plasma levels from the i.v. microdose. Plasma samples were collected through 72 h, and pharmacokinetic parameters were estimated using noncompartmental methods. Postdosing sample analysis demonstrated the consistency of the preparations and allowed the precise calculation of the pharmacokinetic parameters based on the actual injected dose. The absolute bioavailability of venetoclax was estimated at 5.4% under fasting conditions. Venetoclax extraction ratio was estimated to be 0.06 suggesting that the fraction transferred from the enterocytes into the liver is limiting venetoclax bioavailability. The proposed framework can be applied to other highly hydrophobic, poorly water‐soluble compounds that exhibit high nonspecific binding to support the understanding of their absorption and disposition mechanisms and guide formulation development.

Pharmacokinetics and Target Attainment of SQ109 in Plasma and Human-Like Tuberculosis Lesions in Rabbits.

ABSTRACTSQ109 is a novel well-tolerated drug candidate in clinical development for the treatment of drug-resistant tuberculosis (TB). It is the only inhibitor of the MmpL3 mycolic acid transporter in clinical development. No SQ109-resistant mutant has been directly isolated thus far in vitro, in mice, or in patients, which is tentatively attributed to its multiple targets. It is considered a potential replacement for poorly tolerated components of multidrug-resistant TB regimens. To prioritize SQ109-containing combinations with the best potential for cure and treatment shortening, one must understand its contribution against different bacterial populations in pulmonary lesions. Here, we have characterized the pharmacokinetics of SQ109 in the rabbit model of active TB and its penetration at the sites of disease—lung tissue, cellular and necrotic lesions, and caseum. A two-compartment model with first-order absorption and elimination described the plasma pharmacokinetics. At the human-equivalent dose, parameter estimates fell within the ranges published for preclinical species. Tissue concentrations were modeled using an “effect” compartment, showing high accumulation in lung and cellular lesion areas with penetration coefficients in excess of 1,000 and lower passive diffusion in caseum after 7 daily doses. These results, together with the hydrophobic nature and high nonspecific caseum binding of SQ109, suggest that multiweek dosing would be required to reach steady state in caseum and poorly vascularized compartments, similar to bedaquiline. Linking lesion pharmacokinetics to SQ109 potency in assays against replicating, nonreplicating, and intracellular M. tuberculosis showed SQ109 concentrations markedly above pharmacokinetic-pharmacodynamic targets in lung and cellular lesions throughout the dosing interval.

BLZ945 derivatives for PET imaging of colony stimulating factor-1 receptors in the brain

BackgroundThe kinase colony stimulating factor-1 receptor (CSF-1R) has recently been identified as a novel therapeutic target for decreasing tumor associated macrophages and microglia load in cancer treatment. In glioblastoma multiforme (GBM), a high-grade cancer in the brain with extremely poor prognosis, macrophages and microglia can make up to 50% of the total tumor mass. Currently, no non-invasive methods are available for measuring CSF-1R expression in vivo. The aim of this work is to develop a PET tracer for imaging of CSF-1R receptor expression in the brain for future GBM patient selection and treatment monitoring. MethodsBLZ945 and a derivative that potentially allows for fluorine-18 labeling were synthesized and evaluated in vitro to determine their affinity towards CSF-1R. BLZ945 was radiolabeled with carbon-11 by N-methylation of des-methyl-BLZ945 using [11C]CH3I. Following administration to healthy mice, metabolic stability of [11C]BLZ945 in blood and brain and activity distribution were determined ex vivo. PET scanning was performed at baseline, efflux transporter blocking, and CSF-1R blocking conditions. Finally, [11C]BLZ945 binding was evaluated in vitro by autoradiography on mouse brain sections. ResultsBLZ945 was the most potent compound in our series with an IC50 value of 6.9 ± 1.4 nM. BLZ945 was radiolabeled with carbon-11 in 20.7 ± 1.1% decay corrected radiochemical yield in a 60 min synthesis procedure with a radiochemical purity of >95% and a molar activity of 153 ± 34 GBq·μmol−1. Ex vivo biodistribution showed moderate brain uptake and slow wash-out, in addition to slow blood clearance. The stability of BLZ945 in blood plasma and brain was >99% at 60 min post injection. PET scanning demonstrated BLZ945 to be a substrate for efflux transporters. High brain uptake was observed, which was shown to be mostly non-specific. In accordance, in vitro autoradiography on brain sections revealed high non-specific binding. Conclusions[11C]BLZ945, a CSF-1R PET tracer, was synthesized in high yield and purity. The tracer has high potency for the target, however, future studies are warranted to address non-specific binding and tracer efflux before BLZ945 or derivatives could be translated into humans for brain imaging.

Organic Anion-Transporting Polypeptide 1B1/1B3-Mediated Hepatic Uptake Determines the Pharmacokinetics of Large Lipophilic Acids: In Vitro-In Vivo Evaluation in Cynomolgus Monkey.

It is generally presumed that uptake transport mechanisms are of limited significance in hepatic clearance for lipophilic or high passive-permeability drugs. In this study, we evaluated the mechanistic role of the hepato-selective organic anion-transporting polypeptides (OATPs) 1B1/1B3 in the pharmacokinetics of compounds representing large lipophilic acid space. Intravenous pharmacokinetics of 16 compounds with molecular mass ∼400-730 Da, logP ∼3.5-8, and acid pKa <6 were obtained in cynomolgus monkey after dosing without and with a single-dose rifampicin-OATP1B1/1B3 probe inhibitor. Rifampicin (30 mg/kg oral) significantly (P < 0.05) reduced monkey clearance and/or steady-state volume of distribution (VDss) for 15 of 16 acids evaluated. Additionally, clearance of danoprevir was reduced by about 35%, although statistical significance was not reached. A significant linear relationship was noted between the clearance ratio (i.e., ratio of control to treatment groups) and VDss ratio, suggesting hepatic uptake contributes to the systemic clearance and distribution simultaneously. In vitro transport studies using primary monkey and human hepatocytes showed uptake inhibition by rifampicin (100 µM) for compounds with logP ≤6.5 but not for the very lipophilic acids (logP > 6.5), which generally showed high nonspecific binding in hepatocyte incubations. In vitro uptake clearance and fraction transported by OATP1B1/1B3 (ft,OATP1B) were found to be similar in monkey and human hepatocytes. Finally, for compounds with logP ≤6.5, good agreement was noted between in vitro ft,OATP1B and clearance ratio (as well as VDss ratio) in cynomolgus monkey. In conclusion, this study provides mechanistic evidence for the pivotal role of OATP1B-mediated hepatic uptake in the pharmacokinetics across a wide, large lipophilic acid space. SIGNIFICANCE STATEMENT: This study provides mechanistic insight into the pharmacokinetics of a broad range of large lipophilic acids. Organic anion-transporting polypeptides 1B1/1B3-mediated hepatic uptake is of key importance in the pharmacokinetics and drug-drug interactions of almost all drugs and new molecular entities in this space. Diligent in vitro and in vivo transport characterization is needed to avoid the false negatives often noted because of general limitations in the in vitro assays while handling compounds with such physicochemical attributes.

Para-chloro-2-[18F]fluoroethyl-etomidate: A promising new PET radiotracer for adrenocortical imaging.

Introduction: [11C]Metomidate ([11C]MTO), the methyl ester analogue of etomidate, was developed as a positron emission tomography (PET) radiotracer for adrenocortical tumours and has also been suggested for imaging in primary aldosteronism (PA). A disadvantage of [11C]MTO is the rather high non-specific binding in the liver, which impacts both visualization and quantification of the uptake in the right adrenal gland. Furthermore, the short 20-minute half-life of carbon-11 is a logistic challenge in the clinical setting.Objectives: The aim of this study was to further evaluate the previously published fluorine-18 (T1/2=109.5 min) etomidate analogue, para-chloro-2-[18F]fluoroethyl etomidate; [18F]CETO, as an adrenal PET tracer.Methods: In vitro experiments included autoradiography on human and cynomolgus monkey (non-human primate, NHP) tissues and binding studies on adrenal tissue from NHPs. In vivo studies with [18F]CETO in mice, rats and NHP, using PET and CT/MRI, assessed biodistribution and binding specificity in comparison to [11C]MTO.Results: The binding of [18F]CETO in the normal adrenal cortex, as well as in human adrenocortical adenomas and adrenocortical carcinomas, was shown to be specific, both in vitro (in humans) and in vivo (in rats and NHP) with an in vitro Kd of 0.66 nM. Non-specific uptake of [18F]CETO in NHP liver was found to be low compared to that of [11C]MTO.Conclusions: High specificity of [18F]CETO to the adrenal cortex was demonstrated, with in vivo binding properties qualitatively surpassing those of [11C]MTO. Non-specific binding to the liver was significantly lower than that of [11C]MTO. [18F]CETO is a promising new PET tracer for imaging of adrenocortical disease and should be evaluated further in humans.

Preclinical evaluation of [18F]cabozantinib as a PET imaging agent in a prostate cancer mouse model

IntroductionCabozantinib is a tyrosine kinase inhibitor (TKI) approved for the treatment of medullary thyroid cancer, renal cell carcinoma and hepatocellular carcinoma, and is currently in clinical trials for the treatment of prostate cancer and others. It exerts its therapeutic effect mainly through inhibition of the tyrosine kinases MET (hepatocyte growth factor receptor) and VEGFR2 (vascular endothelial growth factor receptor 2), in addition to several other kinases involved in cancer. PET imaging with TKIs such as [18F]cabozantinib could potentially aid in cancer diagnosis and guide treatment. This study aims to evaluate the utility of [18F]cabozantinib as a PET imaging probe in PC3 tumor xenografted mice. Methods[18F]cabozantinib was evaluated in non-tumor and tumor bearing (PC3 xenografted) male mice by ex vivo biodistribution studies and in vivo μPET imaging. Pretreatment studies were performed in the tumor bearing mice with the MET inhibitor PF04217903. Mouse plasma was analyzed with HPLC to quantify radiometabolites. To further evaluate the binding specificity of [18F]cabozantinib, in vitro autoradiography studies on heart and PC3 tumor sections were performed in the presence of authentic cabozantinib or specific MET and VEGFR2 inhibitors. ResultsTissue distribution studies in non-tumor bearing mice revealed slow blood clearance, absence of brain uptake and a high myocardial uptake. In the tumor bearing mice, tumor uptake was low (0.58 ± 0.20% ID/g at 30 min post tracer injection), which was confirmed by μPET imaging. No differences in tissue distribution and kinetics were observed in both biodistributions and μPET studies after pretreatment with the MET inhibitor PF04217903. At 30 min post tracer injection, 60 ± 3% of the recovered radioactivity in plasma in non-tumor bearing mice was present as intact tracer. [18F]cabozantinib binding in vitro to heart and tumor tissues was partly blocked in the presence of selective MET and VEGFR2 inhibitors (up to 40% block). The fraction of non-specific binding was relatively high for both tissues (66% for heart and 39% for tumor). Conclusion[18F]cabozantinib exhibits non-favorable properties as a PET imaging probe, demonstrated by slow excretion kinetics along with low tumor uptake and high non-specific binding in tumor and heart tissue. The results reflect cabozantinibs multi-kinase activity, making PET imaging of tumor specific kinase expression with [18F]cabozantinib challenging.

Field application of an improved protocol for environmental DNA extraction, purification, and measurement using Sterivex filter

Environmental DNA (eDNA) is increasingly popular as a useful non-invasive method to monitor and study biodiversity and community structure in freshwater and marine environments. To effectively extract eDNA from the filter surface is a fundamental factor determining the representativeness of the samples. We improved the eDNA extraction efficiency of an established Sterivex method by 12- to 16-fold using a larger volume of lysis buffer mix coupled with backflushing the cartridges. The DNeasy extraction column could be overloaded when the environmental sample input is high, possibly due to a higher nonspecific binding present in environmental samples, thus resulting in a relatively lower quantity measured. Therefore, we included an internal control DNA in the extraction to monitor the extraction and purification efficiencies in field samples, which is crucial for quantification of original eDNA concentration. The use of Takara Probe qPCR Mix supplemented with protein-based additives improved the robustness of the real time PCR assay on inhibitor-rich environmental samples, but prior purification by Qiagen PowerClean Pro Cleanup kit could be essential for inhibitor-rich water samples, even though the recovery rate was unexpectedly low (average 33.0%). The improved extraction and quantification complement the qualitative analyses including metabarcoding and metagenomics in field application.

Identification and in vitro characterization of C05-01, a PBB3 derivative with improved affinity for alpha-synuclein

The neuropathological hallmark of Parkinsońs disease, multiple system atrophy and dementia with Lewy bodies is the accumulation of α-synuclein. The development of an imaging biomarker for α-synuclein is an unmet need. To date, no selective α-synuclein imaging agent has been identified, though initial studies suggest that the tau tracer [11C]PBB3 displays some degree of binding to α-synuclein. In this study, a series of compounds derived from the PBB3 scaffold were examined using fluorescence imaging and tissue microarrays (TMAs) derived from brain samples with different proteinopathies. One compound, C05-01, was selected based on its higher fluorescence signal associated with Lewy body aggregates compared with other PBB3 analogues. In vitro binding assays using human brain homogenates and recombinant fibrils indicated that C05-01 had higher affinity for α-synuclein (KD/Ki 25 nM for fibrils, Ki 3.5 nM for brain homogenates) as compared with PBB3 (KD 58 nM). In autoradiography (ARG) studies using fresh frozen human tissue and TMAs, [3H]C05-01 displayed specific binding in cases with α-synuclein pathology.C05-01 is the first PBB3 analogue developed as a potential compound targeting α-synuclein. Despite improved affinity for α-synuclein, C05-01 showed specific binding in AD tissue with Amyloid β and tau pathology, as well as relatively high non-specific and off-target binding. Additional efforts are needed to optimize the pharmacological and physicochemical properties of this series of compounds as ligands for α-synuclein. This study also showed that the construction of TMAs from different proteinopathies provides a tool for evaluation of fluorescent or radiolabelled compounds binding to misfolded proteins.