Saturable Binding Research Articles

18F]NP3-627, a candidate PET imaging agent targeting the NLRP3 inflammasome in the central nervous system.

We describe the identification of a candidate positron emission tomography (PET) imaging agent for the NLRP3 protein. NLRP3 plays a critical role in the immune system and has proven a difficult target for the development of imaging agents due to its low and cell-specific expression profile. A recently described series of pyridazine-based inhibitors, with improved permeability and brain-penetration properties, was used as a starting point for the development of a suitable PET imaging agent. Optimization of affinity, non-specific binding and pharmacokinetic properties led to the identification of aminopyridazine (R)-2-(6-((1-cyclopropylpiperidin-3-yl)amino)pyridazin-3-yl)-5-fluoro-3-methylphenol (17b), which meets the preclinical profile of a successful imaging agent, and whose tritiated version demonstrated excellent specificity in a radioligand saturation binding assay, confirming its imaging potential.18F labeling led to [18F]NP3-627, the proposed PET imaging agent.

Copper-saturated chitosan beads (CuSCBs) for Enhanced phosphate removal

This study investigates the sequential use of non-cross-linked chitosan beads (CBs) for copper and phosphate adsorption, emphasizing copper site saturation and selective phosphate binding. Initially, CBs were loaded with copper until saturation was achieved, where all available sites were occupied by copper at an initial concentration of 100 mg/L. These copper-saturated chitosan beads (CuSCBs) were subsequently applied for phosphate adsorption, achieving a maximum adsorption capacity of 85.89 mg/g at pH 5. X-ray Photoelectron Spectroscopy (XPS) analysis revealed a shift in the Cu2O binding energy to lower values upon phosphate adsorption, indicating that phosphate ions interact primarily with the copper ions intercalated on the CBs rather than the chitosan matrix. Additionally, the nitrogen spectra showed no shift, confirming that the amine groups of chitosan do not participate in phosphate binding. The findings suggest a 1:1 interaction ratio between copper and phosphate ions, with each phosphate ion binding to one copper ion, creating a stable and selective adsorption site. The adsorption data fit the Langmuir isotherm and pseudo-second-order kinetic models, suggesting a homogeneous monolayer adsorption mechanism driven by chemisorption, where each copper site on the chitosan surface selectively binds one phosphate ion. Furthermore, the CuSCBs demonstrated minimal copper leaching and retained 90 % of their phosphate adsorption capacity over multiple adsorption–desorption cycles, highlighting their potential as effective and reusable adsorbents for water treatment applications targeting copper and phosphate removal.

Impact of the stiffness of equation of state on saturation properties of asymmetric nuclear matter and Urca cooling in neutron stars

Abstract We consider nuclear equation of state (EOS) based on the semi-realistic M3Y-Paris and M3Y-Reid effective nucleon-nucleon (NN) interactions to assess the influence of the stiffness of the isospinasymmetric nuclear matter (ANM) on its isoscalar and isovector saturation properties, and on the β-stable proton fraction in neutron star (NS) matter. Two parameterizations schemes of the CDM3Y isovector density dependence of the NN interaction are adopted, one is scaled to the isoscalar density dependence and the other (IVF1) is developed independently of it. We found that the stiffer EOS yields larger binding saturation density that decreases with the isospin asymmetry (I), and slightly decreases the maximum asymmetry (Imax) reached for bound ANM. The density-slope (L) of the symmetry energy is found to correlate directly with the stiffness of ANM, meanwhile the symmetryenergy curvature (Ksym) and the quadratic isovector incompressibility coefficient (Kτ ) are found to correlate with it inversely. We found also that the stiffer EOS indicates larger neutron-rich npeμ NS matter under β-equilibrium. Only the CDM3Y-Paris EOSs of isoscalar incompressibility K0 up to 270 MeV and that of CDM3Y-Reid with K0 = 200 MeV indicate allowed direct URCA (DU) cooling process in β-stable NS matter, at densities greater than 3.5 ρ0, based on the IVF1 density dependence. The stiffer EOS proposes a narrower density range for DU process in NS. According to the considered Paris (Reid) forces, muons can emerge in cold β-stable npe matter at ρ ≥ 0.85 ρ0 (0.79 ρ0), which slightly decreases both the neutron richness at NS core and the DU threshold density.

Assessing functional suitability of a lyophilized formulation containing designed ankyrin repeat proteins for radionuclide imaging of HER2/neu overexpression in malignant tumors

Aim. To study in vitro and in vivo the functional suitability of 99mTc-labeled lyophilized formulation containing designed ankyrin repeat protein (DARPin) G3-(GGGS)3Cys for radionuclide imaging of HER2/neu overexpression in malignant tumors.Materials and methods. To create a targeted protein, a modified genetic construct with the sequence encoding DARPin G3-(GGGS)3Cys was used. To generate the experimental probe, we used a lyophilized formulation containing DARPin G3-(GGGS)3Cys with auxiliary substances and 99mTc sodium pertechnetate (500 MBq) incubated at 60 °C for 30 min. Radiochemical purity of 99mTc-G3-(GGGS)3Cys was analyzed by thin-layer radiochromatography. SKOV-3, BT-474, and DU-145 cell lines were used to test binding specificity in vitro. The dissociation constant was determined via a saturation binding assay on SKOV-3 cells with a range of protein concentrations from 0.2 to 40 nM. Nu/j mice bearing HER2-positive SKOV-3 xenografts and HER2-negative Ramos xenografts were used to evaluate the targeting properties and biodistribution.Results. A radiocomplex based on 99mTc and a lyophilized formulation with DARPin G3-(GGGS)3Cys was obtained with the radiochemical purity of more than 96%. Binding of 99mTc-G3-(GGGS)3Cys to the cells was specific (KD 3.9 ± 0.5 nM) and proportional to the level of HER2/neu expression in the cells. The uptake of 99mTc-G3-(GGGS)3Cys in SKOV-3 xenografts was significantly higher than in Ramos xenografts. 99mTc-G3-(GGGS)3Cys demonstrated rapid blood and renal clearance and had low activity in the salivary glands and stomach. Liver uptake was about 5–7%ID/g. In addition, 99mTc-G3-(GGGS)3Cys exhibited very low uptakes in the lungs, muscles, small intestine, and bones.Conclusion. The 99mTc-labeled lyophilized formulation with DARPin G3-(GGGS)3Cys is functionally suitable for imaging HER2/neu overexpression in tumors, as it binds specifically to the receptor, is stable in vivo, and has favorable biodistribution in organs and tissues. The radiocomplex based on 99mTc-G3-(GGGS)3Cys was obtained by a simple method with high radiochemical purity.

12518 Development Of A Four-compartment, Dynamic (In Vivo) Model For Insulin Disposition In Healthy Humans: Use Of Fick’s Law To Model Plasma-interstitial Insulin Flux

Abstract Disclosure: R.I. Dorin: None. C.R. Qualls: None. Introduction: Metabolic elimination of insulin (I) in vivo includes pathways mediated by high-affinity and potentially saturable insulin receptor binding; endogenous insulin secreted into the portal vein is subject to first pass elimination. The rate of insulin flux between vascular (Vp) and interstitial (Vi) compartments is often modeled by first order rate constants; here we analyzed an alternative approach using Fick’s law. Methods: The four-compartment, insulin (I) model is expressed by four differential equations that represent I flow (mass/time) in 4 volumes (V) (or compartments) having 3 corresponding elimination rate functions (α): (i) vascular plasma (Vp), (ii) interstitial (Vi, αi), (iii) hepatic (Vh, αh), and (iv) kidney (Vk, αk). Fick’s law was used to model diffusion of insulin between Vp and Vi; Michaelis-Menten like functions were used for saturable elimination functions (αh and αi); rates of hepatic (HPF) and renal (KPF) plasma flow were from literature. Realistic bounds for solved parameters (insulin permeability constant [k, L/min] and αi [min-[1]]) were developed by test bed analysis of published plasma (Ip) and interstitial (Ii) insulin concentration data. The inverse problem (estimating parameters) used Ip data from insulin-modified, frequently sampled iv glucose tolerance (IM-FSIVGT) studies performed in 40 healthy, non-diabetic women (1). Parameter solutions for individual subjects were obtained by iterative least squares method minimizing differences between model-predicted and measured Ip. Results: For infusion of exogenous I (ZI) at a constant rate (e.g., insulin clamp), reported steady state Ii/Ip data suggest that interstitial insulin elimination (αi) is conditionally saturable. Together with non-steady state data for Ip and Ii, initial estimates of k and αi were obtained. Steady state solutions give rise to four algebraic equations: (1) Ii/Ip = a, where a < 1 is consistent with experimental data, (2) Ih is a reduced version of a linear combination of Ip and endogenous insulin secretion (Zβ), where the factor of reduction (b) is <1, (3) Ik/Ip = d, where d<1, and (4) Ip is a linear combination of (i) ZI and (ii) the reduced (by factor b) Zβ. For Ip, this linear combination of ZI and reduced Zβ is also normalized by factor c (weighted sum of k, HPF, and KPF). Conclusions: (i) The proposed insulin flow model using Fick’s law was advantageous in providing realistic parameters and solutions that are tractable to physiologic interpretation, (ii) a simplified version of this model is generalizable to C-peptide, and (iii) since measurement of Ii is impractical in the clinical setting, the proposed prediction model may provide useful modeling adjunct to personalize pharmacokinetic and pharmacodynamic strategies for insulin administered by subcutaneous, iv, and hybrid closed-loop insulin pump routes. 1. Gower et al. Nutrition & Metabolism (2016) 13:2 Presentation: 6/1/2024

Elucidation of DPP-4 involvement in systemic distribution and renal reabsorption of linagliptin by PBPK modeling with a cluster Gauss-Newton method.

The dipeptidyl peptidase-4 (DPP-4) inhibitor linagliptin (LNG) exhibits target-mediated drug disposition (TMDD) in clinical settings, characterized by saturable binding to plasma soluble DPP-4 (sDPP-4) and tissue transmembrane DPP-4 (tDPP-4). Previous studies have indicated that saturable renal reabsorption of LNG contributes to its nonlinear urinary excretion observed in humans and wild-type mice, but not in Dpp-4 knockout mice. To elucidate the mechanisms underlying these complex phenomena, including DPP-4-related renal reabsorption of LNG, we employed physiologically-based pharmacokinetic (PBPK) modeling combined with a cluster Gauss-Newton method (CGNM). The CGNM facilitated the exploration of parameters in rat and human PBPK models for LNG and the determination of parameter identifiability. Through PBPK-CGNM analysis using reported autoradiography data ([14C]-LNG) in wild-type and Dpp-4-deficient rats, DPP-4-specific distributions of LNG in various tissues were clearly differentiated from nonspecific parts. By fitting to human plasma concentrations and urinary and fecal excretions of LNG after intravenous and oral administrations, multiple unknown PBPK parameters were simultaneously estimated by the CGNM. Notably, the amount of tDPP-4 and the reabsorption clearance for LNG-DPP-4 complexes were identifiable, indicating their critical role in explaining the complex nonlinear pharmacokinetics of LNG. Compared with previous PBPK analyses, the CGNM allowed us to incorporate greater model complexity (e.g., consideration of tDPP-4 expressions and invitro binding kinetics), ultimately resulting in a more accurate reproduction of LNG's TMDD. In conclusion, by considering LNG as a high-affinity probe for DPP-4, comprehensive PBPK-CGNM analyses suggested a dynamic whole-body distribution of DPP-4, including its involvement in the renal reabsorption of LNG.

Insights into the migration mechanism of extracellular antibiotic resistance genes during struvite recovery using synthetic wastewater

In recent, the complexation of extracellular antibiotic resistance genes (eARGs) with environmental particles has been getting significant concerns, since eARGs can consequently disseminate, propagate and pose ecological risks to the environment. This study focused on eARGs complexing with struvite (MgNH4PO4·6H2O) particles in struvite recovery by using synthetic wastewater. The adsorption capacities of eARGs by struvite crystals with different morphologies were firstly examined. Results revealed that struvite crystals possessed the maximum eARGs adsorption capacity of 7.95 × 1012-1.76 × 1013 copies/g. The evolution of struvite morphologies from regular polyhedron to needle-like, coupled with larger BET surface area, resulted in a matching increase relationship of eARGs adsorption. Electrostatic interaction and covalent binding were the predominant forces between eARGs and struvite crystals, attributed to the Mg[H2O]62+ octahedra in the struvite crystallite and the phosphate backbone with its external position in eARGs molecule. The eARGs adsorption in struvite crystallization displayed a “U” curve with the minimum values of 3.57 × 1012-7.28 × 1012 copies/g at pH 8.8, which was ascribed to the excessive existence of Mg2+ ions in the liquid. Despite the gradual increase in the Mg:P molar ratio from 1.0 to 2.5 during crystallization, the abundance of eARGs on recovered solids displayed twice dramatic declines with two or three orders of magnitude lower, which was attributed to the formation and binding saturation of eARGs-Mg chelate, as well as the non-negligible evolution of magnesium species under different pH values. These outcomes provide new insights into the migration behavior of eARGs during struvite recovery from wastewater.

Dually Labeled Neurotensin NTS1R Ligands for Probing Radiochemical and Fluorescence-Based Binding Assays.

The determination of ligand-receptor binding affinities plays a key role in the development process of pharmaceuticals. While the classical radiochemical binding assay uses radioligands, fluorescence-based binding assays require fluorescent probes. Usually, radio- and fluorescence-labeled ligands are dissimilar in terms of structure and bioactivity, and can be used in either radiochemical or fluorescence-based assays. Aiming for a close comparison of both assay types, we synthesized tritiated fluorescent neurotensin receptor ligands ([3H]13, [3H]18) and their nontritiated analogues (13, 18). The labeled probes were studied in radiochemical and fluorescence-based (high-content imaging, flow cytometry, fluorescence anisotropy) binding assays. Equilibrium saturation binding yielded well-comparable ligand-receptor affinities, indicating that all these setups can be used for the screening of new drugs. In contrast, discrepancies were found in the kinetic behavior of the probes, which can be attributed to technical differences of the methods and require further studies with respect to the elucidation of the underlying mechanisms.

The Emission of Internal Conversion Electrons Rather Than Auger Electrons Increased the Nucleus-Absorbed Dose for 161Tb Compared with 177Lu with a Higher Dose Response for [161Tb]Tb-DOTA-LM3 Than for [161Tb]Tb-DOTATATE.

Preclinical data have shown that 161Tb-labeled peptides targeting the somatostatin receptor are therapeutically more effective for peptide receptor radionuclide therapy than are their 177Lu-labeled counterparts. To further substantiate this enhanced therapeutic effect, we performed cellular dosimetry to quantify the absorbed dose to the cell nucleus and compared dose-response curves to evaluate differences in relative biological effectiveness invitro. Methods: CA20948 cell survival was assessed after treatment with [161Tb]Tb- and [177Lu]Lu-DOTATATE (agonist) and with [161Tb]Tb- and [177Lu]Lu-DOTA-LM3 (antagonist) via a clonogenic assay. Cell binding, internalization, and dissociation assays were performed up to 7 d to acquire time-integrated activity coefficients. Separate S values for each type of particle emission (Auger/internal conversion [IC] electrons and β- particles) were computed via Monte Carlo simulations, while considering spheric cells. Once the absorbed dose to the cell nucleus was calculated, survival curves were fitted to the appropriate linear or linear-quadratic model and corresponding relative biological effectiveness was evaluated. Results: Although the radiopeptide uptake was independent of the radionuclide, [161Tb]Tb-DOTATATE and [161Tb]Tb-DOTA-LM3 delivered a 3.6 and 3.8 times higher dose to the nucleus, respectively, than their 177Lu-labeled counterparts on saturated receptor binding. This increased nucleus-absorbed dose was mainly due to the additional emission of IC and not Auger electrons by 161Tb. When activity concentrations were considered, both [161Tb]Tb-DOTATATE and [161Tb]Tb-DOTA-LM3 showed a lower survival fraction than did labeling with 177Lu. When the absorbed dose to the nucleus was considered, no significant difference could be observed between the dose-response curves for [161Tb]Tb- and [177Lu]Lu-DOTATATE. [161Tb]Tb-DOTA-LM3 showed a linear-quadratic dose response, whereas [161Tb]Tb-DOTATATE showed only a linear dose response within the observed dose range, suggesting additional cell membrane damage by Auger electrons. Conclusion: The IC, rather than Auger, electrons emitted by 161Tb resulted in a higher absorbed dose to the cell nucleus and lower clonogenic survival for [161Tb]Tb-DOTATATE and [161Tb]Tb-DOTA-LM3 than for the 177Lu-labeled analogs. In contrast, [161Tb]Tb-DOTATATE showed no higher dose response than [177Lu]Lu-DOTATATE, whereas for [161Tb]Tb-DOTA-LM3 an additional quadratic response was observed. Because of this quadratic response, potentially caused by cell membrane damage, [161Tb]Tb-DOTA-LM3 is a more effective radiopeptide than [161Tb]Tb-DOTATATE for labeling with 161Tb.

Target-specific affinity separation of the bioactive compounds from herbal extract using the spin column packed with the immobilized protein microspheres prior to LC-MS analysis

Excellent pretreatments before instrumental analysis are critical for separation and determination of target compounds for discovery of new drugs from herb medicines. We developed a rapid and highly-selective method to separate the bioactive compounds from herbal extract using protein affinity-selection spin column, which was packed with the new sorbent materials from integrating the recombinant β2-adrenoceptor (β2-AR) directly out of cell lysates onto the surface of microspheres. Protein affinity-selection spin column was placed in a centrifugal tube, where after the non-specific binders were released to the filtrate under the operational centrifugation, the specific binders on the spin column were cleaned with a washing solvent for LC-MS analysis. The known agonists of β2-AR were retained/released on protein affinity-selection spin column but not on control column, demonstrating the method with good recovery (79.4∼95.7 %) and high repeatability (RSD < 3.5 %). The adsorption features of three ligands on the spin column were described best by Prism saturation binding model, and the high-affinity binding and the large binding capacity of the spin column make it feasible to trap the trace analytes effectively. It was applied in separating bioactive compounds from Alstoniae Scholaris extract, two of which were identified as picrinine and oleanolic acid in combination with LC-MS and verified as the potential agonists towards β2-AR though molecular docking and cell experiments. Our study demonstrated that, the spin column with the immobilized protein sorbents in the centrifugal filter device represents a promising tool, enabling rapid and target-specific affinity separation of the bioactive compounds from herbal extract.

Validation of the C-X-C chemokine receptor 3 (CXCR3) as a target for PET imaging of T cell activation

PurposeCXCR3 is expressed on activated T cells and plays a crucial role in T-cell recruitment to the tumor microenvironment (TME) during cell-based and immune checkpoint inhibitor (ICI) immunotherapy. This study utilized a 64Cu-labeled NOTA-α-CXCR3 antibody to assess CXCR3 expression in the TME and validate it as a potential T cell activation biomarker in vivo.ProceduresCXCR3+ cells infiltrating MC38 tumors (B57BL/6 mice, untreated and treated with αPD-1/αCTLA-4 ICI) were quantified using fluorescence microscopy and flow cytometry. A commercial anti-mouse CXCR3 antibody (α-CXCR3) was site-specifically conjugated with 2,2,2-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid (NOTA) and radiolabeled with 64Cu. Saturation binding of [64Cu]Cu-NOTA-α-CXCR3 was investigated using CHO cells stably transfected with murine CXCR3. Biodistribution and PET imaging studies both at baseline and after 1 to 3 cycles of ICI, respectively, were carried out using different molar activities (10 GBq/µmol to 300 GBq/µmol) of [64Cu]Cu-NOTA-α-CXCR3.ResultsFlow cytometry analysis at baseline confirmed the presence of CXCR3 + T-cells in MC38 tumors, which was significantly increased at day five after ICI (treated 33.8 ± 17.4 vs. control 8.8 ± 6.2 CD3+CXCR3+ cells/mg). These results were qualitatively and quantitatively confirmed by immunofluorescence of tumor cryoslices. In vivo PET imaging of MC38 tumor bearing mice before, during and after ICI using [64Cu]Cu-NOTA-α-CXCR3 (Kd = 3.3 nM) revealed a strong dependence of CXCR3-specificity of tracer accumulation in secondary lymphoid organs on molar activity. At 300 GBq/µmol (1.5 µg of antibody/mouse), a specific signal was observed in lymph nodes (6.33 ± 1.25 control vs. 3.95 ± 1.23%IA/g blocking) and the spleen (6.04 ± 1.02 control vs. 3.84 ± 0.79%IA/g blocking) at 48 h p.i. Spleen-to-liver ratios indicated a time dependent systemic immune response showing a steady increase from 1.08 ± 0.19 (untreated control) to 1.54 ± 0.14 (three ICI cycles).ConclusionsThis study demonstrates the feasibility of in vivo imaging of CXCR3 upregulation under immunotherapy using antibodies. However, high molar activities and low antibody doses are essential for sensitive detection in lymph nodes and spleen. Detecting therapy-induced changes in CXCR3+ T cell numbers in tumors was challenging due to secondary antibody-related effects. Nonetheless, CXCR3 remains a promising target for imaging T cell activation, with anticipated improvements in sensitivity using alternative tracers with high affinities and favorable pharmacokinetics.

A radioligand for in vitro autoradiography of CSF1R in post-mortem CNS tissues

BackgroundReactive microglia and recruited peripheral macrophages contribute to the pathogenesis of Alzheimer’s dementia (AD). Monocytes, macrophages and microglia all express the marker colony-stimulating factor 1 receptor (CSF1R). 4-Cyano-N-(4-(4-methylpiperazin-1-yl)-2-(4-methylpiperidin-1-yl)phenyl)-1H-pyrrole-2-carboxamide (1) is a high-affinity antagonist for CSF1R. We report the radiosynthesis of both [3H]1 and [11C]1. The PET imaging properties of [11C]1 in mice and baboon were investigated. [3H]1 was studied in Bmax measurement in post-mortem autoradiography in the frontal cortex, inferior parietal cortex and hippocampus from donors diagnosed with AD and age-matched controls. In vitro binding affinity of 1 was measured commercially. Nor-methyl-1 precursor was radiolabeled with [11C]iodomethane or [3H]iodomethane to produce [11C]1 and [3H]1, respectively. Ex vivo brain biodistribution of [11C]1 was compared in normal mice versus lipopolysaccharide-administered (LPS) murine model of neuroinflammation. Dynamic PET imaging was performed in a healthy male Papio anubis baboon. Post-mortem autoradiography with [3H]1 was performed in frozen sections using a standard saturation binding technique.ResultsCompound 1 exhibits a high in vitro CSF1R binding affinity (0.59 nM). [11C]1 was synthesized with high yield. [3H]1 was synthesized similarly (commercially). Biodistribution of [11C]1 in healthy mice demonstrated moderate brain uptake. In LPS-treated mice the brain uptake of [11C]1 was ~ 50% specific for CSF1R. PET/CT [11C]1 study in baboon revealed low brain uptake (0.36 SUV) of [11C]1. Autoradiography with [3H]1 gave significantly elevated Bmax values in AD frontal cortex versus control (47.78 ± 26.80 fmol/mg vs. 12.80 ± 5.30 fmol/mg, respectively, P = 0.023) and elevated, but not significantly different binding in AD hippocampus grey matter and inferior parietal cortex (IPC) white matter.ConclusionsCompound 1 exhibits a high in vitro CSF1R binding affinity. [11C]1 specifically labels CSF1R in the mouse neuroinflammation, but lacks the ability to efficiently cross the blood–brain barrier in baboon PET. [3H]1 specifically labels CSF1R in post-mortem human brain. The binding of [3H]1 is significantly higher in the post-mortem frontal cortex of AD versus control subjects.

Intravenous ferric carboxymaltose versus oral ferrous sulphate for the treatment of moderate to severe postpartum anaemia in Nigerian women (IVON-PP): protocol for an open-label randomised controlled type 1 hybrid effectiveness-implementation trial

IntroductionPostpartum anaemia is often caused by iron deficiency with onset during the antepartum period and can be exacerbated by excessive blood loss at birth. Its prevalence is estimated as 50–80%...

Non-linear IV pharmacokinetics of the ATR inhibitor berzosertib (M6620) in mice.

The Ataxia Telangiectasia and Rad3-related (ATR) protein complex is an apical initiator of DNA damage response pathways. Several ATR inhibitors (ATRi) are in clinical development including berzosertib (formerly M6620, VX-970). Although clinical studies have examined plasma pharmacokinetics (PK) in humans, little is known regarding dose/exposure relationships and tissue distribution. To understand these concepts, we extensively characterized the PK of berzosertib in mouse plasma and tissues. A highly sensitive LC-MS/MS method was utilized to quantitate berzosertib in plasma and tissues. Dose proportionality was assessed in female BALB/c mice following single IV doses (2, 6, 20 or 60mg/kg). A more extensive PK study was conducted in tumor-bearing mice following a single IV dose of 20mg/kg to evaluate distribution to tissues. PK parameters were calculated by non-compartmental analysis (NCA). A compartmental model was developed to describe the PK behavior of berzosertib. Plasma protein binding was determined in vitro. Increased doses of berzosertib were associated with less than proportional increases in early plasma concentrations and greater than proportional increase in tissue exposure, attributable to saturation of plasma protein binding. Berzosertib extensively distributed into bone marrow, tumor, thymus, and lymph nodes, however; brain and spinal cord exposure was less than plasma. The nonlinear PK of berzosertib displayed here can be attributed to saturation of plasma protein binding and occurred at concentrations close to those observed in clinical trials. Our results will help to understand preclinical pharmacodynamic and toxicity data and to inform optimal dosing and deployment of berzosertib.

A pharmacokinetic framework describing antibiotic adsorption to cardiopulmonary bypass devices.

Cardiopulmonary bypass (CPB) can alter pharmacokinetic (PK) parameters and the drug may adsorb to the CPB device, altering exposure. Cefazolin is a beta-lactam antibiotic used for antimicrobial prophylaxis during cardiac surgery supported by CPB. Adsorption of cefazolin could result in therapeutic failure. An exvivo study was undertaken using CPB devices primed and then dosed with cefazolin and samples were obtained over 1 hour of recirculation. Twelve experimental runs were conducted using different CPB device sizes (neonate, infant, child, and adult), device coatings (Xcoating™, Rheoparin®, PH.I.S.I.O), and priming solutions. The time course of saturable binding, using Bmax (binding capacity), Kd (dissociation constant), and T2off (half-time of dissociation), described cefazolin adsorption. Bmax estimates for the device sizes were neonate 40.0 mg (95% CI 24.3, 67.4), infant 48.6 mg (95% CI 5.97, 80.2), child 77.8 mg (95% CI 54.9, 103), and adult 196 mg (95% CI 191, 199). The Xcoating™ Kd estimate was 139 mg/L (95% CI 27.0, 283) and the T2off estimate was 98.4 min (95% CI 66.8, 129). The Rheoparin® and PH.I.S.I.O coatings had similar binding parameters with Kd and T2off estimates of 0.169 mg/L (95% CI 0.01, 1.99) and 4.94 min (95% CI 0.17, 59.4). The Bmax was small (< 10%) relative to a typical total patient dose during cardiac surgery supported by CPB. A dose adjustment for cefazolin based solely on drug adsorption is not required. This framework could be extended to other PK studies involving CPB.

Synthesis of 64Cu-, 55Co-, and 68Ga-Labeled Radiopharmaceuticals Targeting Neurotensin Receptor-1 for Theranostics: Adjusting InVivo Distribution Using Multiamine Macrocycles.

The development of theranostic radiotracers relies on their binding to specific molecular markers of a particular disease and the use of corresponding radiopharmaceutical pairs thereafter. This study reports the use of multiamine macrocyclic moieties (MAs), as linkers or chelators, in tracers targeting the neurotensin receptor-1 (NTSR-1). The goal is to achieve elevated tumor uptake, minimal background interference, and prolonged tumor retention in NTSR-1-positive tumors. Methods: We synthesized a series of neurotensin antagonists bearing MA linkers and metal chelators. The MA unit is hypothesized to establish a strong interaction with the cell membrane, and the addition of a second chelator may enhance water solubility, consequently reducing liver uptake. Small-animal PET/CT imaging of [64Cu]Cu-DOTA-SR-3MA, [64Cu]Cu-NT-CB-NOTA, [68Ga]Ga-NT-CB-NOTA, [64Cu]Cu-NT-CB-DOTA, and [64Cu]Cu-NT-Sarcage was acquired at 1, 4, 24, and 48 h after injection using H1299 tumor models. [55Co]Co-NT-CB-NOTA was also tested in HT29 (high NTSR-1 expression) and Caco2 (low NTSR-1 expression) colorectal adenocarcinoma tumor models. Saturation binding assay and internalization of [55Co]Co-NT-CB-NOTA were used to test tracer specificity and internalization in HT29 cells. Results: In vivo PET imaging with [64Cu]Cu-NT-CB-NOTA, [68Ga]Ga-NT-CB-NOTA, and [55Co]Co-NT-CB-NOTA revealed high tumor uptake, high tumor-to-background contrast, and sustained tumor retention (≤48 h after injection) in NTSR-1-positive tumors. Tumor uptake of [64Cu]Cu-NT-CB-NOTA remained at 76.9% at 48 h after injection compared with uptake 1 h after injection in H1299 tumor models, and [55Co]Co-NT-CB-NOTA was retained at 60.2% at 24 h compared with uptake 1 h after injection in HT29 tumor models. [64Cu]Cu-NT-Sarcage also showed high tumor uptake with low background and high tumor retention 48 h after injection Conclusion: Tumor uptake and pharmacokinetic properties of NTSR-1-targeting radiopharmaceuticals were greatly improved when attached with different nitrogen-containing macrocyclic moieties. The study results suggest that NT-CB-NOTA labeled with either 64Cu/67Cu, 55Co/58mCo, or 68Ga (effect of 177Lu in tumor to be determined in future studies) and NT-Sarcage labeled with 64Cu/67Cu or 55Co/58mCo may be excellent diagnostic and therapeutic radiopharmaceuticals targeting NTSR-1-positive cancers. Also, the introduction of MA units to other ligands is warranted in future studies to test the generality of this approach.

Population pharmacokinetic and exposure-toxicity analyses of nab-paclitaxel after pegylated recombinant human granulocyte colony-stimulating factor administration in patients with metastatic breast cancer.

This study aimed to establish a population pharmacokinetic (PK) model to evaluate the dynamic relationship between the concentrations of total and unbound paclitaxel, and the exposure-response analysis of albumin-bound paclitaxel (nab-paclitaxel) after pegylated recombinant human granulocyte colony-stimulating factor (PEG-G-CSF) administration in patients with metastatic breast cancer. A total of 653 concentrations corresponding to total paclitaxel and 334 concentrations corresponding to unbound paclitaxel were analyzed in 24 subjects who randomized received a single 260 mg/m2 dose of two nab-paclitaxel formulations with a 21-35-day washout period. PEG-G-CSF was administered to all the patients in each cycle to prevent neutropenia. The exposure-response relationships were evaluated using the exposure to total, albumin-coated, and unbound paclitaxel, as well as the reduction in neutrophil count. The exposure data were analyzed using nonlinear mixed-effect modeling. A linear regression model was used to test the statistical significance of the correlation between percentage of reduction in neutrophil count and exposure. The PK characteristics of total paclitaxel were described using a three-compartment model with first-order elimination, and a mechanism-based model incorporating linear release of nab-paclitaxel and the saturated binding of unbound paclitaxel to plasma components was established. The release ratio of paclitaxel from nab-paclitaxel was estimated to be 4.60% and the maximum unbound fraction (2.76%) was reached at the end of the infusion. The study found that a longer duration of total paclitaxel concentration > 0.19 µmol/L was significantly correlated with a reduction in neutrophil count (r2 = 0.23, P = 0.00062). Specifically, a duration of > 8.6h was a predictor of a decreased neutrophil count. The decrease in neutrophils induced by nab-paclitaxel was significantly correlated with the duration above a total paclitaxel concentration of 0.19 µmol/L despite the use of PEG-G-CSF.

Population pharmacokinetics of total and unbound valemetostat and platelet dynamics in healthy volunteers and patients with non-Hodgkin lymphoma.

Valemetostat is an EZH2/1 inhibitor that has been approved in Japan for the treatment of patients with relapsed/refractory adult T-cell leukemia/lymphoma, based mainly on results from a single-arm phase II trial. It is currently under investigation worldwide for the treatment of other non-Hodgkin lymphomas (NHLs), including peripheral T-cell lymphoma, and for solid tumors. Semi-mechanistic population pharmacokinetic modeling of total and unbound valemetostat and an analysis of the platelet time course during treatment with valemetostat were conducted using data from five clinical trials (two in patients with NHL and three in healthy volunteers). Pharmacokinetic data, including 3162 total/1871 unbound valemetostat observations from 102 patients and 72 healthy volunteers, were described by a three-compartment model with sequential zero-/first-order absorption and saturable binding in the central compartment. Alpha-1-acid glycoprotein (AAG) was the most influential covariate for total valemetostat exposure, yet had little impact on unbound exposure, meaning no dose adjustment was warranted based on AAG levels. The longitudinal platelet data from 101 patients (2313 observations) were adequately described by a modified Friberg model with two proliferation compartments, which characterized unique spontaneous recovery of platelet counts without dose modifications. A model-based simulation quantitatively assessed the proposed dose-adjustment guidance in case of platelet count decreased by comparing the probability of treatment discontinuation due to platelet count decreased with or without the dose adjustment. In summary, the models described observed total and unbound valemetostat concentrations and a unique time course of platelets during treatment, which can justify the clinical dose and provide dose-adjustment guidance.

Pharmacokinetics and pharmacodynamics of itepekimab in adults with moderate-to-severe atopic dermatitis: Results from two terminated phase II trials.

Interleukin-33 (IL-33) is a proinflammatory alarmin cytokine released by damaged epithelial tissue cells that initiates and amplifies both type 1 and type 2 inflammatory cascades. A role for IL-33 in atopic dermatitis (AD; a chronic, relapsing type 2 inflammatory disease of the skin) has been proposed. Itepekimab is a novel human IgG4P monoclonal antibody against IL-33, currently in clinical development for chronic obstructive pulmonary disease (COPD). Two global phase II studies-a dose-ranging itepekimab monotherapy study (NCT03738423) and a proof-of-concept study of itepekimab alone and in combination with dupilumab (NCT03736967)-were conducted in patients with moderate-to-severe AD to assess safety, tolerability, pharmacokinetics, pharmacodynamics, and efficacy; both studies were terminated following an interim analysis of the proof-of-concept study, which failed to demonstrate the efficacy of itepekimab. In these two studies, itepekimab exhibited linear and dose-proportional pharmacokinetics. Pharmacodynamics of total IL-33 indicated that itepekimab saturated binding to the target in serum at 300 mg q2w and q4w doses, and decreased blood eosinophil counts. Concentration-time profiles of itepekimab and total IL-33 were similar for itepekimab with or without dupilumab, and between East Asian and non-East Asian subgroups. Itepekimab was generally well tolerated, both alone and in combination with dupilumab. The lack of clinical efficacy for itepekimab observed in these studies suggests that IL-33 may not be a key pathogenic driver in moderate-to-severe AD.

No evidence of genetic causation between iron and infertility: a Mendelian randomization study.

Observational studies have explored the impact of iron homeostasis on infertility; however, establishing definitive causal relationships remains challenging. This study utilized a two-sample Mendelian randomization approach to investigate the potential causal relationship between iron status and infertility. Four indicators of iron status-serum iron, ferritin, transferrin saturation, and total iron binding capacity, were considered as exposure factors. Infertility was the outcome variable for both men and women. Robust causality was assessed using the primary inverse-variance-weighted method, complemented by three supplementary Mendelian randomization approaches. Sensitivity analyses were performed to enhance the precision and reliability of the results. No statistically significant associations were identified between the four indicators of iron status and infertility. These results remained consistent across multiple Mendelian randomization methodologies. In conclusion, there is no evidence of a genetic causal relationship between iron status and infertility. Nevertheless, this does not preclude the possibility of a connection between iron status and infertility at different mechanistic levels.