Understanding the pharmacokinetic journey of Fc-fusion protein, rhIL-7-hyFc using complementary approach of two analytical methods, accelerator mass spectrometry and ELISA.

Antibody-based therapeutics (ABTs), including monoclonal/polyclonal antibodies and fragment crystallizable region (Fc)-fusion proteins, are increasingly used in disease treatment, driving the global market growth. Understanding the pharmacokinetic (PK) properties of ABTs is crucial for their clinical effectiveness. This study investigated the PK profile and tissue distribution of efineptakin alfa, a long-acting recombinant human interleukin-7 (rhIL-7-hyFc), using enzyme-linked immunosorbent assay (ELISA) and accelerator mass spectrometry (AMS). Totally, four rats were injected intramuscularly with 1 mg/kg of rhIL-7-hyFc containing 14C-rhIL-7-hyFc, which was prepared via reductive methylation. Serum total radioactivity (TRA) and serum rhIL-7-hyFc concentrations were quantified using AMS and ELISA, respectively. The TRA concentrations in organs were determined by AMS. Serum TRA peaked at 10 hours with a terminal half-life of 40 hours. The rhIL-7-hyFc exhibited a mean peak concentration at around 17 hours and a rapid elimination with a half-life of 12.3 hours. Peak concentration and area under the curve of TRA were higher than those of rhIL-7-hyFc. Tissue distribution analysis showed an elevated TRA concentrations in lymph nodes, kidneys, and spleen, indicating rhIL-7-hyFc's affinity for these organs. The study also simulated the positions of 14C labeling in rhIL-7-hyFc, identifying specific residues in the fragment of rhIL-7 portion, and provided the explanation of distinct analytes targeted by each method. Combining ELISA and AMS provided advantages by offering sensitivity and specificity for quantification as well as enabling the identification of analyte forms. The integrated use of ELISA and AMS offers valuable insights for the development and optimization of ABT.

Environmental forensics using stable and radioactive isotopes in naturally attenuated soil after phenol-leakage accidents.

Phenol is a carcinogenic and hazardous chemical used in multiple industries and poses a high risk of chemical spills into the environment. To date, environmental forensic research has not focused on chemically remediated soils. In this study, an advanced environmental forensic analysis was performed on microbial communities and breakdown products of phenol, carbon stable isotopes, and radioactive isotopes in phenol-contaminated soil. As indicators of phenol-spill accidents after natural attenuation, higher δ13C levels and lower 14C/12C ratios were observed in phenol-contaminated soil compared with uncontaminated soil. In addition, 16s rRNA gene analysis revealed that phenol-breakdown products identified by gas chromatography-mass spectrometry and the presence of soil bacteria, such as Nocardioides, Faecalibacterium, and Bacteroides, were indicators of phenol-leakage accidents. Therefore, the proposed environmental forensic strategy is a valuable tool for identifying the location of previously occurring chemical accidents and estimating the ecological impact after the natural attenuation of contaminated soils.

Verification of Therapeutic Effect through Accelerator Mass Spectrometry-Based Single Cell Level Quantification of hESC-Endothelial Cells Distributed into an Ischemic Model.

As the potential of pluripotent stem cell-derived differentiated cells has been demonstrated in regenerative medicine, differentiated vascular endothelial cells (ECs) are emerging as a therapeutic agent for the cardiovascular system. To verify the therapeutic efficacy of differentiated ECs in an ischemic model, human embryonic stem cells (hESCs) are induced as EC lineage and produce high-purity ECs through fluorescence-activated cell sorting (FACS). When hESC-ECs are transplanted into a hindlimb ischemic model, it is confirmed that blood flow and muscle regeneration are further improved by creating new blood vessels together with autologous ECs than the primary cell as cord blood endothelial progenitor cells (CB-EPCs). In addition, previously reported studies show the detection of transplanted cells engrafted in blood vessels through various tracking methods, but fail to provide accurate quantitative values over time. In this study, it is demonstrated that hESC-ECs are engrafted approximately sevenfold more than CB-EPCs by using an accelerator mass spectrometry (AMS)-based cell tracking technology that can perform quantification at the single cell level. An accurate quantification index is suggested. It has never been reported in in vivo kinetics of hESC-ECs that can act as therapeutic agents.

Levosimendan inhibits disulfide tau oligomerization and ameliorates tau pathology in TauP301L-BiFC mice.

Tau oligomers play critical roles in tau pathology and are responsible for neuronal cell death and transmitting the disease in the brain. Accordingly, preventing tau oligomerization has become an important therapeutic strategy to treat tauopathies, including Alzheimer's disease. However, progress has been slow because detecting tau oligomers in the cellular context is difficult. Working toward tau-targeted drug discovery, our group has developed a tau-BiFC platform to monitor and quantify tau oligomerization. By using the tau-BiFC platform, we screened libraries with FDA-approved and passed phase I drugs and identified levosimendan as a potent anti-tau agent that inhibits tau oligomerization. 14C-isotope labeling of levosimendan revealed that levosimendan covalently bound to tau cysteines, directly inhibiting disulfide-linked tau oligomerization. In addition, levosimendan disassembles tau oligomers into monomers, rescuing neurons from aggregation states. In comparison, the well-known anti-tau agents methylene blue and LMTM failed to protect neurons from tau-mediated toxicity, generating high-molecular-weight tau oligomers. Levosimendan displayed robust potency against tau oligomerization and rescued cognitive declines induced by tauopathy in the TauP301L-BiFC mouse model. Our data present the potential of levosimendan as a disease-modifying drug for tauopathies.

Amyloid Against Amyloid: Dimeric Amyloid Fragment Ameliorates Cognitive Impairments by Direct Clearance of Oligomers and Plaques.

Amyloid-ß (Aß) in the form of neurotoxic aggregates is regarded as the main pathological initiator and key therapeutic target of Alzheimer's disease. However, anti-Aß drug development has been impeded by the lack of a target needed for structure-based drug design and low permeability of the blood-brain barrier (BBB). An attractive therapeutic strategy is the development of amyloid-based anti-Aß peptidomimetics that exploit the self-assembling nature of Aß and penetrate the BBB. Herein, we designed a dimeric peptide drug candidate based on the N-terminal fragment of Aß, DAB, found to cross the BBB and solubilize Aß oligomers and fibrils. Administration of DAB reduced amyloid burden in 5XFAD mice, and downregulated neuroinflammation and prevented memory impairment in the Y-maze test. Peptide mapping assays and molecular docking studies were utilized to elucidate DAB-Aß interaction. To further understand the active regions of DAB, we assessed the dissociative activity of DAB with sequence modifications.

In vivo tracking of 14C thymidine labeled mesenchymal stem cells using ultra-sensitive accelerator mass spectrometry.

Despite the tremendous advancements made in cell tracking, in vivo imaging and volumetric analysis, it remains difficult to accurately quantify the number of infused cells following stem cell therapy, especially at the single cell level, mainly due to the sensitivity of cells. In this study, we demonstrate the utility of both liquid scintillator counter (LSC) and accelerator mass spectrometry (AMS) in investigating the distribution and quantification of radioisotope labeled adipocyte derived mesenchymal stem cells (AD-MSCs) at the single cell level after intravenous (IV) transplantation. We first show the incorporation of 14C-thymidine (5 nCi/ml, 24.2 ng/ml) into AD-MSCs without affecting key biological characteristics. These cells were then utilized to track and quantify the distribution of AD-MSCs delivered through the tail vein by AMS, revealing the number of AD-MSCs existing within different organs per mg and per organ at different time points. Notably, the results show that this highly sensitive approach can quantify one cell per mg which effectively means that AD-MSCs can be detected in various tissues at the single cell level. While the significance of these cells is yet to be elucidated, we show that it is possible to accurately depict the pattern of distribution and quantify AD-MSCs in living tissue. This approach can serve to incrementally build profiles of biodistribution for stem cells such as MSCs which is essential for both research and therapeutic purposes.

An Investigation of the Metabolism and Excretion of KD101 and Its Interindividual Differences: A Microtracing Mass Balance Study in Humans.

The absorption, metabolism, and excretion (AME) profiles of KD101, currently under clinical development to treat obesity, were assessed in humans using accelerator mass spectrometry (AMS) after a single oral administration of KD101 at 400 mg and a microdose of 14 C-KD101 at ~ 35.2 µg with a total radioactivity of 6.81 kBq. The mean total recovery of administered radioactivity was 85.2% with predominant excretion in the urine (78.0%). The radio-chromatographic metabolite profiling showed that most of the total radioactivity in the plasma and the urine was ascribable to metabolites. The UDP-glucuronosyltransferase (UGT), including UGT1A1, UGT1A3, and UGT2B7, might have contributed to the interindividual variability in the metabolism and excretion of KD101. The microtracing approach using AMS is a useful tool to evaluate the AME of a drug under development without risk for high radiation exposure to humans.

Nanotracing and cavity-ring down spectroscopy: A new ultrasensitive approach in large molecule drug disposition studies.

New therapeutic biological entities such as bispecific antibodies targeting tissue or specific cell populations form an increasingly important part of the drug development portfolio. However, these biopharmaceutical agents bear the risk of extensive target-mediated drug disposition or atypical pharmacokinetic properties as compared to canonical antibodies. Pharmacokinetics and bio-distribution studies become therefore more and more important during lead optimization. Biologics present, however, greater analytical challenges than small molecule drugs due to the mass and selectivity limitation of mass spectrometry and ligand-binding assay, respectively. Radiocarbon (14C) and its detection methods, such as the emerging 14C cavity ring down spectroscopy (CRDS), thus can play an important role in the large molecule quantitation where a 14C-tag is covalently bound through a stable linker. CRDS has the advantage of a simplified sample preparation and introduction system as compared to accelerator mass spectrometry (AMS) and can be accommodated within an ordinary research laboratory. In this study, we report on the labeling of an anti-IL17 IgG1 model antibody with 14C propionate tag and its detection by CRDS using it as nanotracer (2.1 nCi or 77.7 Bq blended with the therapeutic dose) in a pharmacokinetics study in a preclinical species. We compare these data to data generated by AMS in parallel processed samples. The derived concentration time profiles for anti-IL17 by CRDS were in concordance with the ones derived by AMS and γ-counting of an 125I-labeled anti-IL17 radiotracer and were well described by a 2-compartment population pharmacokinetic model. In addition, antibody tissue distribution coefficients for anti-IL17 were determined by CRDS, which proved to be a direct and sensitive measurement of the extravascular tissue concentration of the antibody when tissue perfusion was applied. Thus, this proof-of-concept study demonstrates that trace 14C-radiolabels and CRDS are an ultrasensitive approach in (pre)clinical pharmacokinetics and bio-distribution studies of new therapeutic entities.

Prediction of pharmacokinetics and drug-drug interaction potential using physiologically based pharmacokinetic (PBPK) modeling approach: A case study of caffeine and ciprofloxacin.

Over the last decade, physiologically based pharmacokinetics (PBPK) application has been extended significantly not only to predicting preclinical/human PK but also to evaluating the drug-drug interaction (DDI) liability at the drug discovery or development stage. Herein, we describe a case study to illustrate the use of PBPK approach in predicting human PK as well as DDI using in silico, in vivo and in vitro derived parameters. This case was composed of five steps such as: simulation, verification, understanding of parameter sensitivity, optimization of the parameter and final evaluation. Caffeine and ciprofloxacin were used as tool compounds to demonstrate the "fit for purpose" application of PBPK modeling and simulation for this study. Compared to caffeine, the PBPK modeling for ciprofloxacin was challenging due to several factors including solubility, permeability, clearance and tissue distribution etc. Therefore, intensive parameter sensitivity analysis (PSA) was conducted to optimize the PBPK model for ciprofloxacin. Overall, the increase in Cmax of caffeine by ciprofloxacin was not significant. However, the increase in AUC was observed and was proportional to the administered dose of ciprofloxacin. The predicted DDI and PK results were comparable to observed clinical data published in the literatures. This approach would be helpful in identifying potential key factors that could lead to significant impact on PBPK modeling and simulation for challenging compounds.

9-[(E)-2-(4-Chloro-phen-yl)ethen-yl]-3,3,6,6-tetra-methyl-2,3,4,5,6,7,8,9-octa-hydro-1H-xanthene-1,8-dione.

In the title compound, C25H27ClO3, each of the cyclo-hexenone rings adopts an envelope conformation, whereas the six-membered pyran ring adopts a flattened boat conformation, with the O and methine C atoms deviating from the plane of the other four atoms. The C=C double bond is in the trans conformation. In the crystal, weak C-H⋯O hydrogen bonds link the mol-ecules into chains running parallel to the b axis.

(E)-N-(3,3-Di-phenyl-allyl-idene)-2-(tri-fluoro-meth-yl)aniline.

In the title compound, C22H16F3N, the C=N bond of the central imine group adopts an E conformation. The dihedral angles between the 2-(tri-fluoro-meth-yl)phenyl ring and the benzene rings are 9.34 (1) and 68.8 (1)°. The imine group displays a C-C-N=C torsion angle of 41.6 (3)°. In the crystal, weak C-H⋯F hydrogen bonds link the mol-ecules into chains parallel to the b-axis direction.

(E)-N-(3,3-Diphenyl-allyl-idene)-4-nitro-aniline.

In the title compound, C(21)H(16)N(2)O(2), the dihedral angles between the mean planes of the 4-nitro-phenyl ring and the two phenyl rings are 57.3 (5) and 16.8 (6)°. The imine group displays a C-C-N-C torsion angle of -24.9 (3)°.

Influence of electrical contacts on the 1/f noise in individual multi-walled carbon nanotubes.

Low-frequency noise of individual MWNTs was investigated with different metal electrodes. Irrespective of the ohmic or non-ohmic voltage-current characteristics, a strong dependence of the noise on the metal contacts was observed. The good electrical contacts with Pd or Pt electrodes enable a quasi-ballistic transport with a bigger exponent in the relation of S1 alpha R4.8, similar to 2D metallic film or graphene. On the other hand, the noise in the case of Cr or Ti electrodes is almost linear with the resistance of the nanotubes, which may be an index of the influence of the contacts in the CNTs.

Analytic model for low-frequency noise in nanorod devices.

In this work analytic model for generation of excess low-frequency noise in nanorod devices such as field-effect transistors are developed. In back-gate field-effect transistors where most of the surface area of the nanorod is exposed to the ambient, the surface states could be the major noise source via random walk of electrons for the low-frequency or 1/f noise. In dual gate transistors, the interface states and oxide traps can compete with each other as the main noise source via random walk and tunneling, respectively.

Effect of rapid thermal annealing on the electrical properties of GaAs Schottky diodes embedded with self-assembled InAs quantum dots.

After rapid thermal annealing (RTA), deep levels were found to be generated in Au/GaAs Schottky diodes embedded with InAs quantum dots grown by migration enhanced molecular beam epitaxy (MEMBE). From the corner frequency of the 1/f2 part of the low-frequency noise specrtral density, the locations of the deep levels were estimated to be 0.58, 0.61, and 0.66 eV below the conduction band edge for the samples without quantum dot layer, with quantum dot layer and capping layer thickness of 0.8 microm, and with quantum dot layer and the capping layer thickness of 0.4 microm, respectively. RTA also lowered the Schottky barrier height.