The antiviral effects of baloxavir marboxil against influenza A virus infection in ferrets.

Background: Baloxavir marboxil (BXM), the oral prodrug of baloxavir acid (BXA), greatly reduces virus titers as well as influenza symptoms of uncomplicated influenza in patients.

Objectives: To investigate the pharmacokinetic profiles of BXA and its efficacy against influenza A virus infection in ferrets.

Methods: Ferrets were dosed orally with BXM (10 and 30 mg/kg twice daily for 1 day), oseltamivir phosphate (OSP) (5 mg/kg twice daily for 2 days) or vehicle to measure the antiviral effects of BXM and OSP.

The 10th Japan Bioanalysis Forum Symposium conference: a report.

The Japan Bioanalysis Forum Symposium was held on 12-14 February 2019 (Yokohama, Japan), in celebration of its 10th anniversary, and over 370 participants from pharmaceutical industries, contractors, academia and regulatory authorities from home and abroad came together in Yokohama. The 3-day symposium particularly aimed to foster collaboration with the scientists surrounding bioanalysts, according to the theme 'Open to the Public.' The symposium also included a broad range of pioneering programs, such as lectures by speakers from DMPK/metabolomics fields, discussions of future bioanalysis and poster presentations by publicly offered presenters as well as the regular ones we had organized.

The ninth Japan Bioanalysis Forum symposium.

The ninth Japan Bioanalysis Forum symposium took place at tower hall Funabori, Tokyo, Japan, between 6 and 8 February, 2018. Bioanalytical scientists from the pharmaceutical industry, CROs, academia and regulatory bodies had many meaningful and relevant discussions on current topics of interest in bioanalysis. The 3-day symposium featured updated perspectives and experiences on regulated bioanalysis of small and large molecules, biomarker measurement and assessment of immunogenicity, as well as new areas of bioanalytical validation such as quantitative polymerase chain reaction(qPCR) and flow cytometry.

Proposal for risk-based scientific approach on full and partial validation for general changes in bioanalytical method.

The guidance and several guidelines on bioanalytical method validation, which were issued by the US FDA, EMA and Ministry of Health, Labour and Welfare, list the 'full' validation parameters; however, none of these provide any details for 'partial' validation. Japan Bioanalysis Forum approved a total of three annual discussion groups from 2012 to 2014. In the discussion groups, members from pharmaceutical companies and contract research organizations discussed the details of partial validation from a risk assessment viewpoint based on surveys focusing on bioanalysis of small molecules using LC-MS/MS in Japan.

Bioanalytical Method for the Determination of Hydroxyproline in Mouse Kidney by High-Performance Liquid Chromatography with Tandem Mass Spectrometric Detection.

An analytical method was developed and validated for the measurement of hydroxyproline (Hyp) levels in mouse kidney by high-performance liquid chromatography with tandem mass spectrometric detection (LC/MS/MS) using an analytical column specially designed for the LC/MS/MS analysis for intact amino acids. Tissue hydrolyzed with hydrochloric acid could be directly injected into the LC/MS/MS, as well as separated and detected using the deuterium labelled Hyp as an internal standard. The calibration curve showed good linearity from 5 to 500 nmol/mg of tissue; the precision and accuracy, including within- and between-run, were less than 6% and within 100 ± 6%, respectively.

Surface Plasmon Resonance Assay of Binding Properties of Antisense Oligonucleotides to Serum Albumins and Lipoproteins.

In the present study, we developed an assay to evaluate the kinetic binding properties of the unconjugated antisense oligonucleotide (ASO) and lipophilic and hydrophilic ligands conjugated ASOs to mouse and human serum albumin, and lipoproteins using surface plasmon resonance (SPR). The lipophilic ligands conjugated ASOs showed clear affinity to the albumins and lipoproteins, while the unconjugated and hydrophilic ligand conjugated ASOs showed no interaction. The SPR method showed reproducible immobilization of albumins and lipoproteins as ligands on the sensor chip, and reproducible affinity kinetic parameters of interaction of ASOs conjugated with the ligands could be obtained.

[Discussion on Partial Validation in Small Molecule Regulated Bioanalysis: Change in Analytical Instruments].

In recent years, the necessity of a bioanalytical method validation has been discussed and guidance/guidelines have been released from regulatory agencies. However, none of these provides any details for partial validation (PV) in case of a partial change in the validated analytical method. Therefore eleven scientists have launched a discussion group (DG) with the approval of Japan Bioanalysis Forum (JBF), and have been discussing PV for chromatographic methods based on survey results of Japanese bioanalysts.

Surface plasmon resonance assay of inhibition by pharmaceuticals for thyroxine hormone binging to transport proteins.

We developed a surface plasmon resonance (SPR) assay to estimate the competitive inhibition by pharmaceuticals for thyroxine (T4) binding to thyroid hormone transport proteins, transthyretin (TTR) and thyroxine binding globulin (TBG). In this SPR assay, the competitive inhibition of pharmaceuticals for introducing T4 into immobilized TTR or TBG on the sensor chip can be estimated using a running buffer containing pharmaceuticals. The SPR assay showed reproducible immobilization of TTR and TBG, and the kinetic binding parameters of T4 to TTR or TBG were estimated.

Binding properties of antimicrobial agents to lipid membranes using surface plasmon resonance.

In the present study, we examined the interaction of antimicrobial agents with four model lipid membranes that mimicked mammalian cell membranes and Gram-positive and -negative bacterial membranes and analyzed the binding kinetics using our surface plasmon resonance (SPR) technique. The selective and specific binding characteristics of antimicrobial agents to the lipid membranes were estimated, and the kinetic parameters were analyzed by application of a two-state reaction model. Reproducible analysis of binding kinetics was observed.

The relationship between in vivo antiviral activity and pharmacokinetic parameters of peramivir in influenza virus infection model in mice.

The purpose of this study was to investigate the relationship between pharmacokinetic (PK) parameters of intravenous (IV) peramivir and in vivo antiviral activity pharmacodynamic (PD) outcomes in a mouse model of influenza virus infection. Peramivir was administrated to mice in three dosing schedules; once, twice and four times after infection of A/WS/33 (H1N1). The survival rate at day 14 after virus infection was employed as the antiviral activity outcome for analysis.

Binding properties of antimicrobial agents to dipeptide terminal of lipid II using surface plasmon resonance.

We developed a surface plasmon resonance (SPR) assay to estimate the interactions of antimicrobial agents with the dipeptide terminal of lipid II (D-alanyl-D-alanine) and its analogous dipeptides (L-alanyl-L-alanine and D-alanyl-D-lactate) as ligands. The established SPR method showed the reproducible immobilization of ligands on sensor chip and analysis of binding kinetics of antimicrobial agents to ligands. The ligand-immobilized chip could be used repeatedly for at least 200 times for the binding assay of antimicrobial agents, indicating that the ligand-immobilized chip is sufficiently robust for the analysis of binding kinetics.

Lipid membrane-binding properties of amphotericin B deoxycholate (Fungizone) using surface plasmon resonance.

In the present study, we developed an assay of interactions of amphotericin B (AmB) deoxycholate, Fungizone, with two model lipid membranes that mimicked mammal cell membranes and fungal membranes. We also analyzed the binding kinetics of the mammal cell membranes and fungal membranes using surface plasmon resonance (SPR). Fungizone showed a higher affinity for the model fungal membrane than that of the model mammal cell membrane, and the binding selectivity of Fungizone in the temperature dependence could be represented by this SPR system.

High-throughput and sensitive assay for amphotericin B interaction with lipid membrane on the model membrane systems by surface plasmon resonance.

In the present study, we developed a high-throughput and sensitive assay for interactions of amphotericin B (AmB) with two model lipid membranes, which mimicked mammal cell membrane and fungal membrane using surface plasmon resonance (SPR). The binding kinetics of AmB to the membrane could be analyzed by multiple sensorgrams obtained at different AmB concentrations, indicating that the binding properties could be clarified for an approximately 7-fold concentration range. AmB showed an approximately 18-fold higher affinity for ergosterol-containing membrane than for cholesterol-containing membrane.

Lipid membrane-binding properties of daptomycin using surface plasmon resonance.

In the present study, we developed an assay of interactions of daptomycin with four model lipid membranes that mimicked mammal cell membranes and gram-positive and negative bacteria membranes. We also analyzed the binding kinetics of the gram-positive bacteria membranes using surface plasmon resonance (SPR). Daptomycin showed a higher affinity for the model gram-positive bacteria membrane than those of the model mammal cell and gram-negative bacteria membranes, and the binding selectivity of daptomycin in the presence of calcium could be represented by this SPR system.

Strongly altered receptor binding properties in PP and NPY chimeras are accompanied by changes in structure and membrane binding.

Neuropeptide Y (NPY) and the pancreatic polypeptide (PP) are members of the neuropeptide Y family of hormones. They bind to the Y receptors with very different affinities: Whereas PP is highly selective for the Y(4) receptor, NPY displays highest affinites for Y(1), Y(2), and Y(5) receptor subtypes. Introducing the NPY segment 19-23 into PP leads to an increase in affinity at the Y(1) and Y(2) receptor subtypes whereas the exchange of this segment from PP into NPY leads to a large decrease in affinity at all receptor subtypes.

Evaluation of the membrane-binding properties of the proximal region of the angiotensin II receptor (AT1A) carboxyl terminus by surface plasmon resonance.

The proximal region of the angiotensin II receptor (AT1A) carboxyl-terminus (known as helix VIII) is important for receptor function. In this study, we used surface plasmon resonance (SPR) to examine the interaction of helix VIII-derived peptides with three model lipid membranes. The membrane-binding properties of these synthetic peptides, as well as a series of peptide analogues with modified amino acid sequences, could be explained by both amino acid sequence and kinetic binding data by SPR.

Studies on the membrane interactions of the cyclotides kalata B1 and kalata B6 on model membrane systems by surface plasmon resonance.

In this study we have demonstrated the interactions of kalata B1 and its naturally occurring analogue kalata B6 with five model lipid membranes and have analyzed the binding kinetics using surface plasmon resonance. Two kalata peptides showed a higher affinity for the phosphatidylethanolamine-containing membranes, indicating that the peptides would bind selectively to bacterial membranes. Also we have optimized the procedure for the immobilization of five liposome mixtures and have shown that the procedure provides reproducible levels of immobilized liposomes and could be used to screen the selective binding of putative antimicrobial peptides to model mammalian or microbial phospholipid membranes.

Lipid membrane-binding properties of tryptophan analogues of linear amphipathic beta-sheet cationic antimicrobial peptides using surface plasmon resonance.

Using a surface plasmon resonance (SPR) system, we investigated the lipid membrane-binding properties of four analogues of the 18-residue linear amphipathic beta-sheet cationic antimicrobial peptide (KIGAKI)3-NH2, each of which contains a single isoleucine-to-tryptophan substitution. The results of the SPR study revealed significant differences in the binding characteristics of the peptides depending upon the position of tryptophan residues. These peptides showed higher binding affinity to membranes containing acidic phospholipids than zwitterionic phospholipids.

Assay of collagenase activity for native triple-helical collagen using capillary gel electrophoresis with laser-induced fluorescence detection.

Three collagenase assays for two native triple-helical collagens have been developed using capillary gel electrophoresis (CGE) with laser-induced fluorescence (LIF) detection in order to discover the matrix metalloproteinases (MMPs) inhibitors. These collagenase assays include measurement of the activities of interstitial collagenase (MMP-1) and neutrophil collagenase (MMP-8) against type I collagen, and collagenase-3 (MMP-13) against type II collagen, and the enzyme activities could be readily measured by determining the 3/4 fragments produced from the cleavage of the native collagens. The highly desired sensitivity of the assays could be achieved, employing a dynamic fluorescence labeling technique with the running buffer containing 0.

Enzyme assay for protein kinase using micellar electrokinetic chromatography with laser-induced fluorescence detection.

Micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection has been developed for a protein kinase assay. This protein kinase assay could readily determine the phosphorylation activity of substrate peptide kemptide using cAMP-dependent protein kinase (PKA) as a model enzyme. Kemptide and phosphorylated kemptide could be reacted with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescence derivatization reagent for LIF detection by directly adding NBD-F into the PKA enzymatic reaction mixture.

Synthesis of acylglucuronides of drugs using immobilized dog liver microsomes octadecylsilica particles coated with phospholipid.

Immobilized dog liver microsome octadecylsilica (ODS) particles coated with phospholipid were developed for the synthesis of acylglucuronides of drugs. The phospholipid-coated ODS particles were readily prepared by stirring a solution containing L-alpha-dipalmitoylphosphatidylcholine with the ODS particles, in which the phospholipid was absorbed on the ODS surfaces by hydrophobic interaction between the acyl group of phospholipid and the otcadecyl group of the ODS particles. Similarly, the microsome-immobilized particles were readily prepared by stirring a buffer solution containing dog liver microsomes with the phospholipid-coated ODS particles, in which the microsomes were immobilized on the phospholipid-coated ODS particles by hydrophobic binding.

Evaluation and application of liquid chromatographic columns coated with 'intelligent' ligands. II. Phospholipid column.

The stationary phases of octadecylsilica (ODS) coated with phospholipid have been developed as a model of artificial lipid membranes for liquid chromatographic columns. An ODS column coated with phospholipid can be readily prepared by recycling a solution containing L-alpha-dipalmitoyl-phosphatidylcholine (DPPC) through an ODS column in a closed loop. DPPC becomes absorbed on the ODS surfaces by hydrophobic interaction between the acyl group of DPPC and the octadecyl group of the ODS surfaces.