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 pharmacokinetic parameters of BXA was determined after single oral dosing of BXM. RESULTS: The maximum plasma concentrations of BXA were observed at 1.50 and 2.00 hours with the two BXM doses, which then declined with an elimination half-life of 6.91 and 4.44 hours, respectively. BXM at both doses remained detectable in the plasma in ferrets, which may be due to higher stability in liver microsomes. BXM (10 and 30 mg/kg twice daily) treatment at Day 1 post-infection (p.i.) reduced virus titers by ≥3 log10 of the 50% tissue culture infective doses by Day 2, which was significantly different compared with vehicle or OSP. Body temperature drops over time were significantly greater with BXM than with vehicle or OSP. Significant reduction in virus titers was also demonstrated when BXM was administrated after symptom onset at Day 2 p.i. compared with vehicle and OSP, although body temperature changes largely overlapped between Day 2 and Day 4. CONCLUSIONS: The results highlight the rapid antiviral action of BXM with post-exposure prophylaxis or therapeutic dosing in ferrets and offer support for further research on prevention of influenza virus infection and transmission.

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. This report summarizes the major topics as a conference report.

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. There were over 260 participants from six countries, with 23 oral and 11 poster presentations, including the outcomes of Japan Bioanalysis Forum discussion groups. This report summarizes the major discussion topics from the conference.

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. This manuscript presents perspectives and recommendations for most conceivable changes that can be made to full and partial validations by members of the discussion groups based on their experiences and discussions at the Japan Bioanalysis Forum Symposium.

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 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. The equilibrium dissociation constants of TTR or TBG measured by SPR did not clearly differ from data reported for other binding assays. To estimate the competitive inhibition of tetraiodothyroacetic acid, diclofenac, genistein, ibuprofen, carbamazepine, and furosemide, reported to be competitive or noncompetitive pharmaceuticals for T4 binding to TTR or TBG, their 50% inhibition concentrations (IC50) (or 80% inhibition concentration, IC80) were calculated from the change of T4 responses in sensorgrams obtained with various concentrations of the pharmaceuticals. Our SPR method should be a useful tool for predicting the potential of thyroid toxicity of pharmaceuticals by evaluating the competitive inhibition of T4 binding to thyroid hormone binding proteins, TTR and TBG.

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. The kinetic binding data of these ASOs to albumin and lipoproteins by SPR were related with the distributions in the whole liver in mice after administration of these conjugated ASOs. The results demonstrated that our SPR method could be a valuable tool for predicting the mechanism of the properties of delivery of conjugated ASOs to the organs.

[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. This document reports the results of discussion on PV for a change of analytical instruments such as: 1) full system (limited to same manufacturer and model); 2) pump; 3) autosampler; and 4) mass spectrometer. The DG members agreed on an outline that validation items required for PV are as follows: calibration curve and reproducibility in case 1); calibration curve, reproducibility, and selectivity in case 2); calibration curve, reproducibility, and carryover in case 3); and nearly full validation items without recovery, dilution integrity, and stability in case 4), in consideration of instrument specification and characteristics of each analytical method. Note that this report does not represent a consensus of all the members of JBF, but is a recommendation from the DG members at this stage. Thus further thought is recommended for future discussions.

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. Vancomyicn, teicoplanin, erythromycin, and linezolid showed little interaction with the four lipid membranes in the SPR system. On the other hand, vancomycin analogues showed interaction with the model lipid membranes in the SPR system. The selective and specific binding characteristics of vancomycin analogues to the lipid membranes are discussed based on data for in vitro antibacterial activities and our data on the binding affinity of the D-alanyl-D-alanine terminus of a pentapeptide cell wall obtained by SPR. The mechanism of antibacterial activity against Staphylococcus aureus and vancomycin-resistant enterococci could be evaluated using the binding affinity obtained with our SPR techniques. The results indicate that the SPR method could be widely applied to predict binding characteristics, such as selectivity and specificity, of many antimicrobial agents to lipid membranes.

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. The relationship between day 14 survival and PK parameters, including area under the concentration-time curve (AUC), maximum concentration (Cmax) and time that drug concentration exceeds IC95 (T(>IC95)), was estimated using a logistic regression model, and model fitness was evaluated by calculation of the Akaike information criterion (AIC) index. The AIC indices of AUC, Cmax and T(>IC95) were about 114, 151 and 124, respectively. The AIC of AUC and T(>IC95) were smaller than that of Cmax. Therefore, both AUC and T(>IC95) were the PK parameters that correlated best with the antiviral activity of peramivir IV against influenza virus infection in mice.

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. In this SPR system, the selective and specific binding characteristics of vancomycin and its analogs to the ligands were estimated and the kinetic parameters were calculated. The kinetic parameters revealed that one of the remarkable binding characteristics was the specific interaction of vancomycin to only the D-alanyl-D-alanine ligand. In addition, the kinetic binding data of SPR showed close correlation with the antimicrobial activity. The SPR data of other antimicrobial agents (e.g., teicoplanin) to the ligands showed correlation with the antimicrobial activity on the basis of the therapeutic mechanism. Our SPR method could be a valuable tool for predicting the binding characteristics of antimicrobial agents to the dipeptide terminal of lipid II.

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. This method also showed reproducible immobilization of model liposome membranes on the sensor chip under two temperature conditions (25 and 37°C). The binding characteristics of the medicinal additive of Fungizone, sodium deoxycholate, were clarified on the assay of interactions of the drug product to the lipid membrane, and it was confirmed that the sodium deoxycholate did not affect the estimation of membrane selectivity of Fungizone. Furthermore, the affinities of AmB and Fungizone were discussed by comparing these affinity constants. The results demonstrate that this newly established SPR method could be applied to estimate the lipid-membrane interaction of Fungizone as a drug product of AmB.

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. We also optimized the procedure for the reproducible immobilization of liposome containing the sterols and the estimation of binding kinetics of AmB to the lipid membranes, and the sensitivity of AmB to membrane interaction was 20-fold higher, compared with the reported method. The throughput of the established method for the binding kinetics characterization was calculated to be 10 compounds a day. The results demonstrate that the established SPR method could be a valuable tool for predicting selective binding to sterol-containing membranes.

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. This method also showed reproducible immobilization of model liposome membranes on the sensor chip, and had a desirable repeatability in the analysis of the binding kinetics to the model gram-positive bacteria membranes. The results demonstrate that this newly established SPR method could be a valuable tool for predicting the binding characteristics of antimicrobial agents to lipid membranes.

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. PP displays a very stable structure in solution, with the N terminus being back-folded onto the C-terminal alpha-helix (the so-called PP-fold). The helix of NPY is less stable and the N terminus is freely diffusing in solution. The exchange of this segment, however, does not alter the PP-fold propensities of the chimeric peptides in solution. The structures of the phospholipid micelle-bound peptides serving to mimic the membrane-bound species display segregation into a more flexible N-terminal region and a well-defined alpha-helical region. The introduction of the [19-23]-pNPY segment into hPP leads to an N-terminal extension of the alpha-helix, now starting at Pro(14) instead of Met(17). In contrast, a truncated helix is observed in [(19)(-)(23)hPP]-pNPY, starting at Leu(17) instead of Ala(14). All peptides display moderate binding affinities to neutral membranes (K(assoc) in the range of 1.7 to 6.8 x 10(4) mol(-)(1) as determined by surface plasmon resonance) with the differences in binding being most probably related to the exchange of Arg-19 (pNPY) by Glu-23 (hPP). Differences in receptor binding properties between the chimeras and their parental peptides are therefore most likely due to changes in the conformation of the micelle-bound peptides.

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. The helix VIII peptides showed a higher affinity for lipid membranes that contained negatively charged phospholipid, rather than zwitterionic phospholipid. The findings of an SPR study may be useful for estimating the cooperative binding of intracellular receptor domains with G proteins and the components of the lipid bilayer.

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. The addition of dimethylsulfoxide to the running buffer was effective in maintaining the solubility of these peptide solutions and obtaining concentration-dependent sensorgrams for the kinetic analysis in this study. The kinetic binding data of SPR correlated closely with both the ability of the peptides to lyse liposomes with the same phospholipid composition and bactericidal activity. The results demonstrate that SPR may be a valuable tool to predict the membrane lytic properties of antimicrobial peptides.

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.

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 0.05% sodium dodecylsulfate and non-covalent fluorescent dye for protein, NanoOrange. The collagen, its 1/4 and 3/4 fragments of type I or II collagen could be separated and detected within the run time of 20 min by CGE mode using the gel buffer (pH 8.8) containing 4% polyacrylamide. Good linearity of the peak area of the 3/4 fragment was obtained over each assay range of collagenase (15-150 ng/tube for MMP-1, 3-30 ng/tube for MMP-8, and 1.5-30 ng/tube for MMP-13, respectively). The relative standard deviation of the peak areas of the 3/4 fragment produced from type II collagen by MMP-13 cleavage was calculated to be less than 13.4%, indicating that the assay was reproducible. Also, IC50 values of three MMPs inhibitors, which were calculated for estimation by the variation of the peak areas of the 3/4 fragments using 90 ng/tube for MMP-1, 30 ng/tube for MMP-8 or 15 ng/tube for MMP-13, were almost consistent with data from other assays. The CGE-LIF method is expected to be very useful for proteinase assay and its application to the estimation of inhibitors because this method enables an assay of collagenase activity using native substrate to be conducted without experimentally troublesome procedure such as preparation of antibody or fluorescence-labeled substrate.

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. These derivatives of substrate and product were separated and detected within the analysis time of 5 min by micellar electrokinetic mode using a mixture of sodium dodecylsulfate and methanol as a running buffer. Good linearity of the peak response of the phosphorylated kemptide was obtained over the range of 1-20 mU/tube of PKA in the assay. The relative standard deviation of the peak areas of the phosphorylated kemptide using 2, 5 and 10 mU/tube of PKA were calculated to <10.4%, indicating that the assay was reproducible. Also, IC(50) values of six PKA inhibitors, the K(i) value and the inhibition pattern of one inhibitor, which were calculated to estimate by the variation of the peak area of the phosphorylated kemptide using 5 mU/tube of PKA, were consistent with the published data. The sensitivity of the assay was higher than that of enzyme-linked immunosorbent assay (ELISA) for PKA phosphorylation activity, as IC(50) values, K(i) value, and the inhibition mechanism of inhibitors could be estimated using one-tenth amounts of PKA, compared with that of ELISA. The MEKC-LIF is expected to be very useful for protein kinase assay and its application to the estimation of inhibitors because this method does not entail experimentally troublesome procedures such as the preparation of antibody or fluorescence-labeled substrate.