Impact of microsampling on toxicological evaluation in rodent safety studies.

Recently, animal welfare has been attracting worldwide attention, and implementation of 3Rs (replacement, reduction, and refinement) is prioritized in every way possible in the drug development. Microsampling, in which small amounts of blood are collected, is attracting attention in this context. ICH S3A Q&A focused on microsampling was published in November 2017 to help accelerate the application of microsampling for toxicokinetic assessment. The increased sensitivity of drug measurement apparatuses such as mass spectrometers has made it possible to measure drug concentrations with small amounts of blood samples. In this review, we summarized the reports on toxicological influence of microsampling in rodents (rats and mice) with or without drug administration or recovery period after blood collection and influences that may arise from differences in the blood sampling site or blood sampling volume. We also summarized some perspectives on further implementation of microsampling in toxicology studies. The use of microsampling in regulatory toxicology studies has gradually increased, although at a lower rate than in discovery studies. Since more animals are used in GLP toxicology studies than in discovery studies, the effect of reducing the number of animals by microsampling is expected to be greater in the toxicology studies. This report aims to promote the application of microsampling to nonclinical studies, as it is beneficial for improving animal welfare and can contribute to the 3Rs.

Strain-induced crystallization and phase separation used for fabricating a tough and stiff slide-ring solid polymer electrolyte.

The demand for mechanically robust polymer-based electrolytes is increasing for applications to wearable devices. Young's modulus and breaking energy are essential parameters for describing the mechanical reliability of electrolytes. The former plays a vital role in suppressing the short circuit during charge-discharge, while the latter indicates crack propagation resistance. However, polymer electrolytes with high Young's moduli are generally brittle. In this study, a tough slide-ring solid polymer electrolyte (SR-SPE) breaking through this trade-off between stiffness and toughness is designed on the basis of strain-induced crystallization (SIC) and phase separation. SIC makes the material highly tough (breaking energy, 80 to 100 megajoules per cubic meter). Phase separation in the polymer enhanced stiffness (Young's modulus, 10 to 70 megapascals). The combined effect of phase separation and SIC made SR-SPE tough and stiff, while these mechanisms do not impair ionic conductivity. This SIC strategy could be combined with other toughening mechanisms to design tough polymer gel materials.

The influence of serial 50 µL microsampling on rats administered azathioprine, the immunosuppressive drug.

According to the ICH S3A Q&A, microsampling is applicable to pharmaceutical drugs and toxicological analysis. Few studies have reported the effect of microsampling on the toxicity of immunotoxicological drugs. The aim of this multicenter study was to evaluate the toxicological effects of serial microsampling on rats treated with azathioprine as a model drug with immunotoxic effects. Fifty microliters of blood were collected from the jugular vein of Sprague-Dawley rats at six time points from day 1 to 2 and 7 time points from day 27 to 28. The study was performed at three organizations independently. The microsampling effect on clinical signs, body weights, food consumption, hematological parameters, biochemical parameters, urinary parameters, organ weights, and tissue pathology was evaluated. Azathioprine-induced changes were observed in certain hematological and biochemical parameters and thymus weight and pathology. Microsampling produced minimal or no effects on almost all parameters; however, at 2 organizations, azathioprine-induced changes were apparently masked for two leukocytic, one coagulation, and two biochemical parameters. In conclusion, azathioprine toxicity could be assessed appropriately as overall profiles even with blood microsampling. However, microsampling may influence azathioprine-induced changes in certain parameters, especially leukocytic parameters, and its usage should be carefully considered.

Effect of microsampling (50 µL) on toxicological evaluation of methapyrilene, a hepatotoxic substance, in a collaborative 28-day study in female rats.

Although microsampling of blood is recommended to promote the 3Rs in toxicokinetic (TK) evaluation, there are few reports applying microsampling in actual toxicity evaluation. Here, we assessed the effects of microsampling on toxicological evaluation of methapyrilene hydrochloride, a hepatotoxic substance. Female SD rats received methapyrilene hydrochloride orally at dose levels of 0 (vehicle), 10, and 30 mg/kg BW, once daily for 4 weeks. Each dose level included a microsampling group and a non-microsampling group (n = 5). In the microsampling groups, blood sampling (50 µL/time point) was performed at 6 time points on day 1 of administration and 7 time points on day 27-28; all the animals underwent necropsy on day 29. Toxicity studies and TK analysis were performed, and through these studies in 2 organizations, cross-organization validation of the effect on toxicity evaluation was conducted. In one organization, microsampling obscured changes in some parameters in hematology due to the administration of methapyrilene hydrochloride. In the other organization, although the relationship between the developing pattern of histopathological findings in the liver and the blood sampling was suspected, it was associated with poor reproducibility; this was considered as a change within a variation range of biological reactions. Each of these phenomena was observed in only one organization without consistency. In both organizations, no effect of blood microsampling was observed in other endpoints. In conclusion, microsampling is considered to be a technique applicable to safety studies of drugs showing hepatotoxicity, as it did not show a marked influence on the toxicological evaluation of methapyrilene hydrochloride.

High-yield one-pot synthesis of polyrotaxanes with tunable well-defined threading ratios over a wide range.

In this work, we report a high-yield one-pot synthesis of polyrotaxane (PR), composed of (2-hydroxypropyl)-α-cyclodextrin (hpCD) and polyethylene glycol (PEG), with well-defined hpCD threading ratios controllable across a wide range from 0.64% to 10%. In hpCD/PEG aqueous solutions, hpCDs are well dispersed and threaded spontaneously into hpCDs to form a pseudo-PR (pPR) structure. The homogeneous dispersion of hpCDs results in a well-defined threading ratio of hpCDs on PEG, which is suggested by the fact that the dispersity of the molecular weight distribution of PR is almost the same as that of pure PEG. The well-defined hpCD threading ratio of the PRs can be controlled over a wide range by tuning the hpCD concentration in the pPR solutions.

Acyl-CoA:diacylglycerol acyltransferase 1 inhibition in the small intestine increases plasma transaminase activity via the activation of protein kinase C pathway.

Acyl-CoAdiacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in the fat absorption step in enterocytes. We previously reported that the pharmacological inhibition of DGAT1 increased plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity in corn oil-loaded rats without any sign of hepatotoxicity. In this study, we investigated this mechanism. We found that this elevation occurred only during the pharmacologically active period of a DGAT1 inhibitor and the magnitude did not depend on the volume of corn oil. In addition, this elevation was not accompanied by increases in ALT or AST mRNA levels in the small intestine and liver. To clarify a lipid component responsible for this elevation, rats were treated with free fatty acids instead of corn oil and no plasma ALT elevation was observed. Next, rats were pretreated with inhibitors of monoacylglycerol acyltransferase 2 and intestinal microsomal triglyceride transfer protein instead of the DGAT1 inhibitor, but no plasma ALT elevation was observed after corn oil loading. Since the results suggested a possible role of diacylglycerol (DAG), which activates protein kinase C (PKC), we measured PKC activity in the small intestine and found that the activity was increased by treatment with the DGAT1 inhibitor and corn oil. Moreover, rats pretreated with a PKC inhibitor in combination with the DGAT1 inhibitor showed suppression of plasma ALT elevation. Taken together, the present results suggest that DAG accumulation induced by pharmacological DGAT1 inhibition and resultant PKC activation in enterocytes are involved in the increase in plasma ALT and AST activity in rats.

Polymer Brush Formation Assisted by the Hierarchical Self-Assembly of Topological Supramolecules.

The development of methods for the polymer brush layer formation on material surfaces to improve the surface properties has been researched for decades. Here, we report a novel approach for the formation of a polymer brush layer on materials and the alteration of the surface properties using a pseudo-polyrotaxane nanosheet (PPRNS). In the PPRNS, ß-cyclodextrin (CD) selectively covered the central poly(propylene oxide)29 segment of the carboxyl-terminated poly(ethylene oxide)75-b-poly(propylene oxide)29-b-poly(ethylene oxide)75 (COOH-EO75PO29EO75) triblock copolymer to form columnar crystals. The EO chains of COOH-EO75PO29EO75 then adopt polymer brush conformations and exhibit an oil-repellent property on the material surfaces. Based on the flexibility derived from the nanosheet structure, the PPRNS showed high adhesion to the Blu-ray disk substrate (1D bending), polystyrene spherical beads (2D bending), and random rough surface of pork skin. The PPRNS is expected to become a new method for obtaining polymer brush layers and improving the surface properties irrespective of the material type.

Tough hydrogels with rapid self-reinforcement.

Most tough hydrogels are reinforced by introducing sacrificial structures that can dissipate input energy. However, because the sacrificial damage cannot rapidly recover, the toughness of these gels drops substantially during consecutive cyclic loadings. We propose a damageless reinforcement strategy for hydrogels using strain-induced crystallization. For slide-ring gels in which polyethylene glycol chains are highly oriented and mutually exposed under large deformation, crystallinity forms and melts with elongation and retraction, resulting both in almost 100% rapid recovery of extension energy and excellent toughness of 6.6 to 22 megajoules per cubic meter, which is one order of magnitude larger than the toughness of covalently cross-linked homogeneous gels of polyethylene glycol.

Molecular Recognition of Fluorescent Probe Molecules with a Pseudopolyrotaxane Nanosheet.

Pseudopolyrotaxane nanosheets (PPRNS) are ultrathin two-dimensional (2D) materials fabricated via supramolecular self-assembly of ß-cyclodextrin (ß-CD) and poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) triblock copolymers. In this study, the molecular loading of various fluorescent probe molecules onto PPRNS was systematically investigated. 1H NMR study for R6G absorption to PPRNS indicated that the small hydrophobic groups, such as the methyl group, of R6G were absorbed by PPRNS. Consistently, the fluorescent probes without methyl groups were not absorbed. These results indicate that PPRNS has a molecular recognition absorption property based on the host-guest interaction of the functional groups on probe molecules and molecular-sized spaces of PPRNS surfaces, which may be vacant ß-CDs and voids between ß-CD columns. The absorbed amount of the molecular probes onto PPRNS was investigated by UV-vis spectra, and the absorption behavior could be described well by the Langmuir absorption isotherm. This is consistent with the suggested model that the probes are absorbed onto the PPRNS surfaces. This study demonstrates that PPRNSs can be applied as adsorbents for toxic compounds, drug delivery systems, and 2D sensors.

Precise control of cyclodextrin-based pseudo-polyrotaxane lamellar structure via axis polymer composition.

Self-assembly of cyclodextrin (CD) with guest polymers has attracted much attention owing to its biocompatibility and accessibility. In this study, we investigate the composition effect of poly(ethylene oxide)m-b-poly(propylene oxide)n-b-poly(ethylene oxide)m (EOmPOnEOm) triblock copolymers on lamellar or plate structures formed by complexation with ß-CD. EO5PO29EO5, EO14PO29EO14, and EO75PO29EO75 show periodic lamellar morphology consisting of single-crystalline pseudo-polyrotaxane (PPR) nanosheets with a thickness equal to the central PO length. This is because ß-CDs selectively cover the PO component and cause the microphase separation between ß-CD and EO layers. The thickness of the EO layers increases linearly with increasing number of EO units, which suggests that the EO chains are constrained into virtual cylinders with the diameter of the ß-CD. This means that we can precisely control the thickness of both the crystal (ß-CD and PO) and the amorphous (EO) layers in the lamellar structure. In contrast, EO2PO29EO2 forms a thin plate structure, where not only PO but also EO chains are covered with ß-CD. Furthermore, the length of the central PO component is necessary to form the lamellar structure with the phase separation between the ß-CD and EO layers. These findings provide a more fundamental understanding to enhance the variety and applicability of CD-based self-assembled materials.

Lack of toxicological influences by microsampling (50 µL) from jugular vein of rats in a collaborative 28-day study.

Due to finalization of the ICH S3A Q&A focusing on microsampling, application of microsampling technique to regular non-clinical animal studies is expected for non-clinical safety assessment of pharmaceuticals. In Europe, microsampling from the tail vein or saphenous vein has often been used, whereas sampling from the jugular vein is thought to be more common for non-clinical studies in Japan. Therefore, we assessed the toxicological effects of serial microsampling from the jugular vein of SD rats in a common 28-day study at 4 independent organizations. Fifty microliter sampling was performed at 6 timepoints on day 1 to 2 and 7 timepoints on day 27 to 28 and its toxicological influences on body weight, food consumption, hematological and clinical chemistry parameters, and organ weights (on day 29 for 3 and day 28 for 1 organizations) were evaluated. The serial microsampling was shown to have no or minimal influences on the assessed parameters. The observed statistical differences for the 18 parameters were sporadic and did not appear to be systemically associated with microsampling. However, the sporadic changes were more often observed in females (14/18 parameters) than in males (6/18), suggesting the possibility that female rats were more susceptible to treatment-based influences. The current results indicate that serial 50 µL sampling from the jugular vein of SD rats had no or very slight toxicological effects, suggesting that this microsampling condition is applicable for toxicokinetic evaluation of non-clinical rat toxicity studies.

Adhesion Force Analysis of Dynamic Polymer Brushes.

Spontaneous surface segregation of amphiphilic diblock copolymers at the water interface from the elastomeric portion was utilized for the fabrication of hydrophilic brushes, named as "dynamic polymer brush". Observation of the dynamic polymer brushes appears only when immersed in water and demands advanced experimental techniques for embedded interfaces such as neutron reflectivity. Measurement of the hydrophobic interaction at the polymer/water interface is not only an alternative method to monitor the brush but also reveals its unique surface properties. We carried out adhesion force measurements using atomic force microscopy with a hydrophobic probe for measuring the hydrophobic interactions of dynamic polymer brushes in water. Dynamic polymer brushes showed reduced hydrophobic interaction, which becomes more significant at higher graft density. Moreover, a unique transitional response to the applied pressure was observed for the dynamic polymer brush: the adhesion force was almost zero at low applied pressure and increased by further increasing the applied pressure. This phenomenon may indicate reallocation or retraction of the block copolymer chains from the contact area by the applied pressure, which are the unique characteristics of nonbound dynamic polymer brush chains. We also conducted adhesion force imaging and proved that dynamic polymer brushes form uniform layers without any defects, irrespective of brush density, which suggests that the interaction between the dynamic polymer brush chains is that of repulsion.

Interfacial Energy Measurement on the Reconstructive Polymer Surface: Dynamic Polymer Brush by Segregation of Amphiphilic Block Copolymers.

Herein, the interfacial energy of a reconstructive polymer surface formed by segregation is analyzed by measuring the change in the size of elastomer thin films floating on water. When a system in which amphiphilic diblock copolymers are mixed with the hydrophobic elastomer is in contact with water, surface reconstruction is triggered by the segregation of copolymers with a gain in the hydration energy of the hydrophilic blocks. The hydrophilic brush layer spontaneously formed at the elastomer-water interface is named the dynamic polymer brush. Although it is anticipated that the interfacial energy will significantly decrease in the dynamic polymer brush system, a direct measurement of the interfacial energy of the reconstructive interface is a challenge. We propose a novel method to measure the interfacial energy of a reconstructive polymer surface by measuring the deformation of elastomer thin films floating on water and apply it to the dynamic polymer brush system. The interfacial energy of the dynamic polymer brush formed by the segregation of amphiphilic diblock copolymers with longer hydrophilic chains drastically decreased to zero due to the high hydration energy of hydrophilic chains. Based on the neutron reflectometry results, the graft density and thickness of the dynamic polymer brush system floating on water were found to be lower than those of the system fixed onto solid substrates. This indicates that the floating system can respond to an external environment with a high degree of freedom (graft density, brush thickness, and interface area).

Molecular Dynamics Simulation and Theoretical Model of Elasticity in Slide-Ring Gels.

In this study, molecular dynamics (MD) simulations were carried out on the uniaxial deformation of slide-ring (SR) networks with slidable cross-links to understand the relationship between the sliding of the cross-linking points and the Young's moduli of SR gels, which are lower than those of covalently cross-linked gels with the same cross-linking densities. The slidability of the cross-links in SR gels was characterized by the rate of change of the segment number between the cross-links, Nslide, estimated by the MD simulation. We have successfully constructed a molecular model for the elasticity of SR gels and proposed a simple equation for the Young's moduli of SR gels as a function of Nslide. The theoretical model was compared with the MD simulation results and experimental data.

Synthesis of Poly(Methyl Methacrylate)-Based Polyrotaxane via Reversible Addition-Fragmentation Chain Transfer Polymerization.

A polyrotaxane (PR) with poly(methyl methacrylate) (PMMA) as the main chain polymer was prepared via reversible addition-fragmentation chain transfer (RAFT) polymerization. Because of the special mechanism of RAFT, the suprastructure of a PMMA-based PR is established by synthesizing inclusion complexes of methyl methacrylate and gamma-cyclodextrin (γCD) into the middle of the poly-N-(3-dimethylamino) propyl methacrylamide segments. The presence of threaded γCD was determined via diffusion ordered spectroscopy from the alignment of the mobility of γCD and the main chain polymer. A PMMA-based PR with 2-20% CD coverage and a molecular weight of 7K-60K g/mol of PMMA-based PR was synthesized with a targeted molecular structure by mediating the RAFT polymerization. The PMMA-based PR prepared in this study is expected to be suitable for wide applications of tough materials with good heat resistance. Moreover, the investigation of this synthetical approach opened possibilities for more variety of PR with controllable properties.

Prolonged Glass Transition due to Topological Constraints in Polyrotaxanes.

Topological constraints in polyrotaxanes significantly affected their glass transition dynamics. The effects of the constraints were systematically studied using a series of different coverage glass-forming polyrotaxanes consisting of a common polymer and threaded ring molecule of varying ratios. Although their ratios were similar and hence exhibited similar Tg values by differential thermal analysis, mechanical relaxation was considerably prolonged with increasing coverage. The relaxation became a two-step process: a faster step at a common temperature near the Tg and another which was prolonged by the coverage increase. Relaxation dynamics analysis revealed that segment motions, which are cooperative translations of different components, freeze at considerably higher temperatures than the Tg with increasing coverage. This suggests that although the rings are released from conventional interactions at the Tg, their cooperative translational motions are significantly constrained by the threading polymers with increasing coverage.

Molecular Dynamics of Polyrotaxane in Solution Investigated by Quasi-Elastic Neutron Scattering and Molecular Dynamics Simulation: Sliding Motion of Rings on Polymer.

In this study, we investigated the molecular dynamics of polyrotaxane (PR), composed of α-cyclodextrins (CDs) and a poly(ethylene glycol) (PEG) axial chain, in solution by means of quasi-elastic neutron scattering (QENS) measurements and full-atomistic molecular dynamics (MD) simulations. From QENS experiments, we estimated the diffusion coefficients of CD and PEG monomers in PR, which are in quantitative agreement with those obtained by MD simulations. By analyzing the simulation results, we succeeded, for the first time, in observing and quantifying the sliding motion of CD along a PEG chain. The diffusion coefficient for the sliding motion is almost 6 times lower than that of the translational diffusion of CD in PR at room temperature. The retardation of the sliding motion is caused by the energy barrier on PEG produced by molecular interactions between CD and PEG. We propose a simple equation to describe the diffusion coefficient of the sliding dynamics in PR by combining the Einstein-Stokes diffusion model and a one-dimensional jump diffusion model. This work provides a general strategy for the molecular designs to control the sliding motion in PR.

Autonomously isolated pseudo-polyrotaxane nanosheets fabricated via hierarchically ordered supramolecular self-assembly.

We succeeded in obtaining autonomously isolated nanosheets consisting of pseudo-polyrotaxane (PPR) fabricated via hierarchically ordered supramolecular self-assembly of ß-cyclodextrin and a poloxamer by introducing charged groups to the axis ends of the poloxamer. The isolated PPR nanosheets exhibited a tunable structural coloration and were aligned using a strong magnetic field.

Drastic Change of Mechanical Properties of Polyrotaxane Bulk: ABA-BAB Sequence Change Depending on Ring Position.

Polyrotaxane (PR), consisting of many ring molecules and an axis polymer, is a typical supramolecular structure with unique topological characteristics. In this study, we demonstrated the drastic change of the macroscopic mechanical properties depending on the ring position of PR in bulk. Poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer was employed as an axis polymer to control the position of ß-cyclodextrin (ß-CD). To transfer the ß-CD positions, hydroxypropyl groups (HPPR) and hydrophobic trimethyl silyl groups (TMS-HPPR), which have hydrophilic and hydrophobic ß-CD, respectively, were synthesized. ß-CDs in HPPR were localized on a central PPO segment and formed crystal domains. The axis polymer of HPPR could not bridge ß-CD crystal domains, resulting in a melt state at high temperature. On the other hand, ß-CDs in TMS-HPPR were transferred to both PEO segments and formed crystal domains. The axis polymer in TMS-HPPR could bridge the ß-CD crystal domains, resulting in an elastic state even at high temperature. We succeeded in demonstrating the potential ability of PR: the macroscopic mechanical properties of PR can be changed from a melt state to an elastic one by manipulating the ring positions.

A thermoresponsive dynamic polymer brush fabricated by the segregation of amphiphilic diblock copolymers.

A highly dense polymer brush was previously fabricated by the spontaneous segregation of amphiphilic diblock copolymers in an elastomer matrix into water and a hydrophobic polymer interface and named a 'dynamic polymer brush'. We fabricated a lower critical solution temperature (LCST)-type thermoresponsive dynamic polymer brush by mixing polyisoprene-b-poly[tri(ethylene glycol)methyl ether methacrylate] (PI-b-PME3MA) into a polystyrene-b-polyisoprene-b-polystyrene (SIS) elastomer. The LCST of PME3MA in water is 52 °C. The structure of the polymer brush was determined at several different temperatures using neutron reflectivity. With increasing temperature, the brush thickness of the LCST-type thermoresponsive dynamic polymer brush decreases, similar to the conventional fixed brush with the LCST-type thermoresponse. However, the graft density of the dynamic polymer brush surprisingly increases with increasing temperature. The change of the brush density of the conventional fixed polymer brush is not allowed. However, we observed for the first time that dynamic polymer brushes uniquely respond to increasing temperature with increasing brush densities.