A New Boron-Rhodamine-Containing Carboxylic Acid as a Sugar Chemosensor.

We propose a boron-rhodamine-containing carboxylic acid (BRhoC) substance as a new sugar chemosensor. BRhoC was obtained by the Friedel-Crafts reaction of 4-formylbenzoic acid and N,N-dimethylphenylboronic acid, followed by chloranil oxidation. In an aqueous buffer solution at pH 7.4, BRhoC exhibited an absorption maximum (Absmax) at 621 nm. Its molar absorption coefficient at Absmax was calculated to be 1.4 × 105 M-1 cm-1, and it exhibited an emission maximum (Emmax) at 644 nm for the excitation at 621 nm. The quantum yield of BRhoC in CH3OH was calculated to be 0.16. The borinate group of BRhoC reacted with a diol moiety of sugar to form a cyclic ester, which induced a change in the absorbance and fluorescence spectra. An increase in the D-fructose (Fru) concentration resulted in the red shift of the Absmax (621 nm without sugar and 637 nm with 100 mM Fru) and Emmax (644 nm without sugar and 658 nm with 100 mM Fru) peaks. From the curve fitting of the plots of the fluorescence intensity ratio at 644 nm and 658 nm, the binding constants (K) were determined to be 2.3 × 102 M-1 and 3.1 M-1 for Fru and D-glucose, respectively. The sugar-binding ability and presence of a carboxyl group render BRhoC a suitable building block for the fabrication of highly advanced chemosensors.

Hydrogen Peroxide-Triggered Conversion of Boronic Acid-Appended Insulin into Insulin and Its Application as a Glucose-Responsive Insulin Formulation.

p-Boronophenylmethoxycarbonyl (BPmoc) is a protecting group for amines that is removable by treatment with hydrogen peroxide (H2O2). We prepared BPmoc-modified insulin (BPmoc-Ins) and subcutaneously injected the formulation into diabetic rats. The results demonstrated that BPmoc effectively sealed the blood glucose (Glc)-lowering effects of Ins. Conversely, coinjection of BPmoc-Ins and Glc oxidase (GOx) resulted in reduced blood Glc levels, indicating that Ins was generated from BPmoc-Ins through the following reactions: oxidation of endogenous Glc by GOx; production of H2O2 accompanied by Glc oxidation; removal of BPmoc residues by H2O2. These results show the potential of BPmoc-Ins for a Glc-responsive Ins release system.

Multinuclear NMR Study on the Formation and Polyol-Induced Deformation Mechanisms of Wormlike Micelles Composed of Cetyltrimethylammonium Bromide and 3-Fluorophenylboronic Acid.

We had previously confirmed a glucose-responsive decrease in the viscosity of cetyltrimethylammonium bromide (CTAB) and phenylboronic acid (PBA) wormlike micelle (WLM) systems. However, the mechanisms of the formation of WLMs and the decrease in viscosity with glucose addition have not been determined. In this study, we elucidated the mechanisms using 3-fluorophenylboronic acid (3FPBA) based on 11B NMR and 19F NMR analyses. The system in 60 mM CTAB/60 mM 3FPBA at pH 7.4 demonstrated high viscoelasticity, and the formation of WLMs in the system was confirmed by rheological characteristics. The 11B NMR spectrum at pH 7.4 revealed that 3FPBA existed in a neutral form with sp2-hybridized boron; however, the 11B signal disappeared in the presence of CTAB. In contrast, 19F NMR studies indicated that the quaternary ammonium ion of CTAB interacts with the phenyl group of 3FPBA in the sp2 form via cation-π interactions. PBA derivatives react with various polyols; thus, we investigated the change in the viscous system after the addition of sugar and sugar alcohols. The viscosity of the WLMs decreased with increased polyol concentration, especially those of fructose and mannitol, in which the decrease was apparent at 40-160 mM polyols. The 19F NMR spectra revealed that polyol addition induced decrease in the sp2 form of 3FPBA and increase in the sp3 form of 3FPBA. Based on the results, we propose the following mechanism of the polyol response: (1) The WLMs are stabilized by CTAB and 3FPBA in the sp2 form using cation-π interactions as the driving force. (2) When polyol is added to the system, the sp2 form of 3FPBA decreases and its sp3 form increases. (3) This change means that the structural component of WLMs decreases, which induces the disruption of WLMs, and the viscosity decreases. The formation and deformation mechanisms of the WLMs determined in this study are notable because 3FPBA interacts as a neutral compound, whereas CTAB often interacts with anionic aromatic compounds to form WLMs. Without 19F NMR measurements, these mechanisms would not have been discovered.

Glucose Responsive Rheological Change and Drug Release from a Novel Worm-like Micelle Gel Formed in Cetyltrimethylammonium Bromide/Phenylboronic Acid/Water System.

A novel glucose (Glc)-responsive gel formed by worm-like micelles (WLMs) has the potential to provide a self-regulating insulin delivery system. We have prepared a WLM gel system using 75 mM cetyltrimethylammonium bromide, 75 mM phenylboronic acid, and water. At pH 9.4, this gel-like system was highly viscous and supported its own weight, and dynamic viscoelasticity measurement indicated that it contained long and entangled WLMs. The visual observation of gels prepared to include >6 mM Glc revealed that these adopted a sol-like appearance, whereas those prepared to include a control compound (2-10 mM diethylene glycol) retained their gel-like appearance. The storage modulus ( G') of this system decreased as the Glc concentration increased (2-10 mM), indicating a gradual shortening of the WLMs. In vitro release was evaluated using a test compound (fluorescein isothiocyanate dextran) in a microsized flow system. By 120 min, the release of this compound from the WLM gel was around 27-fold greater in the presence of 100 mM Glc than without Glc or with 100 mM diethylene glycol. This demonstrated the successful preparation of a WLM gel that showed an altered drug release rate, depending on Glc concentration.

Sugar-Responsive Layer-by-Layer Film Composed of Phenylboronic Acid-Appended Insulin and Poly(vinyl alcohol).

Previous studies have shown that reversible chemical bond formation between phenylboronic acid (PBA) and 1,3-diol can be utilized as the driving force for the preparation of layer-by-layer (LbL) films. The LbL films composed of a PBA-appended polymer and poly(vinyl alcohol) (PVA) disintegrated in the presence of sugar. This type of LbL films has been recognized as a promising approach for sugar-responsive drug release systems, but an issue preventing the practical application of LbL films is combining them with insulin. In this report, we have proposed a solution for this issue by using PBA-appended insulin as a component of the LbL film. We prepared two kinds of PBA-appended insulin derivatives and confirmed that they retained their hypoglycemic activity. The LbL films composed of PBA-appended insulin and PVA were successfully prepared through reversible chemical bond formation between the boronic acid moiety and the 1,3-diol of PVA. The LbL film disintegrated upon treatment with sugars. Based on the results presented herein, we discuss the suitability of the PBA moiety with respect to hypoglycemic activity, binding ability, and selectivity for D-glucose.

A Pseudopolyrotaxane for Glucose-Responsive Insulin Release: The Effect of Binding Ability and Spatial Arrangement of Phenylboronic Acid Group.

A pseudopolyrotaxane (PPRX) comprising 3-carboxy-5-nitrophenylboronic acid modified γ-cyclodextrin (NPBA-γ-CyD) and naphthalene modified polyethylene glycol (Naph-PEG) as a sugar-responsive supramolecular structure is prepared. The binding of sugar by the NPBA group induced disintegration of the Naph-PEG/NPBA-γ-CyD PPRX, allowing the components to be dissolved. The Naph-PEG/NPBA-γ-CyD PPRX exhibited better sensitivity compared to that of a PPRX based on 4-carboxyphenylboronic acid modified γ-cyclodextrin (PBA-γ-CyD). We have previously reported the unique structure of Naph-PEG/PBA-γ-CyD PPRX, which formed an inclusion complex with a single-stranded PEG chain being threaded through the γ-CyD rings, with the remaining internal space being occupied by the sugar-sensing PBA moiety from a neighboring ring, thus shielding it from sugar molecules and reducing the sugar sensitivity of the PPRX. In contrast, structural analyses in this study revealed that the sugar-sensing NPBA moiety in the Naph-PEG/NPBA-γ-CyD PPRX is not included in the neighboring NPBA-γ-CyD. This spatial arrangement and the high affinity of NPBA for sugar contributed to the improved sugar responsivity. The enhanced NPBA-γ-CyD was then applied to a PPRX containing Naph-PEG-appended insulin (Naph-PEG-Ins) that showed an improved response for glucose-induced insulin release.

Effect of Physiological Changes in the Skin on Systemic Absorption of Tacrolimus Following Topical Application in Rats.

Tacrolimus (TL) ointment is a topical treatment for atopic dermatitis, a disease that exhibits various skin conditions. The effect of skin pathologies on the systemic absorption of TL and related side effects remains unknown. This study aimed to investigate factors affecting the cutaneous absorption of TL. We prepared various skin models in hairless rats by tape stripping, injection of prophlogistic material solution (PMS), and continuous subcutaneous adrenaline (Adr) infusion. In vivo absorption studies were conducted, with measurements of transepidermal water loss (TEWL) and skin blood flow as physiological parameters. Very little TL absorption was observed through intact skin. Greater TL absorption was noted in skins with high TEWL values and fully stripped skin with PMS injections. In contrast, Adr infusion, which reduced skin blood flow, resulted in decreased TL absorption through fully stripped skin. Combined use of TL and Adr on skin with PMS injections resulted in suppression of TL absorption. Our results revealed that TL absorption following topical application is affected by alterations in the skin barrier, blood flow, and vascular permeability. We propose an administration plan for TL in a flowchart as a means of preventing systemic side effects.

Preparation of polypseudorotaxanes composed of cyclodextrin and polymers in microspheres.

We prepared polypseudorotaxanes (PPRXs) composed of cyclodextrin (CyD) and polyethylene glycol (PEG) inside microspheres (MSs) by an emulsifying process using polypropylene glycol (PPG) that shows temperature-dependent hydrophilicity changes; PPG is hydrophobic at high temperatures but hydrophilic at low temperatures. An aqueous solution of CyD and PEG was dispersed as droplets in PPG at 60°C then cooled to 0°C to allow water of droplets to transfer into PPG. On removal of water in the droplets, CyD and PEG were left behind as a CyD/PEG PPRX inside the solid-state MSs. Examination of α-, ß-, and γ-CyD revealed that α-CyD was suitable for the formation of PPRX containing PEG in this MS preparation procedure. Interestingly, a new PPRX composed of α-CyD and PPG was formed in the α-CyD MSs when they were prepared in the absence of PEG from the aqueous solution of α-CyD. This MS fabrication procedure can control the size and shape of PPRX particles, and will contribute to the production of new types of CyD inclusion complexes.

Evaluation of the effects of absorption enhancers on Caco-2 cell monolayers by using a pore permeation model involving two different sizes.

We applied a parallel pore permeation model based on the Renkin molecular sieving function by using two different-sized pathways to analyze the permeation-enhancing effects of poly-L-arginine (PLA) or a mixed system of spermine (SPM) and sodium taurocholate (STC). Four paracellular markers were simultaneously applied to Caco-2 cell monolayers, and a set of apparent permeability coefficient (P) values was used to obtain membrane parameters. For PLA treatment, the pore occupancy/length ratio (ε/L) of the large pathways increased while the pore radius (R) did not, suggesting that the number of large pathways for the relatively large hydrophilic molecules in the monolayers could be increased by the addition of PLA. In contrast, application of the mixed system comprising SPM and STC significantly increased not only the R of the large pathways but also ε/L of the small pathways. Such changes in membrane parameters could be related to the enhancing mechanism of these compounds. The simulation curves for molecular weight (MW)-P calculated from the membrane parameters could be used to predict the P of drugs with different MWs.

Sugar-sensitive supramolecular structures based on phenylboronic Acid-modified cyclodextrins.

Supramolecular structures were developed from phenylboronic acid-modified cyclodextrins (PBA-CyDs). The intermolecular interaction between the PBA moiety and the CyD cavity was proved using two dimensional (2D)-NMR and powder X-ray diffraction techniques. PBA-α-CyD formed a head-to-tail supramolecular polymer, whereas PBA-ß-CyD formed a head-to-head dimer. The supramolecular structures were disintegrated in the presence of sugars owing to the resulting boronate sugar interactions.

Caco-2 Cell Sheet Partially Laminated with HT29-MTX Cells as a Novel In Vitro Model of Gut Epithelium Drug Permeability.

The intestinal epithelial Caco-2 cell monolayer is a well-established in vitro model useful for predicting intestinal drug absorption in humans. Coculture models of Caco-2 and goblet-cell-like HT29-MTX cells have been developed to overcome the lack of a mucus layer; however, those models are much leakier compared to the intestinal epithelium. Here, we developed a partially laminated culture model where HT29-MTX cells were superimposed onto a Caco-2 monolayer to overcome this issue. A morphological study showed that the piled HT29-MTX cells were voluntarily incorporated into the Caco-2 monolayer, and mucus production was confirmed via periodic acid-Schiff and mucin protein 2 staining. Permeability was evaluated in terms of transepithelial electrical resistance (TEER) and the apparent permeability of paracellular markers with different molecular sizes. The partially laminated model maintained the high barrier function of the Caco-2 monolayer, whose permeability appeared adjustable according to the HT29-MTX/Caco-2 cell ratio. In contrast, the coculture models showed abnormally high permeability of those markers, correlated with low TEER. Thus, the partially laminated model enabled in vitro recapitulation of effective mucosal barrier function. Consequently, this novel model may be useful as an in vitro high-throughput evaluation system for enteral mucosal permeability and mucus-penetrating efficiency of drugs and nanocarriers.

Fluorometric determination of inulin using 5-quinolineboronic acid and inulinase.

Inulin is a polysaccharide composed mainly of D-fructose units and is the most reliable indicator of the glomerular filtration rate. We have proposed an inulin detection method that involves the hydrolysis of inulin to D-fructose using inulinase and the selective binding of D-fructose from inulin using 5-quinolineboronic acid. In this method, the fluorescence of 5-quinolineboronic acid increases, depending on inulin concentration. For inulin in plasma, the detection and quantitation limits were calculated to be 3.7 and 11 µg/ml, respectively.

The use of an artificial skin model to study transdermal absorption of drugs in inflamed skin.

Studies on drug disposition in inflamed skin are important for safe and effective application of topical drugs. Here, the absorption of flurbiprofen (FP) through inflamed skin was examined in vivo and in a skin-mimicking artificial model system. The model skin system consisted of a silicone membrane acting as a model stratum corneum, laminated dialysis membranes acting as a model of viable skin, and 2 microdialysis probes-one used for determination of FP concentration and one acting as a model vessel. This model system could be used for quantitative evaluation of complicated permeation processes. In the in vivo experiments, FP absorption was suppressed in rats with inflamed skin induced by an intracutaneous injection of a mixed solution of λ-carrageenan, zymosan, and casein. Bovine serum albumin solution was placed between the dialysis membranes in the model skin system to mimic protein leaching in skin; the results suggested that the delayed absorption of FP in inflamed skin was due to binding to serum proteins leaching in the tissue. Such a combination of in vivo experiments and a model skin system is useful for understanding complex phenomena in inflamed and damaged skin and reduces experimental animal use.

[Effects of sperminated pullulans on the pulmonary absorption of insulin].

Sperminated pullulans (SP) having different molecular weights (MWs) were prepared, and the enhancing effect on the pulmonary absorption of insulin in rats was examined. SP acted as enhancers of insulin absorption when a 0.1% solution was applied with insulin simultaneously and their enhancing effects depended on the MW of the SP; the same solutions exhibited low toxicity in the in vivo LDH leaching test. In the in vitro experiments using Calu-3 cells, tight junction-opening effects and a toxic effect of SP in the MTT assay were observed at lower concentrations compared with the in vivo experiments. A mucus layer might interfere with the interaction between SP and the cell surface and might suppress both these effects and toxicity. SP having a high MW will be useful for preparing safe and efficient formulations of peptide and protein drugs. The change in the localization of the tight junction proteins may be related to the permeation-enhancing mechanism of SP.

Analysis of the rat skin permeation of hydrophilic compounds using the Renkin function.

The Renkin function was applied to characterize the penetration pathways through rat skin following different pretreatments. Nonmetabolic oligosaccharides and sugar alcohols, as model hydrophilic compounds, were applied simultaneously to the excised skin to obtain the equivalent cylindrical pore radius (R) and pore occupancy/length ratio (ε/L) for each skin piece. The R and ε/L values obtained were used to construct the simulation curves of the permeability coefficient (P(a))-molecular weight (MW). In the case of full-stripped skin, the P(a) of the model compounds and separately obtained P(a) of 5(6)-carboxyfluorescein (CF) showed good agreement with the simulation curve based on the Renkin function, suggesting that the viable epidermis and dermis in the full-stripped skin contained permeation pathways for hydrophilic compounds like aqueous channels. On the other hand, there was poor agreement of P(a) with the simulation curve for skin pretreated with an ethanol-menthol mixed enhancer system and the observed P(a) of CF in the pretreated skin was twice that calculated. The enhancer system might not be able to create aqueous channels in the lipid layer of the stratum corneum and could increase the permeation of CF in the layer in a different way. The analysis presented here will be useful not only for quantitative evaluation of drug permeation through aqueous channels in treated skins but also for investigation of the mechanism of skin-permeation enhancing techniques.

Cell Adhesive Character of Phenylboronic Acid-Modified Insulin and Its Potential as Long-Acting Insulin.

Phenylboronic acid (PBA) derivatives have attracted substantial attention owing to their unique character of forming dynamic covalent bonds with polyol compounds. Recent studies have shown interactions between PBA and sugar chains on the cell surface; they have interesting applications for sensors and drug delivery systems. In this study, we prepared phenylboronic acid-modified insulin (PBA-Ins) to evaluate its glucose-lowering activity and cell adhesiveness. In the case of intravenous injection, PBA-Ins showed longer glucose-lowering activity than native insulin. We hypothesized that this prolonged effect was the result of the interaction between the PBA moiety and sugar chains on the cell surface. Red blood cells (RBCs) were used as a cell model, and we confirmed PBA-Ins's affinity for RBCs, which induced RBC agglutination. Interestingly, using an alternative PBA-Ins administration route markedly changed its glucose-lowering activity. Unlike the intravenous injection of PBA-Ins, the subcutaneous injection showed a small effect on glucose level, which indicated that a small amount of PBA-Ins was absorbed into the bloodstream. This suggested the importance of investigating the interaction between the PBA moiety and many types of cells, such as adipocytes, in subcutaneous tissues.

Investigation of factors that cause insulin precipitation and/or amyloid formation in insulin formulations.

BACKGROUND: Multiple daily subcutaneous injections (MDSIs) are mainly used for formulating an insulin therapy for diabetic patients; however, they also cause insulin-derived amyloidosis (IDA) and lead to poor glycemic control. In addition, for the continuous subcutaneous insulin infusion system (CSII), precipitation frequently causes catheter occlusion and, if the precipitate in the formulations is amyloid, the injection of the insoluble amyloid into the subcutaneous tissue leads to IDA. The aim of this study was to conduct in vitro experiments and present a situation where insulin formulations cause precipitation and amyloid formation. METHODS: Humulin®R and NovoRapid® were used as model formulations for MDSIs and CSII, respectively. The generation of the precipitation was evaluated by measuring turbidity, and amyloid formation was evaluated by using Thioflavin T. Humulin®R was mixed with saline buffer solutions and glucose solutions to evaluate the effect of dilution. In addition, we created an experimental system to consider the effect of the time course of condition changes, and investigated the effects of insulin concentration, m-cresol existence, and pH change on the generation of the precipitate and amyloid in the formulation. RESULTS: In both the original and diluted formulations, physical stimulation resulted in the formation of a precipitate, which in most cases was an amyloid. The amyloid was likely to be formed at a near neutral pH. On the contrary, although a precipitate formed when the pH was decreased to near the isoelectric point, this precipitate was not an amyloid. Further decreases in pH resulted in the formation of amyloids, suggesting that both the positive and negative charged states of insulin tended to form amyloids. The formulation additive m-cresol suppressed amyloid formation. When additives were removed from the formulation, the amyloid-containing gel was formed in the field of substance exchange. CONCLUSIONS: To consider changes in conditions that may occur for insulin formulations, the relationship between the formation of precipitates and amyloids was demonstrated in vitro by using insulin formulations. From the in vitro study, m-cresol was shown to have an inhibitory effect on amyloid formation.

Multilayered assemblies composed of brilliant yellow and poly(allylamine) for an optical pH sensor.

Multilayered thin films containing poly(allylamine) (PAA) and brilliant yellow (BY) were prepared on a quartz slide by a layer-by-layer (LBL) deposition technique. The UV-visible spectra of the PAA/BY films were sufficiently changed depending upon the pH value of the solution in which the film was immersed. The response of the PAA/BY films was very fast (within a second) upon pH change from 9.0 to 5.0, while the response time was ca. 100 s upon pH change from 5.0 to 9.0.

Recognition of bile acids at cyclodextrin-modified gold electrodes.

Lipoylamino-beta- and gamma-cyclodextrin (LP-beta-CD and LP-gamma-CD, respectively) were adsorbed at the surface of gold electrodes by sulfur-gold bonding. The resultant electrodes exhibited quasi-reversible voltammograms for the redox reaction of Fe(CN)6(3-/4-) in aqueous solutions, with peak-to-peak separation (deltaEp) being 85 mV at 20 mV s(-1) as a potential sweep rate. When bile acids are added to the solution, deltaEp values increased to 200-300 mV with increasing the concentration of bile acids. A Langmuir-type adsorption analyses satisfactorily afforded the binding constants (Ksurf) of the surface-confined LP-beta-CD and LP-gamma-CD with the bile acids. The obtained Ksurf values of LP-gamma-CD are 5.0-50 times larger than the corresponding binding constants of gamma-CD in homogeneous aqueous solutions. Cyclic voltammetric experiments with positively, negatively, and non-charged adamantane derivatives as well as pH titration experiments revealed that the retardation of the electrode reaction of negatively charged Fe(CN)6(3-/4-) caused by bile acids was attributable (1) to electric potential changes due to the accumulation of the negative charges at the electrode surface, and (2) to an increase in the hydrophobicity of the electrode surface due to the binding of hydrophobic bile acids to the LP-beta-CD and LP-gamma-CD membranes.

Sugar-Responsive Pseudopolyrotaxane Composed of Phenylboronic Acid-Modified Polyethylene Glycol and γ-Cyclodextrin.

We have designed a sugar-responsive pseudopolyrotaxane (PPRX) by combining phenylboronic acid-modified polyethylene glycol (PBA-PEG) and γ-cyclodextrin. Phenylboronic acid (PBA) was used as a sugar-recognition motif in the PPRX because PBA reacts with a diol portion of the sugar molecule and forms a cyclic ester. When D-fructose or D-glucose was added to a suspension of PPRX, PPRX disintegrated, depending on the concentration of the sugars. Interestingly, catechol does not show a response although catechol has a high affinity for PBA. We analyzed the response mechanism of PPRX by considering equilibria.