Colonic delivery of docosahexaenoic acid improves impaired glucose tolerance via GLP-1 secretion and suppresses pancreatic islet hyperplasia in diabetic KK-A(y) mice.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that regulates the insulin secretion depending on blood glucose level. Recent studies show that the unsaturated fatty acids can promote GLP-1 secretion from intestinal L-cells. We have shown previously that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) administered into a mouse closed intestinal loop, especially into the colonic segment, stimulate GLP-1 and insulin secretion and have a hypoglycemic effect, suggesting that DHA and EPA have potential as antidiabetic agents. The present study examined the antidiabetic effect of DHA following long-term in vivo delivery to the colon using normal ddY and diabetic KK-A(y) mice. The plasma GLP-1 concentration of KK-A(y) mice increased after long-term DHA administration, and this had a significant hypoglycemic effect. In contrast, although GLP-1 secretion in ddY mice tended to increase after DHA administration, blood glucose concentration did not differ between vehicle- and DHA-treated ddY mice. Immunostaining of the pancreas after long-term DHA administration showed that continuous DHA treatment stimulated ß-cell apoptosis and accordingly suppressed islet cell growth in KK-A(y) mice. Colon targeting of DHA may provide a new strategy for improving impaired glucose tolerance in type 2 diabetes mellitus by stimulating GLP-1 secretion, which may subsequently suppress the compensatory hyperplasia of pancreatic islets.

A statistical approach to the development of a transdermal delivery system for ondansetron.

Transdermal delivery of drugs has gained attention as an alternative to intravenous and oral methods of delivery. However, the skin permeation of drugs is generally poor. To overcome this problem, many permeation enhancers have been developed. In this study, ondansetron hydrogels were prepared, and their skin permeation and pharmacological effects were evaluated in mice. To prepare the hydrogels, a Box-Behnken design was introduced. Fifteen formulations of ondansetron hydrogels composed of hydroxyethylcellulose and hydroxypropylcellulose as gel bases, l-menthol as a penetration enhancer and isopropanol (IPA), N-methyl-2-pyrrolidone (NMP) and water as a solvent were prepared. The quantities of IPA (X(1)), l-menthol (X(2)) and NMP (X(3)) were selected as causal factors. We performed an in vitro skin permeation study and an in vivo skin irritation study with the test hydrogels. The flux and the total irritation score were selected as response variables. The optimal formulation, one that has an appropriate penetration and an acceptable skin irritation score, was estimated using a nonlinear response surface method incorporating thin-plate spline interpolation. The optimal formulation also delivered the desired pharmacological activity. These results indicated the feasibility of delivering ondansetron transdermally.

Structural requirements of penetratin absorption enhancement efficiency for insulin delivery.

Penetratin, a 16-residue peptide, is used widely as a highly efficient delivery carrier for a wide range of poorly permeable therapeutic cargoes. The crucial structural features of penetratin remain unclear, as demonstrated by the difficulties encountered in designing new molecules. The efficiency in enhancing nasal insulin absorption was compared between l-penetratin and 20 of its analogues in rats. We also measured lactate dehydrogenase (LDH) leakage as an indicator of cytotoxicity and scored the histopathological irritation. Substitution of a cationic residue (Arg or Lys) with Leu or addition of tetra-arginine to the C- or N-terminus of penetratin caused considerable reduction in the enhancing efficiency properties of the modified analogues. Mutual exchanging of Arg and Lys in corresponding analogues produced nearly inactive analogues, although changing Arg to Lys in the same analogue produced similar penetratin activity. In addition, activity was impaired markedly upon modification of penetratin within amphiphilic (Trp) or hydrophobic (Ile and Phe) residues. Chain size-modified analogues lacked the ability to induce nasal insulin absorption. In contrast, rearrangement of the modified analogues by C,N-half-exchange and reverse analogues produced activity similar to that of the original penetratin. The enhancing activity was inhibited almost completely upon sequence arrangement of the resulting analogues. Surprisingly, a shuffle (Arg, Lys fix) 2 analogue increased insulin absorption significantly, reaching a relative bioavailability value 1.85-times that of original penetratin. This analogue caused negligible release of LDH in nasal lavage fluid and maintained the integrity of the nasal respiratory epithelium. In conclusion, modulation of amino acid sequences by fixing the cationic residue positions can augment penetratin-enhanced nasal absorption and may lead to improvements in nasal insulin absorption.

Effect of cell-penetrating peptides on the nasal absorption of insulin.

The goal of this study was to evaluate whether cell-penetrating peptides (CPPs) affect the nasal absorption of insulin. L- or D-forms of penetratin, or the L- or D-forms of octaarginine (L- or D-R8), was used as first time for nasal insulin delivery. Furthermore, the concentration of lactate dehydrogenase (LDH) in nasal lavage fluid was determined and a histopathological study of nasal respiratory epithelium was conducted. CPPs dramatically increased nasal insulin absorption, and it was more pronounced for L- and D-penetratin than L- or D-R8. L-penetratin was the most effective promoter of insulin absorption compared with others CPPs. A dose-dependent relationship of L-penetratin and insulin bioavailability was statically significant. The pharmacological availability and bioavailability of nasally administered insulin was up to 76.7% and 50.7% relative to the subcutaneous route, respectively. In contrast, increasing the D-penetratin concentration decreased the efficiency of nasal insulin absorption. There was no significant difference in the release of LDH in nasal lavage fluid and the integrity of nasal respiratory epithelium when L-penetratin was present. In conclusion, these data demonstrate that L-penetratin markedly increased the permeability of insulin across the nasal membrane without causing detectable damage to the integrity of cells in the nasal respiratory mucosa.

Formulation optimization of an indomethacin-containing photocrosslinked polyacrylic acid hydrogel as an anti-inflammatory patch.

Photocrosslinked polyacrylic acid hydrogel, made from polyacrylic acid (PAA) modified with 2-hydroxyethyl methacrylate (HEMA), is a promising candidate adhesive for dermatological patches. In this study, we investigated the further availability of hydrogel as an adhesive for dermatological patches using a hydrogel containing indomethacin (IDM) as a model anti-inflammatory patch. From an orthogonal experimental study, we clarified the relationships between formulation factors and characteristics of model formulation. Formulations with a lower degree of swelling were prepared by increasing the degree of HEMA modification and the addition of Tween 80. Apparent permeation rate was increased by addition of L-menthol and Tween 80. A tendency for higher HEMA modification to be accompanied by the prolongation of the lag time of IDM was observed. To obtain an applicable anti-inflammatory patch, we conducted a formulation optimization study using a novel optimization method, a response-surface method incorporating multivariate spline interpolation (RSM-S). Consequently, a highly functional anti-inflammatory patch in terms of its adhesive properties and bioavailability was successfully obtained. Since a wide range of functions can be fully controlled by manipulating the formulation factors, photocrosslinked polyacrylic acid hydrogel is an attractive candidate adhesive for dermatological patches.

Effect of iontophoresis and switching iontophoresis on skin accumulation of ketoprofen.

The effect of iontophoresis and switching iontophoresis on the skin accumulation of drugs was investigated. An acrylic diffusion cell mounted with electrode cells (bore: 2.2 cm) with circular platinum electrodes (diameter: 2.0 cm) was used for the skin accumulation study. The skin accumulation of fluorescein after non-switching and switching iontophoresis was macroscopically compared with that achieved by passive diffusion (control). Intense fluorescence was observed after the application of non-switching and switching iontophoresis. Furthermore, fluorescence was observed just under the electrode cell and hardly spread in the skin beyond the area of the electrode cell. The skin accumulation of ketoprofen after non-switching and switching iontophoresis was also compared with control data. Although non-switching iontophoresis showed the highest amount of ketoprofen accumulated in skin, skin irritation was observed. Among the various switching intervals, switching iontophoresis using 10-min intervals achieved the highest value, and there was no skin irritation. Furthermore, the amount of ketoprofen accumulated was maintained after switching iontophoresis at 10-min intervals up to 180 min. Since the amount of ketoprofen in skin after switching iontophoresis was greater than that after intermittent iontophoresis, switching iontophoresis should increase the amount of ketoprofen due to enhancement of skin penetration by skin hydration. These findings suggest that switching iontophoresis using an optimal switching interval can prevent skin irritation and enhance drug accumulation in the skin.

In vivo evaluation of Kumazasa extract and chitosan films containing the extract against deep skin ulcer model in rats.

Kumazasa extract (KE) and its whole solid component (EXT)-containing chitosan films produced by drying in air and lyophilization, abbreviated to ND and FD films, respectively, were examined for efficacy and healing features using a deep skin ulcer model in rats. Their effects were compared with those of clinically available dosage forms, Beschitin W, Geben cream and U-PASTA. KE alone exhibited a better effect as compared with other preparations, and FD films also more effective than control in the early stage. Histological analysis showed that KE alone reduced necrosis rapidly and accelerated granulation. ND films delayed healing rate as compared with control. FD films showed histological features between control and KE alone, but tended to delay healing rate in the later period. Thus, reduction rate of wound area and histological features suggested that KE alone should be excellent for the promotion of wound healing. Although FD films were less effective than KE alone, they were superior as to usability such as changing the preparation.

A novel approach using functional peptides for efficient intestinal absorption of insulin.

The aim of this study was to evaluate whether oligoarginine, a cell-penetrating peptide (CPP), can improve intestinal absorption of insulin in rats. Peptides composed of six (R(6)), eight (R(8)) and 10 (R(10)) residues of arginine were used as the CPP. No insulin absorption was observed following administration of insulin solution alone; however, insulin absorption increased dramatically after coadministration of the D-form of R(6) (D-R(6)) and the L-form of R(6) (L-R(6)) in a dose-dependent manner. The effects on insulin absorption were more pronounced for D-R(6) than for L-R(6). Among oligoarginines composed of six, eight, or 10 arginine residues, D-R(8) showed the strongest enhancing effects on insulin intestinal absorption. In contrast, intestinal absorption of other model hydrophilic macromolecules, interferon-beta and fluorescein isothiocyanate-labeled dextran 4400, was not affected by coadministration with oligoarginine. Pretreatment by the effective dose of L-R(6) did not induce lactate dehydrogenase leakage or histological damage, suggesting that oligoarginine has no untoward effect on the intestinal mucosa. Our data demonstrate that coadministration of oligoarginine increases intestinal insulin absorption markedly without causing detectable damage in cellular integrity and that the covalent binding between insulin and oligoarginine is not necessary for this effect. We conclude that oligoarginines are likely to become powerful tools for overcoming the low permeability of insulin through the epithelial cell membrane, the major barrier to oral insulin delivery.

Malignant NK/T-cell lymphoma associated with simian Epstein-Barr virus infection in a Japanese macaque (Macaca fuscata).

A case of spontaneous malignant lymphoma in a Japanese macaque (Macaca fuscata) was pathologically, etiologically and virologically studied. Nasal cavity was involved in the neoplastic lesions in addition to lymphoid and visceral tissues. Histopathological analyses revealed the presence of neoplastic cells classified into histiocytic Hodgkin-like cells and Reed-Sternberg-like cells. Histiocytic Hodgkin-like cells were CD16+ and CD20+, and the CD16+ cells were also positive for simian Epstein-Barr virus (sEBV)-encoded early RNA transcripts. RS-like cells were negative for CD3, CD16 and CD20. Antibodies to early antigen of sEBV were detected, while antibodies to simian T-cell leukemia virus-1 were negative. The case may correspond to EBV-associated nasal type NK/T-cell lymphoma in humans rather than Hodgkin lymphoma.

Effect of mixed micelle formulations including terpenes on the transdermal delivery of diclofenac.

The significant inhibitory action of diclofenac formulated in mixed micelles of lecithin with cholate or deoxycholate was observed on the rat hind paw edema induced by carrageenan. In the primary stage, mixed micelle formulation of deoxycholate was more effective compared with that of cholate. However, in the final term, the inhibitory action was similar in both formulations. In a previous study, the flux of diclofenac was greater in the mixed micelle formulation of deoxycholate compared with that of cholate. It was suggested that the permeation rate of diclofenac through skin was proportional to the pharmacological activity. The hind paw edema was quickly inhibited when cyclic monoterpene such as d-limonene or l-menthol was included in the formulations. All the micelle formulations significantly decreased the value of AUC estimated the hind paw thickness-time profile. This suggests that the micelle formulation of cholate in addition to deoxycholate showed significant anti-inflammatory activity to hind paw edema of rats. Incorporation of d-limonene or l-menthol was more effective on the decrease of AUC. A pharmacological study revealed that micelle formulations were able to reduce the skin irritation of chemicals.