Koichi Isowa

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.

Improving insulin enteral absorption using water-in-oil-in-water emulsion

The purpose of this study was to evaluate the hypoglycemic effects of water-in-oil-in-water (W/O/W) insulin emulsions containing lipoidal absorption enhancer after enteral administration to rats. The hypoglycemic effects of insulin were examined using an in situ loop method in rats. The insulin emulsions prepared with soybean oil, triolein or trilinolein slightly but significantly decreased the serum glucose levels compared to the insulin solution. By addition of 3% limonene or 3% menthol to the triolein emulsion, the hypoglycemic effect of insulin was promoted in the ileum but not in the colon. Strong hypoglycemic effects were observed with the triolein emulsion containing 2% fatty acids such as oleic acid, linoleic acid and linolenic acid. The remarkable enhancing effects occurred more predominately in the colon than in the ileum. The effect of degree of unsaturation of fatty acids was not observed. No tissue damage was noted by light microscopic examination of both regions treated with triolein emulsion, triolein emulsion containing menthol or oleic acid. W/O/W emulsion containing unsaturated fatty acids are able to enhance the ileal and colonic absorption of insulin without tissue damage and may, therefore, be useful in dosage form in enteral delivery system for insulin.

In vivo effects of highly purified docosahexaenoic acid on rectal insulin absorption.

The purpose of this study was to evaluate the effectiveness and the toxicity of polyunsaturated fatty acid, such as oleic acid, eicosapentaenoic acid (DHA), as potential absorption enhancer for rectal delivery of insulin, using a water-in-oil-in water (W/O/W) multiple emulsion. In a single administration study, rectal insulin absorption was enhanced markedly, and marked hypoglycemia was induced by the emulsion incorporating various fatty acids in an insulin dose-related fashion. The pharmacological availability of the emulsion incorporating 2% oleic acid, EPA and DHA was approximately 7.7, 11.0 and 25.4%, respectively. The insulin absorption enhancement effect was not increased in proportion to the amount of DHA in the emulsion, the mean T(max) value of the serum glucose-time curve could be extended to twice that of the emulsion without PF 127. In a multiple administration study, the mean AUC(glucose) values of the emulsion incorporating DHA showed almost the same value on the first and the tenth day. From the morphological appearance of the mucosal surface, the emulsion incorporating DHA induced no or little mucosal damage. Our findings demonstrated that DHA has a strong insulin permeability enhancement effect and little toxicity. Thus, DHA is an attractive candidate as an absorption enhancer for intestinal delivery of insulin.

Multi-objective simultaneous optimization based on artificial neural network in a ketoprofen hydrogel formula containing O-ethylmenthol as a percutaneous absorption enhancer

Abstract The aim of this study was to apply a novel simultaneous optimization technique incorporating an artificial neural network (ANN) to a design of a ketoprofen hydrogel containing O-ethylmenthol (MET). For model formulae, 12 kinds of ketoprofen hydrogels were prepared. The amount of ethanol and MET were selected as causal factors. A percutaneous absorption study in vivo in rats was performed and irritation evoked on rat skin was microscopically judged after the end of the experiments. The rate of penetration (Rp), lag time (tL) and total irritation score (TIS) were selected as response variables. A set of causal factors and response variables was used as tutorial data for ANN and fed into a computer. Nonlinear relationships between the causal factors and the release parameters were represented well with the response surface predicted by ANN. The optimization of the ketoprofen hydrogel was performed according to the generalized distance function method. The observed results of Rp and TIS, which had a lot of influence on the effectiveness and safety, coincided well with the predictions. It was suggested that the multi-objective simultaneous optimization technique incorporating ANN was quite useful for optimizing pharmaceutical formulae when pharmaceutical responses were nonlinearly related to the formulae and process variables.

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.

An antimicrotubule agent, TZT-1027, does not induce neuropathologic alterations which are detected after administration of vincristine or paclitaxel in animal models

One of the major dose-limiting toxicities induced by antimicrotubule antitumor agents such as vinca alkaloids and taxanes is peripheral neuropathy. The neurotoxicity of TZT-1027 (a dolastatin 10 derivative antimicrotubule agent) was thus assessed using the animal models for antimicrotubule agent-induced neurotoxicity. Rabbits were intravenously given TZT-1027 or vincristine weekly for 5 weeks. In the mouse study, TZT-1027, vincristine or paclitaxel was intravenously given every 2 days and/or weekly. Despite the neuropathologic evidence such as myelinated axonal and fiber degeneration in the peripheral nerves and in the sensory tracts of the spinal cord following the treatment with vincristine or paclitaxel, no drug-induced alteration was observed in the TZT-1027 groups. Although there are reports that some other dolastatin derivatives with antimicrotubule activity showed no neurotoxic potential in humans, the present study represents the first demonstration in experimental animals that a dolastatin derivative has no, or at least a lower, neurotoxic potential compared to other antimicrotubule agents.

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.

Evaluation and structure-activity relationship of synthesized cyclohexanol derivatives on percutaneous absorption of ketoprofen using artificial neural network.

The effect of 35 newly synthesized O-ethylmenthol (MET) derivatives on percutaneous absorption of ketoprofen was investigated in rats. In order to understand the relationship between the structure of compounds and promoting activity (structure-activity relationship), an artificial neural network (ANN) was employed. In the in vivo percutaneous absorption study, male Wistar rats, weighing 160-180 g, were used. The apparent penetration rate (Rp) was estimated based on a pharmacokinetic model with a constant rate of penetration through the skin after a lag time. As an index of the promoting activity of each compound, an enhancement factor (Ef), defined as follows, was used: Ef=Rp(with enhancer)/Rp(without enhancer). An irritation evoked on rat skin was microscopically judged at the end of the in vivo percutaneous absorption experiment and evaluated as a total irritation score (TIS). Ef and TIS were selected as output variables to determine the ANN structure. Calculated logP, molecular weight, steric energy (SE), van der Waals area, van der Waals volume, dipole moment, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) were used as factors to determine the structural nature of cyclohexanol derivatives. Among these parameters, logP, SE and LUMO significantly affected the prediction of Ef and TIS. The predicted values of Ef and TIS coincided well with in vivo percutaneous absorption experimental values. However, results observed with a linear regression method were poor compared with the ANN approach. The contribution index of logP was approximately 50% in the prediction of Ef, suggesting that lipophilicity among physicochemical properties contributes most of the promoting activity of these compounds.

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.

Rectal absorption and mucosal irritation of rectal gels containing buprenorphine hydrochloride prepared with water-soluble dietary fibers, xanthan gum and locust bean gum

Rectal gels prepared with water-soluble dietary fibers, xanthan gum and locust bean gum, were evaluated as a vehicle for the rectal administration of buprenorphine hydrochloride (BN-HCI) in rabbits. The maximum plasma concentration of buprenorphine (BN) gradually decreased with increase in the gum concentration. The values of the mean residence time (MRT0–2) increased in proportion to increasing gum concentration. The absorption of BN from rectal gels containing 0.5, 1 and 2% (w/ v) gum compared with those based upon polyethylene glycol (PEG), was more rapid. In particular, the absorption of BN from rectal gels containing 1% (w/v) gum was extremely fast without decreasing the areas under the plasma concentration vs. time curves (AUC0–2). The bioavailabilities obtained in rabbits correlated well with the in vitro release rates determined using dialysis tubing. A histopathological study revealed severe mucosal hyperemia, which was thought to be the main characteristic of rectal irritation induced by PEG-base suppositories. These results suggested that BN-HC1 rectal gels prepared with xanthan gum and locust bean gum were practical rectal preparations with rapid absorption and reduced side effects.