Brief exposure to cycloheximide prior to electrical activation improves in vitro blastocyst development of porcine parthenogenetic and reconstructed embryos.

To investigate the effects of cycloheximide exposure before electrical activation of in vitro-matured porcine oocytes on the subsequent development of parthenogenetic embryos, cumulus-free mature oocytes were exposed to NCSU-23 medium containing cycloheximide (10 microg/mL) for 0, 5, 10, 20, 30 and 60 min, activated by electrical pulse treatment (1.5 kV/cm, 100 micros) and then cultured in PZM-3 for 7 days. To evaluate the effects of cycloheximide on the activation of nuclear transfer embryos, reconstructed embryos were electrically activated by two DC pulses (1.2 kV/cm, 30 micros) before or after exposure to cycloheximide. The reconstructed embryos were allocated into four groups: electrical pulse treatment alone (Ele); exposure to cycloheximide for 10 min followed by electrical activation (CHX+Ele); electrical activation followed by exposure to cycloheximide for 6h (Ele+CHX); exposure to cycloheximide for 10 min, followed by electrical activation and a further exposure to cycloheximide for 6h (CHX+Ele+CHX). The activated reconstructed embryos were cultured in PZM-3 for 6 days. Oocytes treated with 10 min exposure to cycloheximide followed by electrical activation had a significantly higher percentage of blastocyst formation compared to control oocytes and oocytes exposed for > or =30 min. In the reconstructed embryos, the blastocyst development rates of embryos exposed to cycloheximide (CHX+Ele, Ele+CHX and CHX+Ele+CHX) were significantly higher than those of the control group (Ele). Among the cycloheximide-treated groups, the CHX+Ele group had increased development rate and total blastocyst cell number, though these values were not significantly different from those observed in the other cycloheximide-treated groups. To evaluate the quality of NT embryos treated with cycloheximide, apoptosis in blastocysts was analyzed by TUNEL assay. The 10 min exposure to cycloheximide prior to electrical activation significantly reduced cell death compared with longer exposure to cycloheximide after electrical fusion. In conclusion, brief exposure to cycloheximide prior to electrical activation may increase the subsequent blastocyst development rates in porcine parthenogenetic and reconstructed embryos.

Inter- and intralaboratory variation of in vitro diffusion cell measurements: an international multicenter study using quasi-standardized methods and materials.

In vitro measurements of skin absorption are an increasingly important aspect of regulatory studies, product support claims, and formulation screening. However, such measurements are significantly affected by skin variability. The purpose of this study was to determine inter- and intralaboratory variation in diffusion cell measurements caused by factors other than skin. This was attained through the use of an artificial (silicone rubber) rate-limiting membrane and the provision of materials including a standard penetrant, methyl paraben (MP), and a minimally prescriptive protocol to each of the 18 participating laboratories. "Standardized" calculations of MP flux were determined from the data submitted by each laboratory by applying a predefined mathematical model. This was deemed necessary to eliminate any interlaboratory variation caused by different methods of flux calculations. Average fluxes of MP calculated and reported by each laboratory (60 +/- 27 microg cm(-2) h(-1), n = 25, range 27-101) were in agreement with the standardized calculations of MP flux (60 +/- 21 microg cm(-2) h(-1), range 19-120). The coefficient of variation between laboratories was approximately 35% and was manifest as a fourfold difference between the lowest and highest average flux values and a sixfold difference between the lowest and highest individual flux values. Intralaboratory variation was lower, averaging 10% for five individuals using the same equipment within a single laboratory. Further studies should be performed to clarify the exact components responsible for nonskin-related variability in diffusion cell measurements. It is clear that further developments of in vitro methodologies for measuring skin absorption are required.

Electrophysiological studies on the evaluation of absorption enhancers in Caco-2 cells using a microelectrode technique.

We developed a microelectrode technique to characterize the electrophysiological properties in Caco-2 cells, and used it to determine the mechanisms of absorption enhancers. The action of absorption enhancers on the apical membrane of Caco-2 cells was estimated by measuring the apical membrane potential (Vm) with the microelectrode. The Vm value of Caco-2 cells in Hanks' balanced salt solution containing 0.5 mM K+ was 18.9+/-0.8 mV (n=217), and the apical membrane resistance was 49.4+/-1.1 MOhms (n=160). In the electrophysiological study with absorption enhancers, laurylmaltoside (LM) markedly decreased the Vm value, while sodium glycocholate (NaGC) moderately reduced this value, and EDTA did not affect the value. These findings might be associated with their action sites, plasma membrane or tight junction in Caco-2 cell monolayers. In influx and transport studies with these absorption enhancers, LM enhanced the influx of furosemide, which is transported via both the transcellular and paracellular routes into Caco-2 cells, and enhanced its transport to the basolateral side of Caco-2 monolayers more than that of 5(6)-carboxyfluorescein (CF), a paracellular marker. EDTA did not increase the influx of furosemide, and enhanced the transport of furosemide and CF across Caco-2 cell monolayers to the same extent. In contrast, NaGC only slightly increased the influx of furosemide and did not enhance the transport of either furosemide or CF across the Caco-2 monolayers in this study. These findings were well correlated with the effects of these absorption enhancers on the electrophysiological parameters. Therefore, the microelectrode technique might be useful for evaluating the action of absorption enhancers in the plasma membrane at an intact cell level.

Electrophysiological studies on the mechanism for enhanced intestinal transport of water-soluble compounds by antibiotic peptide bacitracin.

Bacitracin is an antibacterial cyclic dodecapeptide produced by Bacillus licheniformin. Besides antibacterial activity, it is reported to have a protease inhibitory activity and an absorption enhancing action. Here we determined the effects of bacitracin on transport of water-soluble dye fluoresceinisothiocyanate (FITC)-dextran across the rat intestinal mucosal membrane using an electrophysiological technique. Bacitracin enhanced the intestinal mucosal-to-serosal transport of FITC-dextran in concentration-dependent and pH-dependent manners. In particular, the addition of bacitracin to the mucosal side led to a remarkable enhancement of FITC-dextran transport across the colonic membrane. Furthermore, its exhibition of transport enhancement required the existence of metal divalent cations, Ca2+ and Mg2+, in the mucosal compartment. Electrophysiological study using voltage-clamp technique revealed that a relatively lower concentration of bacitracin (5 mM) enhanced the transport of 6-carboxyfluorescein via a paracellular pathway in the colonic membrane and higher concentration of bacitracin (20 mM) affects both transcellular and paracellular routes, resulting in significant enhancement of 6-carboxyfluorescein across the colonic membrane. These findings might provide the useful information for enhancing the intestinal transport of poorly absorbable drugs by bacitracin which has multiple functions.

Transport kinetics of leucine enkephalin across Caco-2 monolayers: quantitative analysis for contribution of enzymatic and transport barrier.

In this study, we determined the activities of four aminopeptidases such as aminopeptidase B (APB), M (APM), N (APN) and dipeptidylpeptidase IV (DPP IV) in Caco-2 cells and compared with those in the rat intestinal mucosae. The activities of APB, APM and APN appeared to be highest in rat small intestinal mucosa, while DPP IV activity was much higher in Caco-2 cells than that in the rat intestinal mucosa. Next the inhibitory effects of various protease inhibitors were examined in Caco-2 homogenate. Three tested inhibitors, bacitracin, amastatin and puromycin, effectively inhibited the activities of APM, APN and DPP IV except for APB. Further, we quantitatively evaluated the permeation and degradation properties of leucine enkephalin (Leu-Enk) in the presence or absence of inhibitors in Caco-2 monolayer system. Leu-Enk had a high degradation clearance (CLd) and a low permeation clearance (CLp) in Caco-2 monolayers. This finding indicates that the very rapid degradation of Leu-Enk on the apical side of Caco-2 monolayers was due to aminopeptidases. However, these protease inhibitors besides sodium glycocholate were able to reduce the CLd values markedly, thereby increasing the permeation amount of Leu-Enk across Caco-2 monolayers. In particular, amastatin significantly decreased the CLd value and increased the CLp value. This enhanced CLp value was further increased by the coadministration with an absorption enhancer, EDTA or laurylmaltoside. These findings are relevant to the oral administration of peptide drugs and to developing an efficient oral delivery system.

Enhanced permeability of insulin across the rat intestinal membrane by various absorption enhancers: their intestinal mucosal toxicity and absorption-enhancing mechanism of n-lauryl-beta-D-maltopyranoside.

We have examined the in-vitro permeability characteristics of insulin in the presence of various absorption enhancers across rat intestinal membranes and have assessed the intestinal toxicity of the enhancers using an in-vitro Ussing chamber method. The absorption enhancing mechanism of n-lauryl-beta-D-maltopyranoside was studied also. The permeability of insulin across the intestinal membranes was low in the absence of absorption enhancers. However, the permeability was improved in the presence of enhancers such as sodium glycocholate and sodium deoxycholate in the jejunum, and sodium glycocholate, sodium deoxycholate, n-lauryl-beta-D-maltopyranoside, sodium caprate and ethylenediaminetetraacetic acid (EDTA) in the colon. Overall, the absorption enhancing effects were greater on the colonic membrane than on the jejunal membrane. The intestinal membrane toxicity of these enhancers was characterized using the release of cytosolic lactate dehydrogenase from the colonic membrane. A marked increase in the release of lactate dehydrogenase was observed in the presence of sodium deoxycholate and EDTA. The release of lactate dehydrogenase in the presence of these absorption enhancers was similar to that seen with sodium dodecyl sulphate (SDS), used as a positive control, indicating high toxicity of these enhancers to the intestinal membrane. In contrast, sodium glycocholate and sodium caprate caused minor releases of lactate dehydrogenase, similar to control levels, suggesting low toxicity. In addition, the amount of lactate dehydrogenase in the presence of n-lauryl-beta-D-maltopyranoside was much less than that seen with sodium deoxycholate, EDTA and SDS. Therefore, sodium glycocholate, sodium caprate and n-lauryl-beta-D-maltopyranoside are useful absorption enhancers due to their high absorption enhancing effects and low intestinal toxicity. To investigate the absorption enhancing mechanisms of n-lauryl-beta-D-maltopyranoside, the transepithelial electrical resistance (TEER), voltage clamp experiments and the circular dichroism spectra were studied. n-Lauryl-beta-D-maltopyranoside decreased the TEER values in a dose-dependent manner, suggesting that the enhancer may open the tight junctions of the epithelium, thereby increasing the permeability of insulin via a paracellular pathway. This speculation was supported by the findings that 20 mM n-lauryl-beta-D-maltopyranoside produced a greater increase in the paracellular flux rate than in the transcellular flux rate by the voltage clamp studies. Evaluating the circular dichroism spectra we found that insulin oligomers were not dissociated to monomers by the addition of n-lauryl-beta-D-maltopyranoside, but dissociation did occur with the addition of sodium glycocholate. Thus, the dissociation of insulin was not a major factor in the absorption enhancing effect of n-lauryl-beta-D-maltopyranoside. These findings provide basic information to select the optimal enhancer for the intestinal delivery of peptide and protein drugs including insulin.

Effectiveness and toxicity screening of various absorption enhancers using Caco-2 cell monolayers.

We studied the enhancing and toxic effects of five different absorption enhancers on the transport of FITC-dextran with an average molecular weight of 4000 (FD-4) across Caco-2 cell monolayers, and their enhancing effects were also compared with those in rat intestine. The enhancing and cytotoxic properties of these enhancers were characterized using the following tests: measurement of the permeability coefficients of FD-4 and the transepithelial electrical resistance (TEER) in Caco-2, the release of cytosolic lactate dehydrogenase (LDH) and intracellular mitochondrial dehydrogenase (MDH) activity. All the absorption enhancers increased the permeability of FD-4 across Caco-2 cell monolayers and a good relationship was observed between the enhancement and their toxic effects. However, EDTA and Na-Cap were effective for improving the transport of FD-4 across Caco-2 cells without serious cytotoxicity. At concentrations with low cytotoxicity, various absorption enhancers exihibited reversible effects on the TEER values in Caco-2 cell monolayers, except for 50 mM sodium salicylate (Na-Sal). Moreover, we obtained a good correlation between the enhancement of these enhancers in Caco-2 cell monolayers and in rat large intestine. This finding indicated that the effectiveness of absorption enhancers in the Caco-2 monolayer system was similar to an in vivo rat system. Therefore, the screening system using Caco-2 cell monolayers is useful for examining the effectiveness and toxicity of absorption enhancers.

Effects of various absorption enhancers on the intestinal absorption of water soluble drugs by in vitro Ussing chamber method: correlation with an in situ absorption experiment.

The effect of absorption enhancers on the small and large intestinal absorption of drug in rats was examined using an in vitro modified Ussing chamber method, and the results were compared with those from an in situ absorption experiment. Phenol red was chosen as a model drug, while the absorption enhancers used were sodium glycocholate (Na-GC), sodium taurocholate (Na-TC), sodium deoxycholate (Na-DC), EDTA, sodium salicylate (Na-Sal), sodium caprate (Na-Cap), diethyl maleate (DEM) and N-lauryl-beta-D-maltopyranoside (LM), all used at a concentration of 20 mM. This modified Ussing chamber method showed that Na-DC, EDTA and LM were the most effective absorption enhancers in the large intestine. A good correlation exists between the area under the curve (AUC) (in situ loop model) and the cumulative amount of phenol red absorbed (in vitro modified Ussing chamber method). These results indicated that the in vitro modified Ussing chamber method can be used to evaluate the effects of various absorption enhancers in the intestine.

Effects of protease inhibitors on the absorption of phenol red and fluorescein isothiocyanate dextrans from the rat intestine.

The absorption enhancement effects of three types of protease inhibitors, aprotinin, bacitracin, and soybean trypsin inhibitor, on the small and large intestinal absorption of phenol red (PR) and fluorescein isothiocyanate dextrans (FDs) were examined in rats. Of these protease inhibitors, only bacitracin enhanced the absorption of PR and FDs from the rat small and large intestine. Thus, we suggest that bacitracin has not only a protease-inhibitory but also an absorption-enhancing capability. We examined the effects of various protease inhibitors on intestinal mucosal toxicity by measuring the leakage of Evans blue (EB) from the systemic circulation. Although there was a significant increase in the leakage of EB in the presence of bacitracin, it was considerably less than that in the case of polyoxyethylene 9-lauryl ether (BL-9), which was used as a positive control. Therefore, bacitracin may be a good model adjuvant for improving the intestinal absorption of poorly absorbable drugs because it did not cause serious intestinal mucosal damage, as seen in the case of BL-9.