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.

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SUMMARYThe identity of low-molecular-weight and minor protein spots, appeared in 2-DE patterns of human plasma, was examined. They were not obvious in the patterns of "Type-I" 2-DE (non-denaturing IEF followed by non-denaturing gel electrophoresis), but clearly detected in the patterns of "Type II" 2-DE (non-denaturing IEF followed by SDS gel electrophoresis) at pI 5.5-7.5 and apparent mass 8-40 kDa 1) . The spots were not obviously detected when the IEF gels were kept at low temperature (around 4°C) during electrophoresis, suggesting that they are the proteolysis products of plasma proteins. The minor spots were more obviously detected when human plasma was subjected to ammonium sulfate (AS) fractionation and the 0-35% saturated AS fraction was dialyzed and subjected to Type-II 2-DE. Then the 116 spots on the 2-DE pattern, detected at pI 5-7.5 and apparent mass 8-60 kDa, were excised and subjected to MALDI-MS measurements and the mass spectra were analyzed using the software of peptide mass fingerprinting (PMF) Mascot and ProFound to assign the proteins. Many of the spots were assigned to contain fibrinogen α chain, especially those at pI 5.5-7.5 and apparent mass 8-40 kDa, suggesting that these spots are its fragments. The distribution of the MS-detected peptide fragments suggested that the molecular-mass heterogeneity might be caused by the cleavage of multiple sites on the α chain. Care must be taken to keep the temperature of IEF gels at around 4°C during electrophoresis, when human plasma proteins are subjected to non-denaturing IEF. The absence of the spots of fibrinogen fragments on Type-II 2-DE gels would validate the intactness of plasma proteins. The advantages of micro gel system for the analysis of intact protein mixtures are suggested.

To elucidate the current status of the dosage regimen of digoxin (DX), we reviewed its therapeutic drug monitoring (TDM) data obtained from 50 patients on whom TDM was performed for the first time following the beginning of DX treatment. The serum trough level of DX in 50% the subjects deviated from its therapeutic range (0.5-1.5 ng/ml) because the uniform dosage (0.125-0.148 mg/day) was administered for the patients with various degrees of renal functicns. The serum levels of DX in about 70% of the patients with normal renal function (80 ml/min creatinine clearance. CLcr) were less than its lower limit (0.5 ng/ml), and those in about 70% of patients with severe abnormal renal function (CLcr<40 ml/min) exceeded its upper limit (1.5 ng /ml).To design a simple index for the dosage regimen of DX whith closely corresponded to the renal functions in patients, 286 samples of TDM data obtained from 87 patients who were not coadministered drugs known to alter DX pharmacokinetics were reviewed by dividing then into three groups according to the CLcr values. The mean serum DX levels (0.7-1.2 ng/ml) in each group could be kept at their therapeutic range, as the dosages were respectively 0.25 mg/day (80 CLcr group), 0.125-0.25 mg/day (40

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