Michinori Sakai

Biopharmaceutics: Cytotoxicity of Absorption Enhancers in Caco‐2 Cell Monolayers

This study was performed to evaluate the utility of absorption enhancers with reference to mucosal cell cytotoxicity. Overall assessment of the damage to plasma, lysosomal and nuclear membranes by three absorption enhancers, sodium deoxycholate, sodium caprate and dipotassium glycyrrhizinate, was performed on Caco‐2 cell monolayers.

Effects of absorption enhancers on cytoskeletal actin filaments in Caco-2 cell monolayers

Histomorphological changes of actin filaments, intracellular levels of calcium ion, and amount of released lactate dehydrogenase (LDH) were examined, in order to elucidate the mechanism of action of three absorption enhancers, i.e., sodium caprate (Cap-Na), sodium deoxycholate (Deo-Na), and dipotassium glycyrrhizinate (Grz-K), using Caco-2 cell monolayers. The structure of actin filaments in microvilli was slightly modified by 0.5 %(w/v) Grz-K and was significantly changed by 0.2 %(w/v) Cap-Na, 0.05 %(w/v) Deo-Na, and 0.0015 %(w/v) ionomycin. All of the enhancers, except Grz-K, induced significantly histomorphological changes in the actin filaments on the middle depth and basal side of the cells. Furthermore, the altered structure of the actin filaments in the monolayers was restored after removal of the Cap-Na, Grz-K and ionomycin, but not Deo-Na. Intracellular levels of calcium ion increased in the following order: ionomycin = Cap-Na > Deo-Na. However, the intracellular calcium ion levels decreased by treatment with Grz-K. The changes in transepithelial electric resistance (TEER) at the initial stage of treatment with all enhancers correlated with intracellular calcium ion levels. These results suggest that one of the mechanisms by which these agents exert absorption-enhancing activity involves structural alterations in the cytoskeletal actin filaments which are provoked by changes in intracellular calcium ion levels. Only the monolayers which were treated with 0.05 %(w/v) Deo-Na released a significant amount of LDH and irreversibly altered the structure of actin filaments, thus indicating that Deo-Na might affect the actin filaments not only by increasing intracellular calcium ion level but also by other, presently unknown factors.

IN VITRO AND IN VIVO EVALUATION OF THE ENHANCING ACTIVITY OF GLYCYRRHIZIN ON THE INTESTINAL ABSORPTION OF DRUGS

AbstractPurpose. The enhancing activity of dipotassium glycyrrhizinate (Grz) on the intestinal absorption of drugs has been demonstrated in an in vitro study using Caco-2 cell monolayers and in an invivo absorption study in rats. Methods. The hydrolysis of Grz by luminal content and mucosa of the rat colon was investigated. The absorption-enhancing activity of Grz and its hydrolysates was estimated by changes in transepithelial electrical resistance (TEER) and the permeation of sodium fluorescein (Flu-Na) in Caco-2 cell monolayers. It was further evaluated through the absorption of salmon calcitonin (sCT) in the rat colon. Results. Grz was not hydrolyzed to glycyrrhetinylmonoglucuronide (GrMG) and glycyrrhetinic acid (GA) by colonic mucosa, but, rather by the β-glucuronidase in colonic flora. The hydrolysis of Grz to GrMG was extremely slow and the GrMG produced was rapidly regenerated to GA. Grz and GrMG had no effect on TEER nor on the permeability of Flu-Na across Caco-2 cell monolayers. On the other hand, GA decreased TEER and increased the permeability of Flu-Na in a dose-dependent manner. However, Grz and GrMG enhanced the plasma calcium-lowering effect of sCT after administration in the rat colon. The coadministration of sCT and GA in the rat colon induced the strongest plasma calcium-lowering effect and the highest plasma concentration of sCT. Conclusions. The in vivo enhancing-activity of Grz in the absorption of drugs is dependent on GA, a hydrolysis product of Grz resulting from the action of β-glucuronidase in intestinal flora.

Simultaneous Use of Sodium Deoxycholate and Dipotassium Glycyrrhizinate Enhances the Cellular Transport of Poorly Absorbed Compounds Across Caco‐2 Cell Monolayers

The absorption‐enhancing effect of a combination of sodium deoxycholate and dipotassium glycyrrhizinate in Caco‐2 cell monolayers has been compared with that of the enhancers when used alone, and the mechanism of the enhancement was partially elucidated.

Anti-emetic effects of a novel NK-1 receptor antagonist HSP-117 in ferrets

We have developed a non-peptide compound, HSP-117, antagonist of the tachykinin NK-1 receptor. Binding of 3H-substance P (SP) to the membranes of IM-9 cells was inhibited by the antagonists HSP-117 and CP-99,994, the inhibitory activity of HSP-117 being about 50-fold that of CP-99,994. The SP-induced firing responses of single neuron activity in slices of the nucleus tractus solitarius of ferrets were inhibited by 10 microM HSP-117. Intracerebroventricular injection of HSP-117 significantly inhibited retching and vomiting induced by copper sulphate and morphine and the inhibitory effect of HSP-117 on emesis was greater than that of CP-99,994. These results indicate that (1) HSP-117 is a potent anti-emetic agent, blocking NK-1 receptors in the nucleus tractus solitarius and (2) NK-1 receptors in the nucleus tractus solitarius play an important role in emesis induced by broad-spectrum emetic stimuli.