A.B.J. Noach

Bioadhesive polymers for the peroral delivery of peptide drugs

Abstract Two different classes of bioadhesive excipients which have been approved by the FDA, the anionic charged poly (acrylic acid) derivatives and the cationic charged chitosans, have been investigated with respect to their ability to improve intestinal peptide drug absorption. It was found that both polycarbophil and the chitosan derivatives Daichitosan ® VH and chitosan-glutamate (SeaCure ® + 210) enhance the absorption of the peptide drug 9-desglycinamide, 8-arginine vasopressin (DGAVP) in the vertically perfused intestinal loop model of the rat. Recent studies demonstrated that the two poly (acrylates) polycarbophil and Carbopol ® 934P are able to inhibit the activity of the proteolytic enzyme trypsin at pH 6.7, which may lead to an increased stability of the peptide drug in the intestine. The depletion of Ca 2+ out of the incubation medium due to the Ca 2+ binding properties of the poly (acrylates) is discussed as a possible mechanism of action. Because of the observation that depletion of Ca 2+ can additionally cause an opening of tight junctions, the influence of polycarbophil on the paracellular integrity of Caco-2 monolayers was also investigated by measurements of transepithelial electrical resistance (TEER) as well as by visualization studies using confocal laser scanning microscopy. At pH 4.0, apically applied polycarbophil tended to decrease TEER values stronger than the control solution, whereas at pH 7.0 no pronounced changes of TEER could be observed. At pH 7.4, polycarbophil was only able to increase the paracellular permeability of the hydrophilic model compound fluorescein-isothiocyanate-dextran ( M w 4000) when applied to the basolateral side of the Caco2 cell monolayer. In conclusion, bioadhesive polymers are promising absorption promoting agents for peroral delivery of peptide drugs, and their mechanism of action is probably a combination of inhibiting protease activities and modulating the intestinal epithelial permeability.

Cell-polarity dependent effect of chelation on the paracellular permeability of confluent caco-2 cell monolayers

Abstract To investigate the effect of extracellular chelation at the apical, basolateral or both sides on the resistance and permeability of epithelial cell layers, we used 15 days cultures of a human intestinal adenocarcinoma cell line (Caco-2) and hydrophilic FITC-labeled dextran model compounds of various molecular weights. Transport of these hydrophilic compounds is restricted to the paracellular pathway in which the tight junctions form a barrier. Tight junctions are dependent on extracellular calcium and magnesium for their integrity and function. Calcium and magnesium chelation with 2.5 mM EDTA at the apical and basolateral side of the monolayer resulted in a drastic drop, up to 80% of the initial value, in trans-epithelial electrical resistance after 60 min. Application at the basolateral side resulted in a drop of 40% in resistance, while application on the apical side almost did not give any effect. The same pattern was also found in transepithelial clearance studies with fluorescein-Na and FITC-labeled dextran model compounds with molecular weights ranging from 4000 to 500 000. After 2.5 mM EDTA treatment on both sides a maximal (1400-fold) enhancement in transport clearance occurred for the dextran molecule with molecular weight 20 000 (Stokes-Einstein molecular radius 30 A). For basolateral calcium and magnesium chelation similar results were found, however, with lesser maximal effects. For apical application no transport enhancement could be found with 2.5 mM EDTA. These results have shown that transport of hydrophilic compounds through epithelial monolayers is enhanced more effectively by basolateral application of EDTA than by apical application.

Absorption enhancement of hydrophilic compounds by verapamil in Caco-2 cell monolayers

Caco-2 monolayers were used to determine whether verapamil enhanced the transport of hydrophilic compounds across epithelial cells. Transepithelial electrical resistance (TEER) measurements, as an indicator of the opening of tight junctions, and transport experiments with fluorescein-Na (Flu) and FITC-dextran Mw 4000 (FD-4) were used to assess the effect. (+/-) Verapamil concentrations up to 3 x 10(-4) M increased TEER dose-dependently, whereas from concentrations of 7 x 10(-4) M onwards a dose-dependent drop was found. After removal of verapamil (< 10(-3) M) the effects on TEER were reversible within 30 min. A second administration of verapamil after different time intervals produced a much larger effect on TEER than the first administration. The separate R- and S-enantiomers did not reveal a difference in enantiomer effect. (+/-) Verapamil at 7 x 10(-4) M increased Flu transport about 13-fold and 26-fold after the first and second treatment in the same monolayers, respectively. Transport of FD-4 increased approximately 4-fold and 6-fold after the first and second treatment, respectively. Potential damaging effects were assessed by trypan blue exclusion (cell death) and cell detachment. No cell death occurred at verapamil concentrations of 8.5 x 10(-4) M or lower, whereas cell detachment did not occur within 1 hr at all concentrations used in these experiments. At later times detachment was observed at concentrations of 7 x 10(-4) M and higher. Confocal laser scanning microscopy showed that verapamil opens the paracellular route, thereby enhancing the permeability of hydrophilic compounds. However, relatively high concentrations are needed to achieve this effect and only a narrow concentration range can be used without cytotoxic effects, which limits the potential application of verapamil as an absorption enhancing agent.

Lipid peroxidation and protein thiol depletion are not involved in the cytotoxicity of N-hydroxy-2-acetylaminofluorene in isolated rat hepatocytes

Freshly isolated rat hepatocytes were used to study the mechanism of cell death induced by N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Exposure to 1.0 mM N-OH-AAF resulted in more than 90% cell death (as measured by LDH leakage) of hepatocytes isolated from male rats within 6 hr. Only 36% of the hepatocytes isolated from female rats died within this period. When inorganic sulfate was omitted from the incubation medium, a 6 hr exposure to 1.0 mM N-OH-AAF resulted in only 40% cell death of male hepatocytes. These findings are in accordance with the sex difference and sulfation dependence of N-OH-AAF hepatotoxicity observed in the rat in vivo. N-OH-AAF decreased glutathione (GSH) in male hepatocytes in a concentration-dependent manner. This GSH consumption was only partly dependent on the presence of inorganic sulfate. No lipid peroxidation was observed during N-OH-AAF exposure; N-OH-AAF even prevented endogenous and diethyl maleate (DEM)-induced lipid peroxidation. No reduction of free protein thiol groups was found after exposure to N-OH-AAF, even after 75% cell death had occurred. A reduction of protein thiols after N-OH-AAF exposure was observed in GSH depleted hepatocytes (obtained by DEM plus vitamin E pretreatment). Under these conditions N-OH-AAF-induced cell death occurred earlier. Therefore, GSH protects against protein thiol depletion by N-OH-AAF in control cells. N-OH-AAF-induced cell death was preceded by a loss of intracellular ATP. It is concluded, therefore, that neither lipid peroxidation nor depletion of protein thiols, but possibly loss of intracellular ATP, is involved in the sulfation-dependent cytotoxic mechanism of N-OH-AAF in isolated rat hepatocytes.

Absorption enhancement of a hydrophilic model compound by verapamil after rectal administration to rats.

The use of verapamil as an absorption enhancer for the paracellular route in‐vivo was studied using FITC‐labelled dextran (molecular weight 4000) (FD‐4) as a hydrophilic model compound for transport enhancement.