Transcellular and lipophilic complex-enhanced intestinal absorption of human growth hormone

To evaluate the transcellular mechanism of novel enhancers absorption enhancement of human growth hormone (hGH), by examining the involvement of a P-glycoprotein-like efflux system, changes in membrane fluidity, and membrane damage.

Caco-2 cell monolayers were grown on Snapwell filter supports and placed in a side-by-side diffusion apparatus. Transport in both the apical to basolateral (AP to BL) and basolateral to apical (BL to AP) direction was measured at different temperatures and in the presence of potential inhibitors. Fluorescence anisotropy measurement was used to measure membrane fluidity. The fluorescence anisotropy of DPH- and TMA-DPH-labeled cell suspensions was measured at room temperature. LDH (a measure of cytosolic lactate dehydrogenase) leakage assay was used to evaluate cytotoxicity.

The bi-directional transepithelial fluxes of hGH in the presence of these novel enhancers across Caco-2 cells showed marked asymmetry. Average permeability coefficient values obtained in the apical to basolateral (AP to BL) direction were lower than those of the reverse (BL to AP) direction. On the other hand, the fluxes for hGH alone were symmetric. When P-gp-like efflux inhibitors were included in the transport medium, the permeability coefficient value of BL to AP direction was significantly decreased while the transport was increased in the reverse direction in the presence of novel enhancers. In addition, lowering the temperature to 25 degrees C completely eliminated the asymmetry of hGH transport in the presence of novel enhancers. It was also shown by fluorescence anisotropy that these novel enhancers alone only slightly increased membrane fluidity. On the other hand, upon addition of hGH to the novel enhancers, the cell membrane showed a dramatic change as compared to treatment with novel enhancers alone. The results from the LDH assay showed that the novel enhancers and/or hGH did not cause cell damage, at least up to 1 hour, and the damage seen at the 2 hour point is also much lower than other known enhancers.

This study shows that human growth hormone alone cannot be transported across Caco-2 cells, except in small quantities, by passive diffusion, but in the presence of novel enhancers, human growth hormone permeation is substantial. In addition, the asymmetry of transport of the complexed hGH appears to be due to a P-gp-like efflux system. Assuming that the present substrate specificity of the P-gp-like efflux system shows the same preference for hydrophobic molecules as p-gp, the present work also indirectly shows that human growth hormone has become more lipophilic in the presence of these novel enhancers. Furthermore, membrane fluidity data also supports the premise that these novel enhancers interact and stabilize hGH, to make them more hydrophobic and easier to be transported through cell membranes.