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

Purpose. To evaluate the transcellular mechanism of novel enhancers absorpt ion enhancement of human growth hormone (hGH), by examining the involvement of a P-glycoprotein-like efflux system, changes in membrane fluidity, and membrane damage. Methods. Caco-2 cell monolayers were grown on Snapwell(R) filter supports a nd placed in a side-by-side diffusion apparatus. Transport in both the apic al to basolateral (AP to BL) and basolateral to apical (BL to AP) direction was measured at different temperatures and in the presence of potential in hibitors. Fluorescence anisotropy measurement was used to measure membrane fluidity. The fluorescence anisotropy of DPH- and TMA-DPH-labeled cell susp ensions was measured at room temperature. LDH (a measure of cytosolic lacta te dehydrogenase) leakage assay was used to evaluate cytotoxicity. Results. The bi-directional transepithelial fluxes of hGH in the presence o f these novel enhancers across Caco-2 cells showed marked asymmetry. Averag e 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-lik e 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 nov el enhancers. In addition, lowering the temperature to 25 degrees C complet ely eliminated the asymmetry of hGH transport in the presence of novel enha ncers. It was also shown by fluorescence anisotropy that these novel enhanc ers alone only slightly increased membrane fluidity. On the other hand, upo n addition of hGH to the novel enhancers, the cell membrane showed a dramat ic 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 poin t is also much lower than other known enhancers. Conclusions. This study shows that human growth hormone alone cannot be tra nsported across Caco-2 cells, except in small quantities, by passive diffus ion, but in the presence of novel enhancers, human growth hormone permeatio n is substantial. In addition, the asymmetry of transport of the complexed hGH appears to be due to a P-gp-like efflux system.(3) Assuming that the pr esent substrate specificity of the P-gp-like efflux system shows the same p reference for hydrophobic molecules as p-gp, the present work also indirect ly shows that human growth hormone has become more lipophilic in the presen ce of these novel enhancers. Furthermore, membrane fluidity data also suppo rts the premise that these novel enhancers interact and stabilize hGH, to m ake them mon hydrophobic and easier to be transported through cell membrane s.