Influence of physicochemical parameters on intestinal absorption of pharmacological substances with Caco-2 cells, an in vitro model of the intestinal epithelium : application to Desferrioxamine B
(2004)
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- Authors
- Supervisors
- Schneider, Yves-Jacques
- Abstract
- In addition to pharmacological activity, the bioavailability of a drug candidate, that includes solubility, absorption, distribution, metabolism and excretion, is an important feature for the development of a new potent drug. The introduction of modern technologies having resulted in a serious increase in the number of these drug candidates, rapid assessment of the potential of a new molecule is of prime importance. On one hand, it is known that physicochemical properties of molecules can affect their bioavailability, and particularly their intestinal absorption. On the other hand, the chelation of iron in overloaded patients is still a problem, principally be due to the poor oral bioavailability of the Desferrioxamine B (DFO), the drug that is clinically used since several decades. Because of this lack of effectiveness when given orally, the molecule has to be administrated subcutaneously, which is a mode of administration very constraining and expensive. Without wanting in any manner to develop a new orally active drug, the aim of this study was to investigate if chemical modifications of the DFO-molecule, by changing its physicochemical properties, could affect its cellular pharmacology and, particularly, its intestinal absorption and cellular accumulation. The interest of DFO in this perspective is the presence of a terminal amine function accessible to chemical modification and not implicated in the iron complexation. The first part of this study describes therefore the choice of the DFO derivatives, that were the tritiated dimethylated (diMe-) and acetylated (Ac-) derivatives and the fluorescent NBD- and FITC-conjugates, as well as their synthesis. The iron-complexed forms of these molecules constituted four additional compounds for our analysis. In the second part of this thesis, we have examined the intestinal absorption of the DFO derivatives that were synthesised, with the Caco-2 cell culture system, validated and widely used as an in vitro model of the intestinal barrier. The interest of this model is that it integrates numerous important parameters implicated in the intestinal absorption and/or metabolism, i.e. intestinal enzymes, ionic and active transporters as well as drug efflux systems, revealed by the transport of reference compounds on the culture conditions used in our laboratory. The transport of the DFO derivatives across Caco-2 cell monolayers was therefore measured in the directions of absorption (apical to basolateral passage) and excretion (basolateral to apical passage), and was compared to that of simultaneously incubated [14C]-mannitol that is a classical marker for the paracellular route. The obtained results showed that the dimethyl-, acetyl- and FITC-derivatives pass through the cell monolayer exactly as mannitol, indicating a probable paracellular transport. However, for an unknown reason, the excretion of both FITC-conjugates was weaker than that of mannitol. The NBD-derivatives presented a higher transport rate than mannitol in both directions, even higher for the iron-complexed form than for the iron-free one. Furthermore, the excretion of both compounds was higher than their absorption, resulting in a net excretory flux. A transcellular, diffusion-mediated route is therefore proposed to occur, with the recognition of the apical P-glycoprotein (P-gp) by these NBD-conjugates being suggested to be responsible for their net excretory flux. These transport results were then compared and correlated with the various theoretical models of prediction of intestinal permeability, based on the measured and/or calculated physicochemical properties of these molecules. Whereas quite good correlations were observed for reference compounds tested in the same culture conditions, no correlation could be found between any of the analysed physicochemical properties, or combinations of it, for our synthesised DFO derivatives. All the models led to an underestimation of the permeability of our conjugates. Nevertheless, this analysis revealed that an important parameter governing the passage of these high molecular weight compounds could be their lipophilicity, with the more lipophilic NBD-derivatives passing more than the less lipophilic dimethyl-, acetyl- and FITC-conjugates. Indeed, these theoretical models are largely based on compounds that undergo a transcellular transport, and could not be as adequate for compounds transported by the paracellular route or by carrier-mediated or drug efflux systems. In the third part of this work, the effects of some experimental conditions, i.e. addition of methylamine, passage at 4°C, energy depletion, addition of verapamil or EGTA, were assayed and seem to confirm the preliminary results : a paracellular passage for diMe-, Ac- and FITC-DFO/FO and a transcellular passage implying a P-gp-mediated efflux for the NBD-derivatives. The fourth part of this thesis is devoted to the study of the intracellular accumulation of the DFO derivatives in Caco-2 cells. From the amount of compounds recovered cell-associated at 4°C, we showed that membrane adsorption is likely to occur, governed by the hydrogen bonding capacity and by the net charge of the molecules at the considered pH. In addition, intracellular accumulation could result from adsorptive and/or fluid phase endocytosis of the more hydrophilic compounds, whereas the more lipophilic ones could additionally diffuse within the cells. Furthermore, NBD-DFO presented also a polarity in its uptake, that was lost when the P-gp inhibitor verapamil or metabolic inhibitors were added. This seems to confirm that this compound could be a substrate of the P-gp, limiting its intracellular accumulation. However, NBD-FO accumulated itself more than all the other compounds. It is suggested that this molecule could have a very high affinity for an unknown intracellular compartment or entity. By this way, the capacity of the P-gp for this molecule could be exceeded, explaining the absence of polarity of the uptake and the absence of effect of verapamil on the accumulation but not on the transcellular transport of this molecule. Intracellular localisation of the fluorescent derivatives by confocal laser scanning microscopy has helped us to make these hypotheses. Finally, in the fifth part of this study, the accumulation of our series of DFO derivatives was studied in cultured J774 macrophages that are non polarised cells likely to concentrate molecules and to represent an important target for iron deposition. The profiles of accumulation and the clearance rates at steady state were measured. The results obtained are quite similar to those obtained with Caco-2 cells. However, the rapid saturation of an intracellular site, yet unknown, seems to limit the accumulation of the NBD-conjugates in these cells. In conclusion, modification of the physicochemical properties of this family of compounds, by chemical modification, may lead to an improved intestinal absorption, as in the case of the NBD-derivatives, even though the fact that these molecules could be substrates of the P-gp could be a limiting factor. Also, if only the theoretical models of prediction of intestinal absorption are considered, they fail to correctly predict the measured intestinal absorption of all these derivatives. It is therefore proposed that the in vitro experimental approach with Caco-2 cells would be more relevant for the study of the oral absorption, even though solutions have to be suggested to encounter the need for high throughput screening. This model, by solving the intestinal transport mechanisms, would therefore also help to optimise the chemical structure of potent drug candidates.
- Affiliations
Citations
Hody, M. (2004). Influence of physicochemical parameters on intestinal absorption of pharmacological substances with Caco-2 cells, an in vitro model of the intestinal epithelium : application to Desferrioxamine B. https://hdl.handle.net/2078.5/97489