Mechanistic study of physicochemical and biochemical processes affecting intestinal absorption of the sesquiterpene lactone nobilin from multi-component systems in the Caco-2 model

The overall aim of this work was to investigate the intestinal absorption of the sesquiterpene lactone nobilin from the flowers of Anthemis nobilis L. (Chamomillae romanae flos) in the in vitro Caco-2 model and identify strategies for its improvement. The influence of physicochemical processes i.e., stability and solubility and biochemical processes i.e., bioconversion and membrane permeation by passive diffusion and carrier mediated transport on nobilin absorption were elucidated to gain a basic mechanistic understanding of the absorption of the compound. Physiologically based multi-compartment kinetic modeling was employed to delineate the influence of simultaneously acting phenomena. Based on this understanding, possibilities were finally explored to improve absorption of nobilin utilizing plant extract, formulation, and post-prandial application approaches. The thesis is divided into one theoretical part and three experimental studies, each addressing other aspects of physicochemical and biochemical processes affecting nobilin absorption.
The first study focused on chemical degradation of nobilin and its solubility under conditions of concurrent degradation and the effect of biorelevant media used in the in vitro measurement of intestinal absorption. Purely aqueous medium (aq-TMCaco), fasted and fed state simulated intestinal fluid (FaSSIF-TMCaco and FeSSIF-TMCaco), and two liposomal formulations (LiposomesFaSSIF and LiposomesFeSSIF) with the same lipid concentration as FaSSIF-TMCaco and FeSSIF-TMCaco were used. Degradation products were identified by NMR and X-ray crystallography and the order of reaction kinetics was determined. Solubility was deduced with a mathematical model encompassing dissolution and degradation kinetics that took into account particle size distribution of the solid material. Degradation mechanism of nobilin involved water catalyzed opening of the lactone ring and transannular cyclization resulting in five degradation products. Degradation followed first order kinetics in aq-TMCaco and FaSSIF-TMCaco and higher order kinetics in FeSSIF-TMCaco and the two liposomal formulations while degradation in the latter media was reduced. Solubility of purified nobilin increased in the order: aq-TMCaco < FaSSIF-TMCaco < LiposomesFaSSIF < FeSSIF-TMCaco < LiposomesFeSSIF. Improvement of stability and solubility of nobilin was consistent with incorporation of the molecule into colloidal lipid particles. The developed kinetic model is proposed to be a useful tool for deducing solubility of a highly unstable compound with a wide particle size distribution.
In the second study, the role of bioconversion and carrier mediated efflux of conjugation products in the absorption mechanism of nobilin in the Caco-2 model and the impact of the extract of Chamomillae romanae flos and its ingredients on bioconversion and efflux was investigated. Permeation and bioconversion parameter values were deduced by means of kinetic multi-compartment modeling. Nobilin exhibited high permeability, low absorption and fast bioconversion producing glucuronide, cysteine conjugate, and glutathione conjugate that were transported by P-gp (the first two), apical MRP2 and basal MRP3 and possibly MRP1 out of the cell. Inhibition of efflux resulted in diminished bioconversion and improved absorption. The extract increased the relative fraction absorbed primarily by directly inhibiting bioconversion but also by reducing efflux. This effect of the extract was only partly explained by its individual ingredients. The transport-bioconversion interplay is shown to be a possible mechanism for increasing absorption of a compound undergoing extensive bioconversion. Plant extracts may increase absorption by this mechanism in addition to metabolic enzyme inhibition.
The focus of the third study was the effect of biorelevant media and two lipid based formulations (SMEDSS1 and SMEDDS2) on in vitro absorption of nobilin applied as a pure compound or as a full ethanolic extract of Chamomillae romanae flos. Permeation and bioconversion parameters were deduced by means of kinetic multi-compartment modeling. FaSSIF-TMCaco reduced apical efflux of all conjugates but did not affect nobilin absorption while FeSSIF-TMCaco increased absorption which was attributed to the increased chemical stability in the medium and decreased bioconversion while permeability coefficient was also decreased. SMEDDS1 increased and SMEDDS2 decreased absorption due to a decrease and an increase of bioconversion, respectively, although both formulations improved chemical stability and decreased permeability coefficient. The effect of FeSSIF-TMCaco, SMEDDS1, and SMEDDS2 is consistent with an incorporation of nobilin into colloidal lipid particles while FeSSIF-TMCaco, SMEDDS1 appeared to inhibit and SMEDDS2 to promote enzymatic reaction in the cell. In combination with the extract, no additional effect was observed. Formulation and transport media influence, therefore, in vitro absorption by changing bioconversion, chemical stability and permeation parameters.
This work gives an insight in the absorption mechanism of nobilin and shows the effect of physicochemical and biochemical processes on absorption and possibilities to improve it. With multi-compartment modeling simultaneous processes which took place during absorption were unraveled and their contribution to absorption was elucidated. This technique allowed studying the effect of external factors such as inhibitors of transporters, plant extract, formulations, and biorelevant media on individual processes, on the interplay between these processes and further on the entire absorption mechanism. The work contributes towards raising the awareness that intestinal absorption is a complex puzzle of different processes which can be influenced by external factors and which bear a high potential for interaction.