Various combinations of digestive lipases were tested in vitro under conditions simulating the earlier phases of gastrointestinal lipolysis in the stomach and the duodenum. A solid/liquid test meal was mixed first with either human gastric juice or a solution containing gastric lipase, followed by either the addition of human pancreatic juice and bile or the addition of a solution containing pancreatic lipase, colipase, and bile salts. The rate of lipolysis and the composition of the lipolysis products were assessed as a function of time after lipid extraction and analysis by thin-layer chromatography coupled to flame ionization detection. The lipolytic potential of a crude rabbit gastric extract (RGE) associated with porcine pancreatic extract (PPE) was assessed and compared with the rates of lipolysis of the meal triacylglycerols by human digestive lipases recorded under the same in vitro conditions. RGE combined with PPE appeared to be a good substitute for human gastric and pancreatic lipases. RGE and PPE could therefore be used to simulate the gastrointestinal lipolysis of various foods and emulsions in vitro, as well as that of pharmaceutical lipid formulations.
There is a growing interest in the optimization of dietary emulsions for monitoring postprandial lipid metabolism in the frame of preventing metabolic diseases. Using various emulsions, we investigated in a systematic scheme the combination of (i) in vitro gastrointestinal lipolysis and (ii) absorption and metabolism of lipolysis media in Caco-2 cells. Four emulsions based on either milk fat olein (OL) or rapeseed oil (RA) as the dispersed phase and either lecithin (LE) or sodium caseinate (CA) as the emulsifier were tested. After a sequential incubation of these emulsions with gastric and pancreatic enzymes, lipolysis media were incubated with Caco-2 cells, after dilution (1 : 20) to maintain the barrier integrity. Both gastric and duodenal lipolysis levels were similar to values reported in vivo and the rates of lipolysis were higher with LE-stabilized emulsions than with CA-stabilized emulsions (P < 0.05). TAG secretion by Caco-2 cells was found to be higher using (i) duodenal vs. gastric media (P < 0.001) and (ii) emulsions stabilized with CA vs. LE (P < 0.01). Consistently, gene expression of both FABP2 and FATP4 induced by the duodenal media was (i) higher than that with gastric media (P < 0.001) and (ii) faster than that with model mixed micelles. Using gastric media, TAG secretion of Caco-2 cells after 12 h was higher with RA than with OL (P < 0.001). Moreover, the RA-CA emulsion increased the size of secreted lipoprotein particles (514 nm vs. 61 to 130 nm; P < 0.01). In conclusion, it was possible to observe distinct responses in the lipid metabolism of Caco-2 cells incubated with lipolysis media obtained from different dietary emulsions digested by gastrointestinal lipases in vitro.
Diffuse intrinsic pontine gliomas (DIPG) carry a particularly poor prognosis, with median survival shorter than one year. Recent efforts aimed at traversing the blood-brain barrier have explored the capability of convection-enhanced delivery (CED) as a possible alternative to systemic delivery. Accurate pharmacokinetic information (distribution and clearance) is problematic since most agents have no intrinsic imaging capacity. This information however is critical in designing optimal infusion parameters and monitoring response. The mainstay of current practice is to rely on imaging surrogates to estimate molecular kinetics. These imaging tracers, however, have no homologous bioactive profile with the therapeutic molecule. Our objective of the current work was to perform direct labeling using a positron-emitting [ 18 F]F 2 B-moiety that could be imaged by PET/CT. In the initial phase of this project, we first coupled the kinase-inhibitor dasatinib, a therapeutic chosen for its ability to tolerate the alteration of its molecularstructure. In vitro cell viability assays were performed to confirm that our modifications did not affect drug bioactivity. Labelled therapeutics were then delivered via CED to the frontal lobe in mice. We observed that the clearance properties vary greatly depending on the functional group added to the dasatinib backbone, with both distribution and clearance following injection being group-specific. We subsequently repeated these modifications with panobinostat. We observed that panobinostat distributes very differently from dasatinib, despite their similar molecular weight, both spatially (i.e. brain regions involved) and temporally (different clearance following infusion). This data suggests that surrogate tracers may not adequately estimate pharmacokinetics of locally delivered therapeutic compounds. If chemically possible, PET-imaging affords a more representative assessment of drug kinetics which would be more beneficial in defining dosing and scheduling. Our findings demonstrate the feasibility of designing theranostic compounds that are suitable for accurate pharmacokinetic monitoring with CED.
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