Interaction between hydroxyethyl starch and propofol: computational and laboratorial study

<div><p>Background: Hydroxyethyl starch (HES) is one of the most used colloids for intravascular volume replacement during anesthesia. Aim: To investigate the existence of a chemical interaction between HES and the anesthetic propofol by <i>in vitro</i> propofol dosing, computational docking, and examination of a complex between propofol and HES by infrared (IR), ultraviolet (UV), and ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. Methods: Ten samples with human plasma mixed with HES or lactated Ringers (<i>n</i> = 5 for each fluid) were prepared, and the propofol free fraction was quantified until 50 min, using gas chromatography-mass spectrometry. The docking study was performed between HES and propofol and compared with controls. The binding affinities between HES and the small molecules were evaluated by binding free energy approximation (ΔGb, kJ mol⁻¹). The IR, UV, and NMR spectra were measured for propofol, HES, and a mixture of both obtained by the kneading method. Results: Propofol concentrations were significantly lower in the HES samples than in the LR samples (<i>p</i> = .021). The spectroscopic characterization of propofol combined with HES revealed differences in spectra and docking studies reinforced a potential interaction between propofol and HES. Conclusions: Propofol and HES form a complex with different physical-bio-chemical behavior than the single drugs, which may be an important drug interaction. Further studies should evaluate its clinical effects.</p></div

RedRif docked on P-glycoprotein.

<p>a) Top and b) side views. c) Detailed view of RedRif interactions with residues on P-gp. Hydrogen interactions are represented with yellow dashes. Other interactions are represented with pink dashes.</p

Rif and RedRif’s effect on P-glycoprotein expression.

<p>Cells were exposed to 10 µM Rif or RedRif and P-gp expression was evaluated by western blot after 24, 48 and 72 h of exposure, using C219 anti-P-gp antibody. Rif significantly increased P-gp expression after 72 h while RedRif induced a significant increase in P-gp expression from 48 h on. Results refer to mean ± SD of 3 or 4 independent experiments. Differences between treated and untreated cells were estimated using two-way ANOVA followed by Bonferroni’s multiple comparison <i>post hoc</i> test. ***p<0.001 and ****p<0.0001 <i>vs</i>. control.</p

Reversal of RedRif-induced P-glycoprotein protective effect against paraquat cytotoxicity.

<p><b>a</b>) Effect of P-gp blockade by potent P-gp inhibitor, GF120918, in cells simultaneously exposed for 48h to RedRif and PQ, with (black bars; dashed line) or without (grey bars; filled line) GF120918. <b>b</b>) All RedRif-induced protective effect was mediated by P-gp as no differences were observed between PQ-only and RedRif+PQ+GF120918-treated cells. Two-way ANOVA was performed to estimate the differences between RedRif+PQ or PQ (black bars) and RedRif+PQ+GF120918 treatment (grey bars) for each PQ concentration. Concentration–response curves were fitted using least squares as the fitting method, and the comparisons between the curves obtained in the presence and the absence of GF120918 (bottom, top and EC₅₀) were made using the extra sum-of-squares F test. At least 3 independent experiments were performed in triplicate. Significant differences were observed in the presence of GF120918. *p<0.05, **p<0.01 and ***p<0.001 for differences related to the presence of GF120918 and for differences between the curves; $p<0.05 for differences in EC₅₀<i>vs</i>. control.</p

RedRif’s effect on paraquat-induced cytotoxicity.

<p>The NR uptake assay was performed to assess RedRif’s effect in PQ cytotoxicity in a <b>a</b>) simultaneous exposure to RedRif and PQ for 48 h – study of P-gp activation effect, and <b>b</b>) 24 h, <b>c</b>) 48 h and <b>d</b>) 72 h of exposure to RedRif before exposure to PQ – study of P-gp induction effect. RedRif’s protective effect against PQ-induced cytotoxicity was more significant in the simultaneous exposure assay. Two-way ANOVA was performed to estimate the differences between control (black bars) and RedRif-treated (grey bars) cells for each PQ concentration. Concentration–response curves shown as inserts were fitted using least squares as the fitting method, and the comparisons between the curves obtained in the presence and the absence of RedRif (bottom, top and EC₅₀) were made using the extra sum-of-squares F test. At least 3 independent experiments were performed in triplicate. **p<0.01 and ***p<0.001 and ****p<0.0001 for differences between control and RedRif-treated cells for each PQ concentration and for differences between the curves <i>vs</i>. control; $p<0.05 and$ $p<0.01 for differences in EC₅₀<i>vs</i>. control.</p

Reaction scheme for the synthesis of Rif’s derivatives.

<p>Rifampicin (Rif) was used as a model compound for the synthesis of three new derivatives on the search for new P-glycoprotein activators/inducers. A reduced derivative (RedRif), a peracetylated derivative (PerAcRif) and a mono-methoxylated derivative (MeORif) were obtained, as represented in the scheme. RT stands for room temperature.</p

RedRif’s effect on P-glycoprotein activity – rhodamine 123 efflux ratio.

<p>P-gp activity is proportional to the ratio between Rho123 intracellular fluorescence from inhibited (+CyA) and non-inhibited (-CyA) cells. A significant increase in P-gp activity was found in RedRif-treated cells after 24 (P-gp activation effect) and 72h (P-gp induction effect) of exposure. Rif did not significantly change P-gp activity. Results refer to mean ± SD of at least 3 independent experiments performed in triplicate. Differences between treated and untreated cells were estimated using two-way ANOVA followed by Bonferroni’s multiple comparison <i>post hoc</i> test. ***p<0.001 <i>vs</i>. control.</p

Development of Novel Rifampicin-Derived P-Glycoprotein Activators/Inducers. Synthesis, <i>In Silico</i> Analysis and Application in the RBE4 Cell Model, Using Paraquat as Substrate

<div><p>P-glycoprotein (P-gp) is a 170 kDa transmembrane protein involved in the outward transport of many structurally unrelated substrates. P-gp activation/induction may function as an antidotal pathway to prevent the cytotoxicity of these substrates. In the present study we aimed at testing rifampicin (Rif) and three newly synthesized Rif derivatives (a mono-methoxylated derivative, MeORif, a peracetylated derivative, PerAcRif, and a reduced derivative, RedRif) to establish their ability to modulate P-gp expression and activity in a cellular model of the rat’s blood–brain barrier, the RBE4 cell line P-gp expression was assessed by western blot using C219 anti-P-gp antibody. P-gp function was evaluated by flow cytometry measuring the accumulation of rhodamine123. Whenever P-gp activation/induction ability was detected in a tested compound, its antidotal effect was further tested using paraquat as cytotoxicity model. Interactions between Rif or its derivatives and P-gp were also investigated by computational analysis. Rif led to a significant increase in P-gp expression at 72 h and RedRif significantly increased both P-gp expression and activity. No significant differences were observed for the other derivatives. Pre- or simultaneous treatment with RedRif protected cells against paraquat-induced cytotoxicity, an effect reverted by GF120918, a P-gp inhibitor, corroborating the observed P-gp activation ability. Interaction of RedRif with P-gp drug-binding pocket was consistent with an activation mechanism of action, which was confirmed with docking studies. Therefore, RedRif protection against paraquat-induced cytotoxicity in RBE4 cells, through P-gp activation/induction, suggests that it may be useful as an antidote for cytotoxic substrates of P-gp.</p> </div