Ana Pilipović

Antitumor activity of newly synthesized oxo and ethylidene derivatives of bile acids and their amides and oxazolines

HIGHLIGHTSRegio‐ and stereoselective synthesis of bile acid C‐7 ethylidene derivatives.21 bile acid, amide, oxazoline or/and ethylidene derivatives were prepared.Antiproliferative activity of compounds was tested against tumor cell lines.Strong antitumor activity of derivatives against MDA‐MB‐231, PC3 or HeLa cell lines. ABSTRACT Bile acid derivatives with modifications in side chain and modifications on steroid skeleton were synthetized and their antitumor activity against five human cancer cell lines was investigated. Modifications in side chain include amid group, formed in reaction with 2‐amino‐2‐methylpropanol, and 4,4‐dimethyloxazoline group, obtained after cyclization of amides. In the steroid skeleton oxo groups were introduced in position 7 (2, 2a, 2b) and 7,12 (3, 3a, 3b). Ethylidene groups were introduced regio‐ and stereoselectively on C‐7, and/or without stereoselectivity on C‐3 by Wittig reaction. By combination of these modifications, a series of 19 bile acid derivatives were synthesized. Compounds containing both C‐7 ethylidene and C‐12 carbonyl groups (6, 6a, 6b) shown very good antitumor activity with IC50 < 5 &mgr;M. Altering carboxylic group to amide or oxazoline group has positive effect on cytotoxicity. Different molecular descriptors were determined in silico and after principal component analysis was found that molecular descriptor BLTF96 can be used for fast assessment of experimental cytotoxicity of bile acid derivatives.

pKa values of hyodeoxycholic and cholic acids in the binary mixed micelles sodium-hyodeoxycholate-Tween 40 and sodium-cholate-Tween 40: Thermodynamic stability of the micelle and the cooperative hydrogen bond formation with the steroid skeleton.

Graphical abstract Figure. No Caption available. HighlightsFor bile acids HD and C pKa values increase in mixed micelles with Tween 40.Increase of &Dgr;pKa of HD and C related to pKa of free HD and C is a measure of mixed micelles stability.H‐bonds between steroids OH‐Tween 40 polyoxyethylene groups: aggregate stability.Cooperative hydrogen bond formation between HD and Tween 40. Abstract Due to a relatively small size of bile acid salts, their mixed micelles with nonionic surfactants are analysed. Of the special interests are real binary mixed micelles that are thermodynamically more stable than ideal mixed micelles. Thermodynamic stability is expressed with an excess Gibbs energy (GE) or over an interaction parameter (&bgr;ij). In this paper sodium salts of cholic (C) and hyodeoxycholic acid (HD) in their mixed micelles with Tween 40 (T40) are analysed by potentiometric titration and their pKa values are determined. Examined bile acids in mixed micelles with T40 have higher pKa values than free bile acids. The increase of &Dgr;pKa acid constant of micellary bound C and HD is in a correlation with absolute values of an interaction parameter. According to an interaction parameter and an excess Gibbs energy, mixed micelle HD–T40 are thermodynamically more stable than mixed micelles C–T40. &Dgr;pKa values are higher for mixed micelles with Tween 40 whose second building unit is HD, related to the building unit C. In both micellar systems, &Dgr;pKa increases with the rise of a molar fraction of Tween 40 in binary mixtures of surfactants with sodium salts of bile acids. This suggests that, &Dgr;pKa can be a measure of a thermodynamic stabilization of analysed binary mixed micelles as well as an interaction parameter. &Dgr;pKa values are confirmed by determination of a distribution coefficient of HD and C in systems: water phase with Tween 40 in a micellar concentration and 1‐octanol, with a change of a pH value of a water phase. Conformational analyses suggests that synergistic interactions between building units of analysed binary micelles originates from formation of hydrogen bonds between steroid OH groups and polyoxyethylene groups of the T40. Relative similarity and spatial orientation of C3 and C6 OH group allows cooperative formation of hydrogen bonds between T40 and HD – excess entropy in formation of mixed micelle. If a water solution of analysed binary mixtures of surfactants contains urea in concentration of 4 M significant decreases of an interaction parameter value happens which confirms the importance of hydrogen bonds in synergistic interactions (urea compete in hydrogen bonds).

Comparison of dissolution profiles and serum concentrations of two lamotrigine tablet formulations.

AbstractObjective: The aim of this study was to investigate the extent of variations in lamotrigine serum concentrations between two immediate-release tablet formulations. Data were compared with in vitro difference and similarity tests on dissolution profiles of the two formulations. Methods: Dissolution characteristics of formulations A (reference) and B (test) were evaluated at three points spanning the physiologic pH range (pH 1.2, pH 4.5, pH 6.8). A model-independent approach of difference (f1) and similarity (f2) tests were applied to dissolution data. A clinical study was performed with 16 patients who were divided into two groups — one group received formulation A (n=9) and the other received formulation B (n=7). Lamotrigine steady-state concentrations were determined by high-performance liquid chromatography on a reverse-phase column. Results: There were no statistically significant differences in lamotrigine serum concentrations between the two groups, although formulation B had slightly higher mean concentration values (formulation A: 3.97±4.1 μg/mL; formulation B: 5.78±2.7 μg/mL). Dissolution profiles of the two formulations were similar in the pH 1.2 dissolution medium; however, the dissolution profiles of formulation B were outside the dissolution limit (≥85% at 15 minutes) in the pH 4.5 and 6.8 dissolution media. Conclusions: No significant changes in the serum concentrations of lamotrigine were seen between the two investigated formulations. There is no evidence to suggest that the differences in dissolution profiles at pH 4.5 and pH 6.8 affect the therapeutic efficacy of the formulations. It is evident that the doses of test formulation given to the patients were higher as a consequence of common assumption that generic products have a lower absorption rate, which is proven unnecessary in this study. This investigation was a pilot study and thus further investigations with a larger sample size are necessary to determine if there is a connection between dissolution profiles and the therapeutic effect of investigated formulations.

Importance of reversed-phase chromatographic parameters in predicting biopharmaceutical and pharmacokinetic descriptors on the group of androgen derivatives

Abstract Nowadays the standard measure of lipophilicity, the logarithm of n‐octanol‐water partition coefficient, logP, is proposed to be replaced with chromatographic techniques. Chromatography techniques (reversed phase thin layer chromatography RPTLC and reversed phase thin layer chromatography RPHPLC) are the most widely used alternatives to the shake flask method. However, it is shown that, by changing the temperature or concentration of organic modifier in the chromatography experiment, it is possible to derive data matrix of retention parameters from which, by principle component analysis, structural characteristics of the examined molecules can be gained. The question may be asked which of the chromatography experimentally obtained and calculated parameters: capacity factor k, &Dgr;Gx (the change in Gibbs energy of binding of molecule for stationary phase), &Dgr;Hx (the change in enthalpy of binding of molecule for stationary phase) or &Dgr;Sx (the change in the entropy of binding of molecule for stationary phase) is the most suitable in describing hydrophobicity. The canonical correlation analysis (CCA) method is used to evaluate the importance of the n functions in explaining the variance of molecular descriptors connected to pharmaceutical processes and wherein molecules hydrophobicity is expressed and possible differences between molecular descriptors with realistic conformations of the analyzed molecules steroid skeleton are discussed. Conformational analysis showed that structure of steroid skeleton in hydrophobicity is most completely described with k or &Dgr;Gx, and connection between conformation of the steroid skeleton and hydrophobicity to a lesser extent is projected on temperature dependence on &Dgr;Hx and similarly on &Dgr;Sx, so in describing molecules hydrophobicity it is necessary to observe entropic as well as enthalpic contribution together, expressed with &Dgr;Gx function. Canonical conformation analysis (CCA) showed that hydrophobicity contained in &Dgr;Gx and k explains 61% of variance represented in in silico descriptors. Analyzed molecular descriptors, derived from different molecules fragments don’t map conformational specifics of those molecules in small groups so recommendation is to use them complementary with chromatographic data in describing hydrophobicity. Graphical abstract Figure. No Caption available.