Deoxycholic acid derivatives as inhibitors of P-glycoprotein-mediated multidrug efflux.

Deoxycholic acid derivatives were designed as P-glycoprotein (Pgp, ABCB1) inhibitors. Thus the synthesis and the biological activity of methyl deoxycholate derivatives 5-10 and their ether analogs 15-20 have been reported. The potency of these compounds to modulate Pgp-mediated MDR was evaluated through daunorubicin accumulation and potentiation of doxorubicin cytotoxicity in K562/R7 multidrug resistant cells overexpressing Pgp. In parallel, their intrinsic toxicity was appreciated on K562 sensitive cells. Methyl 12α-[(2R or 2S) tetrahydro-2H-pyran-2-yloxy]-3-oxo-5ß-cholan-24-oate 9b has shown a good efficiency as a Pgp inhibitor and a low intrinsic toxicity. Therefore, this derivative constitutes a new lead compound which can be used as a starting point to improve the design of non-toxic Pgp modulators.

Synthesis of new steroidal inhibitors of P-glycoprotein-mediated multidrug resistance and biological evaluation on K562/R7 erythroleukemia cells.

A simple route for improving the potency of progesterone as a modulator of P-gp-mediated multidrug resistance was established by esterification or etherification of hydroxylated 5α/ß-pregnane-3,20-dione or 5ß-cholan-3-one precursors. X-ray crystallography of representative 7α-, 11α-, and 17α-(2'R/S)-O-tetrahydropyranyl ether diastereoisomers revealed different combinations of axial-equatorial configurations of the anomeric oxygen. Substantial stimulation of accumulation and chemosensitization was observed on K562/R7 erythroleukemia cells resistant to doxorubicin, especially using 7α,11α-O-disubstituted derivatives of 5α/ß-pregnane-3,20-dione, among which the 5ß-H-7α-benzoyloxy-11α-(2'R)-O-tetrahydropyranyl ether 22a revealed promising properties (accumulation index 2.9, IC50 0.5 µM versus 1.2 and 10.6 µM for progesterone), slightly overcoming those of verapamil and cyclosporin A. Several 7α,12α-O-disubstituted derivatives of 5ß-cholan-3-one proved even more active, especially the 7α-O-methoxymethyl-12α-benzoate 56 (accumulation index 3.8, IC50 0.2 µM). The panel of modulating effects from different O-substitutions at a same position suggests a structural influence of the substituent completing a simple protection against stimulating effects of hydroxyl groups on P-gp-mediated transport.

A low alpha-linolenic intake during early life increases adiposity in the adult guinea pig.

BACKGROUND: The composition of dietary fatty acids (FA) during early life may impact adult adipose tissue (AT) development. We investigated the effects of alpha-linolenic acid (ALA) intake during the suckling/weaning period on AT development and metabolic markers in the guinea pig (GP). METHODS: Newborn GP were fed a 27%-fat diet (w/w %) with high (10%-ALA group), moderate (2.4%-ALA group) or low (0.8%-ALA group) ALA content (w/w % as total FA) until they were 21 days old (d21). Then all animals were switched to a 15%-fat diet containing 2% ALA (as total FA) until 136 days of age (d136). RESULTS: ALA and docosapentaenoic acid measured in plasma triglycerides (TG) at d21 decreased with decreasing ALA intake. Total body fat mass was not different between groups at d21. Adipose tissue TG synthesis rates and proliferation rate of total adipose cells, as assessed by 2H2O labelling, were unchanged between groups at d21, while hepatic de novo lipogenesis was significantly 2-fold increased in the 0.8%-ALA group. In older GP, the 0.8%-ALA group showed a significant 15-%-increased total fat mass (d79 and d107, p < 0.01) and epididymal AT weight (d136) and tended to show higher insulinemia compared to the 10%-ALA group. In addition, proliferation rate of cells in the subcutaneous AT was higher in the 0.8%-ALA (15.2 +/- 1.3% new cells/5d) than in the 10%-ALA group (8.6 +/- 1.7% new cells/5d, p = 0.021) at d136. AT eicosanoid profiles were not associated with the increase of AT cell proliferation. CONCLUSION: A low ALA intake during early postnatal life promotes an increased adiposity in the adult GP.

Development of a radioimmunoassay for the measurement of Bisphenol A in biological samples.

Bisphenol A (BPA) is widely used in the manufacturing of polycarbonate plastic food and drink packaging. Possessing a weak estrogenic activity, BPA is listed among a growing list of endocrine disrupting compounds. In this study, a polyclonal anti-BPA antibody was obtained by immunization with BPA-monocarboxymethylether covalently linked to BSA. The antibody demonstrates negligible cross-reactivity with most analogous BPA phenolic structures, and no cross-reactivity with endogenous steroids. An extraction step with ethyl acetate minimized matrix effects and allowed the BPA measurement in plasma and other biological samples. Recovery after loading test was 96 +/- 4% and dilution tests had a linear profile (r2 > 0.93). The limit of detection of the BPA RIA was 0.08 microg L(-1) with an IC50 of 1.25 microg L(-1). The intra- and inter-assay coefficients of variation were 5.6 and 8.6%, respectively at a BPA concentration of 0.7 microg L(-1) and 6.9 and 5.7% at a BPA concentration of 1.3 microg L(-1). A significant correlation was found between the values obtained by the RIA and HPLC-MS (r2 = 0.92) or HPLC coupled to a fluorescence detector (r2 = 0.80). In conclusion, we described a BPA-RIA that is a suitable tool for evaluating human exposure to BPA.

Functional characterization of an anti-estradiol antibody by site-directed mutagenesis and molecular modelling: modulation of binding properties and prominent role of the V(L) domain in estradiol recognition.

The high-affinity monoclonal anti-estradiol antibody 9D3 presents a specificity defect towards estradiol-3-sulphate and 3-glucuronide conjugates incompatible with use in direct immunoassays. The corresponding single-chain variable fragment (scFv), cloned and produced in E. coli, exhibited a 10-fold lower affinity for estradiol (K(a)=1.2 x 10(9) M (-1)) and a slightly increased specificity defect for the 3-position. Site-directed mutagenesis revealed critical residues involved in estradiol recognition and produced mutants exhibiting up to a 3-fold increase of the binding affinity for estradiol and up to a 2-fold decrease of the cross-reactivity with estradiol-3-sulphate. A comparative model of the antibody 9D3-estradiol complex was built in which the estradiol D-ring is buried into the binding pocket while the 3-, 6- and 7-positions are solvent exposed, agreeing with the lack of specificity for these three positions. Two potential alternative orientations of the A-ring, one close to CDR H3 and L2 loops, and the other one close to CDR H2 and L3 loops, have been considered for the docking of estradiol, none of which could be unambiguously privileged taking into account data from cross-reactivity measurements, photolabelling and mutagenesis studies. For both orientations, estradiol is stabilized by hydrogen bonding of the 17beta-OH group with TyrL36, His89 and GlnH35 in the first case, or TyrL36, only, in the second case and by van der Waals contacts from TyrL91 with alpha- or beta-face of estradiol, respectively, and from ValH95 and GlyH97 with the opposite face. To elucidate the molecular basis of antibody 9D3 specificity, as compared with that of another anti-estradiol antibody 15H11, single variable domains (V(H) and V(L)) and scFv hybrids have been constructed. The binding activity of V(L)9D3 as well as the specificity of the V(L)9D3/V(H)15H11 hybrid, both similar to antibody 9D3, revealed a prominent role of V(L) in estradiol recognition. These findings establish premises for antibody engineering to reduce cross-reactivity, especially with estradiol-3-conjugates.