In vitro modulation of multidrug resistance by pregnane steroids and in vivo inhibition of tumour development by 7α-OBz-11α(R)-OTHP-5ß-pregnanedione in K562/R7 and H295R cell xenografts.

Synthetic progesterone and 5α/ß-pregnane-3,20-dione derivatives were evaluated as in vitro and in vivo modulators of multidrug-resistance (MDR) using two P-gp-expressing human cell lines, the non-steroidogenic K562/R7 erythroleukaemia cells and the steroidogenic NCI-H295R adrenocortical carcinoma cells, both resistant to doxorubicin. The maximal effect in both cell lines was observed for 7α-O-benzoyloxy,11α(R)-O-tetrahydropyranyloxy-5ß-pregnane-3,20-dione 4. This modulator co-injected with doxorubicin significantly decreased the tumour size and increased the survival time of immunodeficient mice xenografted with NCI-H295R or K562/R7 cells.

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.

Intravenous injection of human sex steroid hormone-binding globulin in mouse decreases blood clearance rate and testicular accumulation of orally administered [2-125I]iodobisphenol A.

Bisphenol A, an environmental compound with estrogenic activity, has been shown to bind human sex steroid hormone-binding globulin (hSHBG), the main plasma transport protein which regulates the metabolism of androgens and estrogens and limits their access to target organs. The present study was conducted to determine whether physiologically relevant concentrations of hSHBG can influence the blood clearance rate of bisphenol A and its accumulation in the testes. A radioactive [2-125I]iodobisphenol tracer was synthesized with an association constant (Ka) for binding to hSHBG of 0.14 +/- 0.01 x 10(6) M(-1) at 37 degrees C, a value much lower than for [2-125I]iodoestradiol, which was also synthesized. We used i.v. injection of immunopurified hSHBG in adult male mice to maintain hSHBG levels within the physiologically possible range for humans (27-267 nM) before gavage administration of [2-125I]iodobisphenol or [2-125I]iodoestradiol, for measuring the blood clearance rate of radioactive signal in blood samples taken during the following 120 min. Testicular accumulation of radioactivity was measured 24 h and 48 h after gavage of [2-125]iodobisphenol A. In mice receiving immunopurified hSHBG or vehicle, the time-dependent blood clearance of radioactivity exhibited a bi-exponential decrease which indicated alpha-diffusion and beta-elimination phases for both radioactive ligands. The presence of circulating hSHBG significantly and dose-dependently lowered the clearance rate of radioactivity. However, much higher circulating levels of hSHBG were required to retard the blood clearance of [2-125I]iodobisphenol A as compared to those required for [2-125I]iodoestradiol, in keeping with the important difference in their respective Ka value for binding to SHBG. In addition, mice treated with hSHBG exhibited significantly (P = 0.036) reduced testicular accumulation of radioactivity 24 h and 48 h after ingestion of [2-125I]iodobisphenol A. Provided that the binding properties of bisphenol A for hSHBG are not substantially different from those measured for [2-125I]iodobisphenol A, these findings suggest that, although hSHBG binds 2-mono-iodobisphenol A with a relatively low binding affinity, high enough concentrations of circulating hSHBG (range concentrations between 85 and 267 nM) are potentially able to exert a protective effect against exposure to bisphenol A.

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.