Anti-estrogen interaction with uterine estrogen receptors. Studies with a radiolabeled anti-estrogen (CI-628)

Radiolabeled anti-estrogen α-[4-pyrrolidinoethoxy]phenyl-4- methoxy-α'-nitrostilbene ([³H]CI-628) of high specific activity (16 Ci/mmol) and radiochemical purity (>95%) has been prepared by catalytic tritium-iodine exchange and purified by alumina column chromatography. Its interaction with estrogen receptors in immature (day 20 to 24) rat uterus is studied in vitro and in vivo. [³H]CI-628 (CI) competes with estra-1,3,5(10-triene-3,17β-diol (estradiol (E₂)) for binding to specific cytoplasmic estrogen receptors, but has a lower binding affinity (CI: K(d) = 1.7 x 10^-9 M; E₂: K(d) = 1 x 10^-10 M, at 0-4°, determined at equilibrium by charcoal/dextran adsorption). Both [³H]CI and [³H]E₂ have similar rates of association with the receptor (CI: k(+1) = 5.5 x 10⁵ M^-1 s^-1; E₂: k(+1) = 4.4 x 10⁵ M^-1 s^-1, at 0-4°), but [³H]CI dissociates much more rapidly (CI: k(-1) = 2.6 x 10^-4 s^-1, t(1/2) = 44 min; E₂: k(-1) = 2.4 x 10^-6 s^-1, t(1/2) = 80 h, at 0-4°). Different sedimentation profiles are seen for the cytoplasmic receptor complexes with [³H]CI and with [³H]E₂ on low salt sucrose density gradients: over 90% of the [³H]E₂ receptor complexes sediment at 8.0 S, whereas 70% of the [³H]CI receptor complexes (estrogen-compatible sites) sediment at 4.5 S, only 30% sedimenting a 8.0 S. After administration of [³H]CI in vivo, radioactivity can be found associated with specific estrogen-receptor sites in uterine nuclei. Salt-extracted nuclear receptor [³H]CI complexes sediment at 5.4 S on 0.4 M KCl sucrose gradients as do [³H]E₂ nuclear receptor complexes. Thin layer chromatographic analysis of ethyl acetate extracts of the nuclear receptor peak fractions from sucrose gradients shows that a polar metabolite of CI-628 is selectively bound to the 5.4 S receptor. Pharmacokinetic studies reveal that high levels of unmetabolized CI persist in serum and uterus for long periods of time (half-cleared in 18 to 24 hr), whereas E₂ is much more rapidly cleared (half-cleared in 30 min). These results indicate that in many respects, [³H]CI-628 and [³H]E₂ interact with the estrogen receptor in a parallel manner; however, certain properties of the antiestrogen receptor complex are different from those of the estradiol receptor complex. Further, it is likely that the prolonged in vivo activity of this antiestrogen derives, at least in part, from its slow rate of clearance and that the agent active in vivo may be a metabolite of CI-628.