IDR-90104

The HPLC methods described here for the assay and purity test of Estredox (E2CDS), a molecule with a redox-based, brain-targeted chemical delivery system for estradiol, allow reliable conclusions to be made on the potency and purity of API and E2CDS/HPCD complex samples. Extensive work was done to isolate and characterize the major, potential contaminants, and ensure the required stability of solutions of E2CDS, an inherently labile compound by design. Both the sample solvent and the eluent were thoroughly tested to avoid unwanted changes in sample solutions during analyses. The 12 minute isocratic assay method at 220 or 360 nm is simple, well-founded, highly precise and accurate. Purity profiling of E2CDS raised several problems in detection, stability and accuracy, owing to the fact that the pattern of the UV spectra and the stability of the compound and those of the potential contaminants often differed greatly. As a result of meticulous analysis of the UV spectra and the factors influencing the behaviour, in solution, of the compounds concerned, the 20 minute gradient method developed for the purity test, at 220 nm, of E2CDS and E2CDS/HPCD complex samples has proved to be a reliable means of adequately resolving 15-20 peaks of known and unknown compounds, and establishing the purity of various E2CDS samples. Sample impurity can be expressed as area % at 220 nm, and/or as approximate w/w % (if needed), since the relative response factors, at 220 nm, of the 6 major, potential contaminants have also been determined.

Estredox is a novel brain-targeted delivery system for estradiol (E2). The mechanism of this estradiol-chemical delivery system (E2-CDS) is based on an interconvertible dihydropiridine <--> pyridinium salt carrier (targetor) attached to E2. After administration of the E2-CDS, the targetor moiety is oxidized to a quaternary pyridinium salt (E2-Q+). Here we demonstrate that a single i.v. injection with E2-CDS (3 mg/kg) resulted in sustained presence of E2-Q+ in three various brain regions. The sustained and gradual release of estradiol from E2-Q+ is reflected by the time-course of plasma estrogen level. At the end of repeated administration of E2-CDS (daily once 0.3 mg/kg i.v. for 10 consecutive days) we found a sharp decrease in the levels of plasma estradiol followed by a gradual decrease. The levels of E2-Q+ in the investigated brain regions decreased gradually from the first post-treatment day, however, a detectable amount of E2-Q+ was still present in the hypothalamus, striatum, and cortex even on the 24th post-treatment day. Strikingly different plasma estradiol levels were found in the groups of orchidectomized rats that received daily i.v. injections of estradiol benzoate (E2-BZ). The plasma estradiol levels in these animals were much higher compared to E2-CDS-treated animals throughout the treatment period but the level sharply dropped immediately after the treatments. In contrast to the E2-CDS-treated animals there was no estradiol in any of the brain regions of E2-BZ-treated rats on the 1st and 2nd post-treatment day. All of these data are in line with the long-lasting pharmacological effects of E2-CDS-treatment on estrogen-mediated functions in castrated rats and give further experimental support for brain-targeting estrogen-treatment approach as opposed to the traditional estrogen replacement therapy.