Separation of stereoisomers of 7-oxa-bicyclo[2.2.1]heptene sulfonate (OBHS), a Selective Estrogen Receptor Modulator (SERM), via chiral stationary phases using SFC/UV and SFC/MS.

The enantiomeric separation of a racemate of 7-oxa-bicyclo[2.2.1]heptene sulfonate (OBHS) derivatives, a Selective Estrogen Receptor Modulator (SERM), was obtained using supercritical fluid chromatography in tandem with UV and mass spectrometry (SFC/UV and SFC/MS, respectively). Supercritical CO2 modified with methanol or isopropyl alcohol was used with isopropylamine (IPAm), trimethylamine (TEA), or trifluoroacetic acid (TFA) as an additive to obtain the enantiomers separations. Both Chiralpak IC and IA were evaluated for the separation of enantiomers. Results showed enantiomers separation can be achieved in less than 5 min with a resolution greater than 1 and 0.9, respectively, for the different OBHS derivatives (compounds A and B) using supercritical CO2 modified with 40% isopropyl alcohol containing 0.25% IPAm and IC column applying isocratic conditions. Similar conditions were used with the semi-preparative Chiralpak IC column to isolate more than 50 mg of each enantiomer. SFC/MS and SFC/UV results showed pure enantiomers were isolated. Method development via SFC was much simpler than those reported in the literature using HPLC.

Specific stereochemistry of OP-1074 disrupts estrogen receptor alpha helix 12 and confers pure antiestrogenic activity.

Complex tissue-specific and cell-specific signaling by the estrogen receptor (ER) frequently leads to the development of resistance to endocrine therapy for breast cancer. Pure ER antagonists, which completely lack tissue-specific agonist activity, hold promise for preventing and treating endocrine resistance, however an absence of structural information hinders the development of novel candidates. Here we synthesize a small panel of benzopyrans with variable side chains to identify pure antiestrogens in a uterotrophic assay. We identify OP-1074 as a pure antiestrogen and a selective ER degrader (PA-SERD) that is efficacious in shrinking tumors in a tamoxifen-resistant xenograft model. Biochemical and crystal structure analyses reveal a structure activity relationship implicating the importance of a stereospecific methyl on the pyrrolidine side chain of OP-1074, particularly on helix 12.

Simplifying and expanding analytical capabilities for various classes of doping agents by means of direct urine injection high performance liquid chromatography high resolution/high accuracy mass spectrometry.

So far, in sports drug testing compounds of different classes are processed and measured using different screening procedures. The constantly increasing number of samples in doping analysis, as well as the large number of substances with doping related, pharmacological effects require the development of even more powerful assays than those already employed in sports drug testing, indispensably with reduced sample preparation procedures. The analysis of native urine samples after direct injection provides a promising analytical approach, which thereby possesses a broad applicability to many different compounds and their metabolites, without a time-consuming sample preparation. In this study, a novel multi-target approach based on liquid chromatography and high resolution/high accuracy mass spectrometry is presented to screen for more than 200 analytes of various classes of doping agents far below the required detection limits in sports drug testing. Here, classic groups of drugs as diuretics, stimulants, ß2-agonists, narcotics and anabolic androgenic steroids as well as various newer target compounds like hypoxia-inducible factor (HIF) stabilizers, selective androgen receptor modulators (SARMs), selective estrogen receptor modulators (SERMs), plasma volume expanders and other doping related compounds, listed in the 2016 WADA prohibited list were implemented. As a main achievement, growth hormone releasing peptides could be implemented, which chemically belong to the group of small peptides (<2kDa) and are commonly determined by laborious and time-consuming stand-alone assays. The assay was fully validated for qualitative purposes considering the parameters specificity, robustness (rRT: <2%), intra- (CV: 1.7-18.4 %) and inter-day precision (CV: 2.3-18.3%) at three concentration levels, linearity (R2>0.99), limit of detection (0.1-25ng/mL; 3'OH-stanozolol glucuronide: 50pg/mL; dextran/HES: 10µg/mL) and matrix effects.

Smart coumarin-tagged imprinted polymers for the rapid detection of tamoxifen.

A signalling molecularly imprinted polymer was synthesised for easy detection of tamoxifen and its metabolites. 6-Vinylcoumarin-4-carboxylic acid (VCC) was synthesised from 4-bromophenol to give a fluorescent monomer, designed to switch off upon binding of tamoxifen. Clomiphene, a chlorinated analogue, was used as the template for the imprinting, and its ability to quench the coumarin fluorescence when used in a 1:1 ratio was demonstrated. Tamoxifen and 4-hydroxytamoxifen were also shown to quench coumarin fluorescence. Imprinted and non-imprinted polymers were synthesised using VCC, methacrylic acid as a backbone monomer and ethylene glycol dimethacrylate as cross-linker, and were ground and sieved to particle sizes ranging between 45 and 25 µm. Rebinding experiments demonstrate that the imprinted polymer shows very strong affinity for both clomiphene and tamoxifen, while the non-imprinted polymer shows negligible rebinding. The fluorescence of the imprinted polymer is quenched by clomiphene, tamoxifen and 4-hydroxytamoxifen. The switch off in fluorescence of the imprinted polymer under these conditions could also be detected under a UV lamp with the naked eye, making this matrix suitable for applications when coupled with a sample preparation system.

Quantification of tamoxifen in pharmaceutical formulations using micellar liquid chromatography.

This paper describes a micellar liquid chromatographic method used to analyze tamoxifen (TAMO) in pharmaceutical formulations, while focusing in its interesting features. Solid samples were solved in a micellar solution, irradiated at 254 nm, filtered and injected. Extraction steps were avoided and thus expediting the procedure. Tamoxifen was resolved in <5 min, using a mobile phase containing 0.15 M sodium dodecyl sulfate-7% pentanol at pH 3, running at 1.5 mL/min under an isocratic mode at 40°C through a C18 column. Detection was achieved by fluorescence by excitation at 260 nm and emission at 380 nm. The validation was performed following the requirements of the International Conference on Harmonization (ICH) Tripartite Guidelines in terms of: specificity, sensitivity, calibration range (0.2 - 20 mg/L), accuracy (98.8 - 101.7%), precision (<1.5%) and robustness (<6.2%). The method was applied to quantify TAMO in TAMO citrate tablets supplied in Spain, and was found appropriate for the quality control of TAMO formulations.

The first electrochemical MIP sensor for tamoxifen.

We present an electrochemical MIP sensor for tamoxifen (TAM)-a nonsteroidal anti-estrogen-which is based on the electropolymerisation of an O-phenylenediamine‒resorcinol mixture directly on the electrode surface in the presence of the template molecule. Up to now only "bulk" MIPs for TAM have been described in literature, which are applied for separation in chromatography columns. Electro-polymerisation of the monomers in the presence of TAM generated a film which completely suppressed the reduction of ferricyanide. Removal of the template gave a markedly increased ferricyanide signal, which was again suppressed after rebinding as expected for filling of the cavities by target binding. The decrease of the ferricyanide peak of the MIP electrode depended linearly on the TAM concentration between 1 and 100 nM. The TAM-imprinted electrode showed a 2.3 times higher recognition of the template molecule itself as compared to its metabolite 4-hydroxytamoxifen and no cross-reactivity with the anticancer drug doxorubucin was found. Measurements at +1.1 V caused a fouling of the electrode surface, whilst pretreatment of TAM with peroxide in presence of HRP generated an oxidation product which was reducible at 0 mV, thus circumventing the polymer formation and electrochemical interferences.

Development of a simple, fast, and accurate method for the direct quantification of selective estrogen receptor modulators using stable isotope dilution mass spectrometry.

A rapid analytical procedure was developed to quantify major selective estrogen receptor modulators (SERMs) simultaneously using stable isotope dilution mass spectrometry (SID-LCMS). Two novel isotopically labeled (SIL) analogues of natural SERMs, genistein and daidzein, were synthesized using a H/D exchange reaction mechanism. Computational chemistry coupled with MS and NMR data confirmed the site and mechanism of deuteration. The SIL analogues, which were mono- and dideutero substituted at the ortho positions, exhibited minimal deuterium isotope effects and were stable under the employed sample preparation protocol and MS analysis. An isotopic overlap correction was successfully employed to improve the accuracy and precision of the analytical method. The developed method, which was found to be sensitive, selective, precise and accurate, could be a valuable tool for research focused on determining the bioavailability of individual SERMs.

SERMs and SARMs: detection of their activities with yeast based bioassays.

Selective estrogen receptor modulators (SERMs) and selective androgen receptor modulators (SARMs) are compounds that activate their cognate receptor in particular target tissues without affecting other organs. Many of these compounds will find their use in therapeutic treatments. However, they also will have a high potential for misuse in veterinary practice and the sporting world. Here we demonstrate that yeast estrogen and androgen bioassays can be used to detect SERMs and SARMs, and are also useful screening tools to investigate their mode of action. Six steroidal 11beta-substituents of E2 (SERMs) and some arylpropionamide- and quinoline-based SARMs were tested. In addition, 7 compounds previously tested on AR agonism and determined as inactive in the yeast androgen bioassay, while QSAR modelling revealed strong binding to the human androgen receptor, are now shown to act as AR antagonists.

Gradient HPLC analysis of raloxifene hydrochloride and its application to drug quality control.

A rapid, sensitive and selective method for the determination of raloxifene hydrochloride (RLX) in pure drug and in tablets was developed using gradient high performance liquid chromatography (HPLC). The devised method involved separation of RLX on a reversed phase Hypersil ODS column and determination with UV detection at 284 nm. The standard curve was linear (R = 0.999) over the concentration range of 50-600 microg mL-1 with a detection limit of 0.04 microg mL-1 and a quantification limit of 0.16 microg mL-1. Intra-day and inter-day precision and accuracy of the method were established according to the current ICH guidelines. Intra-day RSD values at three QC levels (250, 450 and 550 microg mL-1) were 0.2-0.5%, based on the peak area. The intra-day relative error (er) was between 0.2 and 0.5%. The developed method was successfully applied to the determination of RLX in tablets and the results were statistically compared with those obtained by a literature method. Accuracy, evaluated by means of the spike recovery method, was the excellent with percent recovery in the range 97.7-103.2 with precision in the range 1.6-2.2%. No interference was observed from the co-formulated substances. The method was economical in terms of the time taken and the amount of solvent used.

HPLC analysis of raloxifene hydrochloride and its application to drug quality control studies.

Raloxifene hydrochloride is a selective estrogen receptor modulator and is currently being used for prevention of osteoporosis in postmenopausal women. In this article, a high performance liquid chromatography (HPLC) method for detection of raloxifene hydrochloride was developed and validated using an ultraviolet (UV) and coulometric detectors. Limit of quantification (LOQ) was 0.336 and 0.610 mg L(-1) for coulometric and ultraviolet detectors, respectively. Acceptable accuracy (93.1-100.3%) as well as intra- and inter-day precision (CV

Spectrophotometric analysis of raloxifene hydrochloride in pure and pharmaceutical formulations.

Two simple and sensitive spectrophotometric methods (A and B) for the determination of raloxifene hydrochloride in bulk samples and pharmaceutical formulations are described. Method A is based on the oxidation of the drug with ferric chloride and coupling with potassium ferric cyanide. Method B is based on reduction of the drug with Fehling's reagent. Bluish green color formed in method A absorbs at 735 nm and brown color produced in method B absorbs at 430 nm.

Environmental fate and chemistry of raloxifene hydrochloride.

Raloxifene hydrochloride is a selective estrogen receptor modulator (SERM) used for the prevention and treatment of osteoporosis in women. Excretion of raloxifene occurs through the feces of patients. Raloxifene has the potential to be discharged into waste treatment systems after therapeutic use. Raloxifene hydrochloride was investigated using a battery of studies designed to describe its physical/chemical characteristics and define its fate in the environment. The mean measured solubility of raloxifene hydrochloride (+/- standard deviation) was 345.2 +/- 15.6 microg/ml, 13.3 +/- 0.6 microg/ml, 0.9224 +/- 0.015 microg/ml, and 627.4 +/- 132.0 microg/ml in aqueous buffers at pH 5, 7, and 9 and in unbuffered water, respectively. Raloxifene exhibited a mean molar absorptivity of 34,000 and a wavelength absorbance maximum at 287 nm for pH 5 and 7 aqueous buffer solutions and 297 nm at pH 9. Mean measured Kow values were 516 +/- 17, 1,323 +/- 91, and 1,556 +/- 135 at pH 5, 7, and 9, respectively. After 5 d at 50 degrees C, raloxifene hydrolyzed 8.02, 10.61, and 23.81% in pH 5, 7, and 9 aqueous buffers, respectively. In a 28-d hydrolysis study at 25 degrees C, the calculated first-order hydrolysis rates were 6.92 x 10(-4), 1.70 x 10(-3), and 7.66 x 10(-3)/d, and the corresponding half-lives were 1,001, 410, and 90 d in pH 5, 7, and 9 aqueous buffers, respectively. Raloxifene sorbed significantly to sewage treatment solids with Freundlich isotherm adsorption coefficients K between 2,000 and 3,000. Raloxifene degraded rapidly in the presence of sewage solids. In a system containing 0.470 g/L sludge solids, the raloxifene biodegradation rate and half-life were 0.0966/h and 7.17 h, respectively. In a 28-d aerobic-aquatic biodegradation study containing 30 mg/L sludge solids, the raloxifene biodegradation rate and half-life were 0.0188/d and 37 d, respectively. Given the fate and behavior of raloxifene in these studies, it is anticipated that raloxifene would rapidly dissipate in the environment.

Engineering of a mouse for the in vivo profiling of estrogen receptor activity.

In addition to their well known control of reproductive functions, estrogens modulate important physiological processes. The identification of compounds with tissue-selective activity will lead to new drugs mimicking the beneficial effects of estrogen on the prevention of osteoporosis and cardiovascular or neurodegenerative diseases, while avoiding its detrimental proliferative effects. As an innovative model for the in vivo identification of new selective estrogen receptor modulators (SERMs), we engineered a mouse genome to express a luciferase reporter gene ubiquitously. The constructs for transgenesis consist of the reporter gene driven by a dimerized estrogen-responsive element (ERE) and a minimal promoter. Insulator sequences, either matrix attachment region (MAR) or beta-globin hypersensitive site 4 (HS4), flank the construct to achieve a generalized, hormoneresponsive luciferase expression. In the mouse we generated, the reporter expression is detectable in all 26 tissues examined, but is induced by 17beta-estradiol (E2) only in 15 of them, all expressing estrogen receptors (ERs). Immunohistochemical studies show that in the mouse uterus, luciferase and ERs colocalize. In primary cultures of bone marrow cells explanted from the transgenic mice and in vivo, luciferase activity accumulates with increasing E(2) concentration. E2 activity is blocked by the ER full antagonist ICI 182,780. Tamoxifen shows partial agonist activity in liver and bone when administered to the animals. In the mouse system here illustrated, by biochemical, immunohistochemical, and pharmacological criteria, luciferase content reflects ER transcriptional activity and thus represents a novel system for the study of ER dynamics during physiological fluctuations of estrogen and for the identification of SERMs or endocrine disruptors.