Lactic Acid Bacteria-Derived Postbiotics as Adjunctive Agents in Breast Cancer Treatment to Boost the Antineoplastic Effect of a Conventional Therapeutic Comprising Tamoxifen and a New Drug Candidate: An Aziridine-Hydrazide Hydrazone Derivative.

Breast cancer is associated with high mortality and morbidity rates. As about 20-30% of patients exhibiting ER-positive phenotype are resistant to hormonal treatment with the standard drug tamoxifen, finding new therapies is a necessity. Postbiotics, metabolites, and macromolecules isolated from probiotic bacteria cultures have been proven to have sufficient bioactivity to exert prohealth and anticancer effects, making them viable adjunctive agents for the treatment of various neoplasms, including breast cancer. In the current study, postbiotics derived from L. plantarum and L. rhamnosus cultures were assessed on an in vitro breast cancer model as potential adjunctive agents to therapy utilizing tamoxifen and a candidate aziridine-hydrazide hydrazone derivative drug. Cell viability and cell death processes, including apoptosis, were analyzed for neoplastic MCF-7 cells treated with postbiotics and synthetic compounds. Cell cycle progression and proliferation were analyzed by PI-based flow cytometry and Ki-67 immunostaining. Postbiotics decreased viability and triggered apoptosis in MCF-7, modestly affecting the cell cycle and showing a lack of negative impact on normal cell viability. Moreover, they enhanced the cytotoxic effect of tamoxifen and the new candidate drug toward MCF-7, accelerating apoptosis and the inhibition of proliferation. This illustrates postbiotics' potential as natural adjunctive agents supporting anticancer therapy based on synthetic drugs.

Interaction of neutral and protonated Tamoxifen with the DPPC lipid bilayer using molecular dynamics simulation.

Tamoxifen as an antiestrogen is successfully applied for the clinical treatment of breast cancer in pre- and post-menopausal women. Due to the side effects related to the oral administration of Tamoxifen (such as deep vein thrombosis, pulmonary embolism, hot flushes, ocular disturbances and some types of cancer), liposomal drug delivery is recommended for taking this drug. Drug encapsulation in a liposomal or lipid drug delivery system improves the pharmacokinetic and pharmacodynamic properties. In this regard, we carried out 200-ns molecular dynamics (MD) simulations for three systems (pure DPPC and neutral and protonated Tamoxifen-loaded DPPC). Here, DPPC is a model lipid bilayer to provide us with conditions like liposomal drug delivery systems to investigate the interactions between Tamoxifen and DPPC lipid bilayers and to estimate the preferred location and orientation of the drug molecule inside the bilayer membrane. Properties such as area per lipid, membrane thickness, lateral diffusion coefficient, order parameters and mass density, were surveyed. With insertion of neutral and protonated Tamoxifen inside the DPPC lipid bilayers, area per lipid and membrane thickness increased slightly. Also, Tamoxifen induce ordering of the hydrocarbon chains in DPPC bilayer. Analysis of MD trajectories shows that neutral Tamoxifen is predominantly found in the hydrophobic tail region, whereas protonated Tamoxifen is located at the lipid-water interface (polar region of DPPC lipid bilayers).

Challenging Approach to the Development of Novel Estrogen Receptor Modulators Based on the Chemical Properties of Guaiazulene.

Tamoxifen, a therapeutic agent for breast cancer, has been associated with genetic polymorphisms in the metabolism of N,N-dialkylaminoethyl substituent, which plays an important role in the expression of selective estrogen receptor modulator (SERM) activity. To solve this problem, we developed a novel estrogen receptor (ER) modulator, Az-01, on the basis of the aromaticity, dipole moment, and isopropyl group of guaiazulene. Az-01 showed four-fold lower binding affinity for ER than E2 but had similar ER-binding affinity to that of 4-hydroxytamoxifen (4-HOtam). Unlike tamoxifen, Az-01 acted as a partial agonist with very weak estrogenic activity at high concentrations when used alone, and it showed potent anti-estrogenic activity in the presence of E2. The cell proliferation and inhibition activities of Az-01 were specific to ER-expressing MCF-7 cells, and no effect of Az-01 on other cell proliferation signals was observed. These findings are important for the development of new types of SERMs without the N,N-dialkylaminoethyl substituent as a privileged functional group for SERMs.

Design and synthesis of marine sesterterpene analogues as novel estrogen receptor α degraders for breast cancer treatment.

Targeted protein degradation using small molecules is an intriguing strategy for drug development. The marine sesterterpene compound MHO7 had been reported to be a potential ERα degradation agent. In order to further improve its biological activity, two series of novel MHO7 derivatives with long side chains were designed and identified as novel selective estrogen receptor down-regulators (SERDs). The growth inhibition activity of the novel SERD compounds were significantly affected by the type and length of the side chain. Most of the derivatives were significantly more potent than MHO7 against both drug-sensitive and drug-resistant breast cancer cells. Among them, compound 16a, with IC50 values of 0.41 µM against MCF-7 cell lines and 9.6-fold stronger than MHO7, was the most potential molecule. A whole-genome transcriptomic analysis of MCF-7 cells revealed that the mechanism of 16a against MCF-7 cell was similar with that of MHO7. The estrogen signaling pathway was the most affected among the disturbed genes, but the ERα degradation activity of 16a was observed higher than that of MHO7. Other effects of 16a were confirmed similar with MHO7, which means that the basic mechanisms of the derivatives are the same with the ophiobolin backbone, i.e. the degradation of ERα is mediated via proteasome-mediated process, the induction of apoptosis and the cell cycle arrest at the G1 phase. Meanwhile, a decrease of mitochondrial membrane potential and an increase of cellular ROS were also detected. Based on these results, as a novel modified ophiobolin derived compound, 16a may warrant further exploitation as a promising SERD candidate agent for the treatment of breast cancer.

Design and Characterization of Maltoheptaose-b-Polystyrene Nanoparticles, as a Potential New Nanocarrier for Oral Delivery of Tamoxifen.

Tamoxifen citrate (TMC), a non-steroidal antiestrogen drug used for the treatment of breast cancer, was loaded in a block copolymer of maltoheptaose-b-polystyrene (MH-b-PS) nanoparticles, a potential drug delivery system to optimize oral chemotherapy. The nanoparticles were obtained from self-assembly of MH-b-PS using the standard and reverse nanoprecipitation methods. The MH-b-PS@TMC nanoparticles were characterized by their physicochemical properties, morphology, drug loading and encapsulation efficiency, and release kinetic profile in simulated intestinal fluid (pH 7.4). Finally, their cytotoxicity towards the human breast carcinoma MCF-7 cell line was assessed. The standard nanoprecipitation method proved to be more efficient than reverse nanoprecipitation to produce nanoparticles with small size and narrow particle size distribution. Moreover, tamoxifen-loaded nanoparticles displayed spherical morphology, a positive zeta potential and high drug content (238.6 ± 6.8 µg mL-1) and encapsulation efficiency (80.9 ± 0.4 %). In vitro drug release kinetics showed a burst release at early time points, followed by a sustained release profile controlled by diffusion. MH-b-PS@TMC nanoparticles showed higher cytotoxicity towards MCF-7 cells than free tamoxifen citrate, confirming their effectiveness as a delivery system for administration of lipophilic anticancer drugs.

Elucidation of Agonist and Antagonist Dynamic Binding Patterns in ER-α by Integration of Molecular Docking, Molecular Dynamics Simulations and Quantum Mechanical Calculations.

Estrogen receptor alpha (ERα) is a ligand-dependent transcriptional factor in the nuclear receptor superfamily. Many structures of ERα bound with agonists and antagonists have been determined. However, the dynamic binding patterns of agonists and antagonists in the binding site of ERα remains unclear. Therefore, we performed molecular docking, molecular dynamics (MD) simulations, and quantum mechanical calculations to elucidate agonist and antagonist dynamic binding patterns in ERα. 17ß-estradiol (E2) and 4-hydroxytamoxifen (OHT) were docked in the ligand binding pockets of the agonist and antagonist bound ERα. The best complex conformations from molecular docking were subjected to 100 nanosecond MD simulations. Hierarchical clustering was conducted to group the structures in the trajectory from MD simulations. The representative structure from each cluster was selected to calculate the binding interaction energy value for elucidation of the dynamic binding patterns of agonists and antagonists in the binding site of ERα. The binding interaction energy analysis revealed that OHT binds ERα more tightly in the antagonist conformer, while E2 prefers the agonist conformer. The results may help identify ERα antagonists as drug candidates and facilitate risk assessment of chemicals through ER-mediated responses.

Searching for an ideal SERM: Mining tamoxifen structure-activity relationships.

The repurposing of old drugs for new treatments has recently garnered increased attention in the face of new diseases and declining productivity of the pharmaceutial industry. This report draws attention to potential opportunities hiding in plain sight within the SAR of off-patent drugs. Herein we explore the untapped potential of Selective Estrogen Receptor Modulators (SERMs). SERMs are a class of molecules that have been highly influential in the treatment of estrogen receptor-positive breast cancers. However, the most commonly prescribed SERM, tamoxifen, has been found to increase the risk of endometrial cancer. Another SERM, raloxifene, does not increase incidence of endometrial cancer, but has been abandoned as a breast cancer treatment. We report the design, synthesis, and evaluation of an unexplored tamoxifen substitution pattern which mimics the geometry of raloxifene to confer its favorable pharmacodynamics. This substitution pattern was found to maintain excellent binding affinity to estrogen receptor-α.

Heterodimeric GW7604 Derivatives: Modification of the Pharmacological Profile by Additional Interactions at the Coactivator Binding Site.

(E/Z)-3-(4-((E)-1-(4-Hydroxyphenyl)-2-phenylbut-1-enyl)phenyl)acrylic acid (GW7604) as a derivative of (Z)-4-hydroxytamoxifen (4-OHT) was linked by diaminoalkane spacers to molecules that are known binders to the coactivator binding site (benzimidazole or thioxo-quinazolinone scaffolds). With this modification, an optimization of the pharmacological profile was achieved. The most active thioxo-quinazolinone derivative 16 showed extraordinarily high affinity to the estrogen receptor (ER) ß (RBA = 110%), inhibited effectively the coactivator recruitment (IC50 = 20.88 nM (ERα) and 28.34 nM (ERß)), acted as a pure estradiol (E2) antagonist in a transactivation assay (IC50 = 18.5 nM (ERα) and 7.5 nM (ERß)), and downregulated the ERα content in MCF-7 cells with an efficacy of 60% at 1 µM. The cytotoxicity was restricted to hormone-dependent MCF-7 (IC50 = 4.2 nM) and tamoxifen-resistant MCF-7TamR cells (IC50 = 476.6 nM). The compounds bearing a thioxo-quinazolinone moiety can therefore be assigned as pure E2-antagonistic selective ER degraders/downregulators. By contrast, the benzimidazole derivatives acted solely as pure antagonists without degradation of the ER.

Replacing the Z-phenyl Ring in Tamoxifen® with a para-Connected NCN Pincer-Pt-Cl Grouping by Post-Modification †.

Post-modification of a series of NCN-pincer platinum(II) complexes [PtX(NCN-R-4)] (NCN = [C6H2(CH2NMe2)2-2,6]-, R = C(O)H, C(O)Me and C(O)Et), X = Cl- or Br-) at the para-position using the McMurry reaction was studied. The synthetic route towards two new [PtCl(NCN-R-4)] (R = C(O)Me and C(O)Et) complexes used above is likewise described. The utility and limitations of the McMurry reaction involving these pincer complexes was systematically evaluated. The predicted "homo-coupling" reaction of [PtBr(NCN-C(O)H-4)] led to the unexpected formation of 3,3',5,5'-tetra[(dimethylamino)methyl]-4,4'-bis(platinum halide)-benzophenone (halide = Br or Cl), referred to hereafter as the bispincer-benzophenone complex 13. This material was further characterized using X-ray crystal structure determination. The applicability of the pincer complexes in the McMurry reaction is shown to open a route towards the synthesis of tamoxifen-type derivatives of which one phenyl ring of Tamoxifen® itself is replaced by an NCN arylplatinum pincer fragment. The newly synthesized derivatives can be used as potential candidates in anti-cancer drug screening protocols. Two NCN-arylpincer platinum tamoxifen type derivatives, 5 and 6, were successfully synthesized and of 5 the separation of the diastereomeric E-/Z-forms was achieved. Compound 6, which is the pivaloyl protected NCN pincer platinum hydroxy-Tamoxifen® derivative, was obtained as a mixture of E-/Z-isomers. The new derivatives were further analyzed and characterized with 1H-, 13C{1H}- and 195Pt{1H}-NMR, IR, exact mass MS and elemental analysis.

A tamoxifen receptor within a voltage-gated sodium channel.

Voltage-gated sodium channels are targets for many analgesic and antiepileptic drugs whose therapeutic mechanisms and binding sites have been well characterized. We describe the identification of a previously unidentified receptor site within the NavMs voltage-gated sodium channel. Tamoxifen, an estrogen receptor modulator, and its primary and secondary metabolic products bind at the intracellular exit of the channel, which is a site that is distinct from other previously characterized sodium channel drug sites. These compounds inhibit NavMs and human sodium channels with similar potencies and prevent sodium conductance by delaying channel recovery from the inactivated state. This study therefore not only describes the structure and pharmacology of a site that could be leveraged for the development of new drugs for the treatment of sodium channelopathies but may also have important implications for off-target health effects of this widely used therapeutic drug.

Concept Design, Development and Preliminary Physical and Chemical Characterization of Tamoxifen-Guided-Mesoporous Silica Nanoparticles.

Conventional chemotherapies used for breast cancer (BC) treatment are non-selective, attacking both healthy and cancerous cells. Therefore, new technologies that enhance drug efficacy and ameliorate the off-target toxic effects exhibited by currently used anticancer drugs are urgently needed. Here we report the design and synthesis of novel mesoporous silica nanoparticles (MSNs) equipped with the hormonal drug tamoxifen (TAM) to facilitate guidance towards estrogen receptors (ERs) which are upregulated in breast tumours. TAM is linked to the MSNs using a poly-ʟ-histidine (PLH) polymer as a pH-sensitive gatekeeper, to ensure efficient delivery of encapsulated materials within the pores. XRD, HR-TEM, DLS, SEM, FT-IR and BET techniques were used to confirm the successful fabrication of MSNs. The MSNs have a high surface area (>1000 m2/g); and a mean particle size of 150 nm, which is an appropriate size to allow the penetration of premature blood vessels surrounding breast tumours. Successful surface functionalization was supported by FT-IR, XPS and TGA techniques, with a grafting ratio of approximately 29%. The outcomes of this preliminary work could be used as practical building blocks towards future formulations.

A Bioinformatics Study of the Involved Mechanisms in Relapse and Drug Resistance of Tamoxifen-Treated Breast Cancer.

BACKGROUND: Breast cancer is currently among the most common causes of mortality in women. Estrogen and its subsequent signaling pathways play an important role in the occurrence of breast cancer relapse. Tamoxifen is the most common breast cancer treatment option in ER+ patients, which acts as an adjuvant endocrinotherapy with X-ray and surgery. This approach is recommended as the first-line treatment and has increased the survival rate of breast cancer patients and reduced the relapse cases. However, we can observe resistance to tamoxifen and relapse cases in one-third of patients treated with this drug, which has become a major concern. OBJECTIVE: The precise mechanisms of relapse and resistance to tamoxifen have not yet been identified and were explored in this study. METHODS: Microarray profiles of relapse and relapse-free patients were investigated to explain the processes leading to relapse and possibly to tamoxifen resistance. RESULTS: According to the preliminary analysis, 1460 genes showed increased expression while 1132 genes showed decreased expression. According to our default for inclusion (-2LogFC≥ + 2), 36 genes had increased expression (upregulated) while 33 genes had decreased expression (down-regulated). CONCLUSION: It seems that the mechanisms of resistance and relapse are multifactorial, and tumor cells induce relapse and resistance to tamoxifen through cell proliferation, survival, invasion, angiogenesis, extracellular matrix secretion, pump and membrane changes, and immune evasion.

Formulation Development of Tamoxifen Loaded Lipid Nanoparticle by Taguchi (L12 (211)) Orthogonal Array Design.

BACKGROUND: A better understanding of the biopharmaceutical and physicochemical properties of drugs and the pharmaco-technical factors would be of great help for developing pharmaceutical products. But, it is extremely difficult to study the effect of each variable and interaction among them through the conventional approach. METHODS: To screen the most influential factors affecting the particle size (PS) of lipid nanoparticle (LNPs) (solid lipid nanoparticle (SLN) and nanostructured lipid carrier (NLC)) for poorly watersoluble BCS class-II drug like tamoxifen (TMX) to improve its oral bioavailability and to reduce its toxicity to tolerable limits using Taguchi (L12 (211)) orthogonal array design by applying computer optimization technique. RESULTS: The size of all LNPs formulations prepared as per the experimental design varied between 172 nm and 3880 µm, polydispersity index between 0.033 and 1.00, encapsulation efficiency between 70.8% and 75.7%, and drug loading between 5.84% and 9.68%. The study showed spherical and non-spherical as well as aggregated and non-aggregated LNPs. Besides, it showed no interaction and amorphous form of the drug in LNPs formulation. The Blank NLCs exhibited no cytotoxicity on MCF-7 cells as compared to TMX solution, SLNs (F5) and NLCs (F12) suggest that the cause of cell death is primarily from the effect of TMX present in NLCs. CONCLUSIONS: The screening study clearly showed the importance of different individual factors significant effect for the LNPs formulation development and its overall performance in an in-vitro study with minimum experimentation thus saving considerable time, efforts, and resources for further in-depth study.

Tamoxifen Delivery System Based on PEGylated Magnetic MCM-41 Silica.

Magnetic iron oxide containing MCM-41 silica (MM) with ~300 nm particle size was developed. The MM material before or after template removal was modified with NH2- or COOH-groups and then grafted with PEG chains. The anticancer drug tamoxifen was loaded into the organic groups' modified and PEGylated nanoparticles by an incipient wetness impregnation procedure. The amount of loaded drug and the release properties depend on whether modification of the nanoparticles was performed before or after the template removal step. The parent and drug-loaded samples were characterized by XRD, N2 physisorption, thermal gravimetric analysis, and ATR FT-IR spectroscopy. ATR FT-IR spectroscopic data and density functional theory (DFT) calculations supported the interaction between the mesoporous silica surface and tamoxifen molecules and pointed out that the drug molecule interacts more strongly with the silicate surface terminated by silanol groups than with the surface modified with carboxyl groups. A sustained tamoxifen release profile was obtained by an in vitro experiment at pH = 7.0 for the PEGylated formulation modified by COOH groups after the template removal. Free drug and formulated tamoxifen samples were further investigated for antiproliferative activity against MCF-7 cells.

Cholesterol-conjugated bovine serum albumin nanoparticles as a tamoxifen tumor-targeted delivery system.

In the present study, we introduced cholesterol (CLO)-conjugated bovine serum albumin nanoparticles (BSA NPs) as a new system for indirect targeting drug delivery. Tamoxifen, as an anticancer drug, was loaded on BSA NPs (BSA-TAX NPs); CLO was then conjugated to the BSA-TAX NPs surface for the targeted delivery of NPs system, by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxy succinimide carbodiimide chemistry (CLO-BSA-TAX NPs). The physicochemical properties, toxicity, in vitro, and in vivo biocompatibility of the BSA NPs system were characterized on cancer cell lines (4T1). The results revealed that the BSA NPs system has a regular spherical shape and negative zeta-potential values. The drug release of BSA NPs system has shown controlled and pH-dependent drug release behavior. BSA NPs system was biocompatible but it was potentially toxic on the cancer cell line. The CLO-BSA-TAX NPs exhibited higher toxicity against cancer cell lines than other NPs formulation (BSA NPs and BSA-TAX NPs). It can be concluded that the CLO, as an indirect targeting agent, enhances the toxicity and specificity of NPs system on cancer cell lines. It could potentially be suitable approaches to targeting the tumors in clinical cancer therapy.

Antimicrobial, Antitumor and Side Effects Assessment of a Newly Synthesized Tamoxifen Analog.

BACKGROUND: Tamoxifen citrate is a very prevalent drug marketed under several trade names like Apo-Tamox, Nolvadex, Tamec, Tamizam, and Tamoplex. This molecule is approved by the FDA for breast cancer treatment. Some studies have shown that tamoxifen has anti-tuberculosis and antiparasitic activities. Like any drug, tamoxifen possesses side effects, more or less dangerous. AIMS: Basically, this work is a comparative study that aims to: primarily compare the antimicrobial and antitumor activities of tamoxifen and a newly synthesized tamoxifen analog; and to determine the molecule with lesser side effects. METHODS: Three groups of mice were injected with tamoxifen citrate and compound 2(1,1-bis[4-(3- dimethylaminopropoxy)phenyl]-2-phenyl-but-1-ene dihydrochloride) at doses corresponding to C1 (1/10), C2 (1/50), and C3 (1/100) to compound 2 lethal dose (LD50 = 75 mg/kg) administered to adult mice. A group of noninjected mice served as a study control. RESULTS: Experimental results suggest that compound 2 has better antitumor and antimicrobial activity than tamoxifen citrate besides its lower toxicity effects. CONCLUSION: The results obtained from the present study confirmed the antitumor and antimicrobial effect of tamoxifen citrate and its hematological side effects. Compound 2 seems to be more effective than tamoxifen citrate for antitumor and antimicrobial treatment while having less hematological side effects and less disruption of the blood biochemical parameters. These findings encourage us to perform further studies on compound 2 and test it for other therapeutic uses for which tamoxifen was found effective.

A Virion-Based Assay for Glycoprotein Thermostability Reveals Key Determinants of Filovirus Entry and Its Inhibition.

Ebola virus (EBOV) entry into cells is mediated by its spike glycoprotein (GP). Following attachment and internalization, virions traffic to late endosomes where GP is cleaved by host cysteine proteases. Cleaved GP then binds its cellular receptor, Niemann-Pick C1. In response to an unknown cellular trigger, GP undergoes conformational rearrangements that drive fusion of viral and endosomal membranes. The temperature-dependent stability (thermostability) of the prefusion conformers of class I viral fusion glycoproteins, including those of filovirus GPs, has provided insights into their propensity to undergo fusion-related rearrangements. However, previously described assays have relied on soluble glycoprotein ectodomains. Here, we developed a simple enzyme-linked immunosorbent assay (ELISA)-based assay that uses the temperature-dependent loss of conformational epitopes to measure thermostability of GP embedded in viral membranes. The base and glycan cap subdomains of all filovirus GPs tested suffered a concerted loss of prefusion conformation at elevated temperatures but did so at different temperature ranges, indicating virus-specific differences in thermostability. Despite these differences, all of these GPs displayed reduced thermostability upon cleavage to GP conformers (GPCL). Surprisingly, acid pH enhanced, rather than decreased, GP thermostability, suggesting it could enhance viral survival in hostile endo/lysosomal compartments. Finally, we confirmed and extended previous findings that some small-molecule inhibitors of filovirus entry destabilize EBOV GP and uncovered evidence that the most potent inhibitors act through multiple mechanisms. We establish the epitope-loss ELISA as a useful tool for studies of filovirus entry, engineering of GP variants with enhanced stability for use in vaccine development, and discovery of new stability-modulating antivirals.IMPORTANCE The development of Ebola virus countermeasures is challenged by our limited understanding of cell entry, especially at the step of membrane fusion. The surface-exposed viral protein, GP, mediates membrane fusion and undergoes major structural rearrangements during this process. The stability of GP at elevated temperatures (thermostability) can provide insights into its capacity to undergo these rearrangements. Here, we describe a new assay that uses GP-specific antibodies to measure GP thermostability under a variety of conditions relevant to viral entry. We show that proteolytic cleavage and acid pH have significant effects on GP thermostability that shed light on their respective roles in viral entry. We also show that the assay can be used to study how small-molecule entry inhibitors affect GP stability. This work provides a simple and readily accessible assay to engineer stabilized GP variants for antiviral vaccines and to discover and improve drugs that act by modulating GP stability.

Development and validation of a high throughput bioanalytical UPLC-MS/MS method for simultaneous determination of tamoxifen and sulphoraphane in rat plasma: Application to an oral pharmacokinetic study.

Tamoxifen (TAM) is the choice of a drug approved by the Food and Drug Administration (FDA) for the treatment of estrogen-positive receptor (ER+) breast cancer. Sulphoraphane (SFN), a natural plant antioxidant compound, also acts on estrogen-positive breast cancer receptor. Thus, a combination of TAM with SFN is preferred as it helps to minimize the drug-related toxicity and increases the therapeutic efficacy by providing synergistic anticancer effects of both drugs. In the present study, a new simple, sensitive, precise, and selective UPLC-MS/MS method was developed for the simultaneous quantification of tamoxifen and sulphoraphane using propranolol as an internal standard (IS) in rat plasma. Chromatographic separation was achieved on reverse phase Acquity UPLC BEH C18 column (50 mm × 2.1 mm, i.d., 1.7 µm) with an isocratic mobile phase composed of solvent A (0.1% formic acid in acetonitrile) and B (0.1% formic acid in water) (80:20, v/v) at a flow-rate of 0.4 mL/min. The detection and quantification of analytes was performed on Waters ZsprayTM Xevo TQD using selected-ion monitoring operated under a positive electrospray ionization mode. The transitions were m/z = 372.0 [M+H]+ → 71.92 for tamoxifen, m/z = 177.9 [M+H]+ → 113.9 for sulphoraphane and m/z = 260.3 [M+H]+ → 116.1 for propranolol. The method was linear over the concentration range of 8-500 ng/mL (r2 = 0.9996) for tamoxifen, 30-2000 ng/mL (r2 = 0.9998) for sulphoraphane with insignificant matrix effect and high extraction recovery on spiked quality control (QC) samples. The intra- and inter-batch precisions and accuracy were within the acceptable limits, and both the analytes were found to be stable throughout the short term, long term and freeze thaw stability studies. The validated method was successfully applied for the simultaneous estimation of TAM and SFN in an oral pharmacokinetic study in female Wistar rats. This developed UPLC-MS/MS method could be a valuable tool for future pharmacokinetic interaction, therapeutic drug monitoring and pharmacokinetic characterization of novel formulations.

Identification and targeting of selective vulnerability rendered by tamoxifen resistance.

BACKGROUND: The estrogen receptor (ER)-positive breast cancer represents over 80% of all breast cancer cases. Even though adjuvant hormone therapy with tamoxifen (TMX) is saving lives of patients with ER-positive breast cancer, the acquired resistance to TMX anti-estrogen therapy is the main hurdle for successful TMX therapy. Here we address the mechanism for TMX resistance and explore the ways to eradicate TMX-resistant breast cancer in both in vitro and ex vivo experiments. EXPERIMENTAL DESIGN: To identify compounds able to overcome TMX resistance, we used short-term and long-term viability assays in cancer cells in vitro and in patient samples in 3D ex vivo, analysis of gene expression profiles and cell line pharmacology database, shRNA screen, CRISPR-Cas9 genome editing, real-time PCR, immunofluorescent analysis, western blot, measurement of oxidative stress using flow cytometry, and thioredoxin reductase 1 enzymatic activity. RESULTS: Here, for the first time, we provide an ample evidence that a high level of the detoxifying enzyme SULT1A1 confers resistance to TMX therapy in both in vitro and ex vivo models and correlates with TMX resistance in metastatic samples in relapsed patients. Based on the data from different approaches, we identified three anticancer compounds, RITA (Reactivation of p53 and Induction of Tumor cell Apoptosis), aminoflavone (AF), and oncrasin-1 (ONC-1), whose tumor cell inhibition activity is dependent on SULT1A1. We discovered thioredoxin reductase 1 (TrxR1, encoded by TXNRD1) as a target of bio-activated RITA, AF, and ONC-1. SULT1A1 depletion prevented the inhibition of TrxR1, induction of oxidative stress, DNA damage signaling, and apoptosis triggered by the compounds. Notably, RITA efficiently suppressed TMX-unresponsive patient-derived breast cancer cells ex vivo. CONCLUSION: We have identified a mechanism of resistance to TMX via hyperactivated SULT1A1, which renders selective vulnerability to anticancer compounds RITA, AF, and ONC-1, and provide a rationale for a new combination therapy to overcome TMX resistance in breast cancer patients. Our novel findings may provide a strategy to circumvent TMX resistance and suggest that this approach could be developed further for the benefit of relapsed breast cancer patients.

Synthesis of Tamoxifen-Artemisinin and Estrogen-Artemisinin Hybrids Highly Potent Against Breast and Prostate Cancer.

In the search for new and effective treatments of breast and prostate cancer, a series of hybrid compounds based on tamoxifen, estrogens, and artemisinin were successfully synthesized and analyzed for their in vitro activities against human prostate (PC-3) and breast cancer (MCF-7) cell lines. Most of the hybrid compounds exhibit a strong anticancer activity against both cancer cell lines - for example, EC50 (PC-3) down to 1.07 µM, and EC50 (MCF-7) down to 2.08 µM - thus showing higher activities than their parent compounds 4-hydroxytamoxifen (afimoxifene, 7; EC50 =75.1 (PC-3) and 19.3 µM (MCF-7)), dihydroartemisinin (2; EC50 =263.6 (PC-3) and 49.3 µM (MCF-7)), and artesunic acid (3; EC50 =195.1 (PC-3) and 32.0 µM (MCF-7)). The most potent compounds were the estrogen-artemisinin hybrids 27 and 28 (EC50 =1.18 and 1.07 µM, respectively) against prostate cancer, and hybrid 23 (EC50 =2.08 µM) against breast cancer. These findings demonstrate the high potential of hybridization of artemisinin and estrogens to further improve their anticancer activities and to produce synergistic effects between linked pharmacophores.