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A new amphiphilic copolymer have been synthesized starting from the hydrosoluble polyaspartylhydrazide (PAHy) polymer, by grafting both hydrophilic PEG(2000) chains and hydrophobic palmitic acid (C(16)) moieties on polymer backbone, and the structure of obtained PAHy-PEG(2000)-C(16) copolymer have been characterized by 2D (1)H/(13)C NMR experiments. PAHy-PEG(2000)-C(16) copolymer showed the ability of self-assembling in aqueous media giving a core-shell structure and resulted potentially useful for encapsulating and dissolving hydrophobic drug. The formation of micellar core-shell structure has been investigated by 2D (1)H NMR NOESY experiments. The presence of cross-peaks for protons of C(16) and PAHy portions, indicated that the two domains are in close proximity forming micelle core. The critical aggregation concentration (CAC) values of PAHy-PEG(2000)-C(16) amphiphilic graft copolymer was determined in water by fluorescence technique, and it was demonstrated that PAHy-PEG(2000)-C(16) micelles are well suited to be micellar vehicle of highly hydrophobic molecules. Therefore, anticancer drug tamoxifen, used as a model hydrophobic molecule, was loaded into PAHy-PEG(2000)-C(16) micelles obtaining an increase of drug solubility of about 3000 times. Transmission electron microscopy (TEM) observations showed the spherical morphology of micelles formed by PAHy-PEG(2000)-C(16) copolymer with a mean diameter of about 30nm, as confirmed also by dynamic light scattering (DLS) studies. Finally, in vitro cell viability studies were carried out on human breast cancer cells (MCF-7) testing the pharmacological activity of tamoxifen-loaded PAHy-PEG(2000)-C(16) micelles, in comparison with free tamoxifen at different drug concentrations, demonstrating that tamoxifen-loaded PAHy-PEG(2000)-C(16) micelles exhibited a concentration-dependent cytotoxic activity., (Copyright 2010 Elsevier B.V. All rights reserved.)

Tamoxifen is a selective estrogen receptor modulator widely used in oncology and reproductive endocrinology. In order to decrease its non-desirable effects and elucidate mechanisms of action, permanently charged tamoxifen derivatives (PCTDs) have been reported. Whether PCTDs have genomic effects remains controversial. Since the clinical relevance of tamoxifen, the necessity to have new anticancer drugs, and in order to gain insights into the mechanisms of action of PCTDs, we obtained six quaternary ammonium salts derived from tamoxifen including three new compounds. We characterized them by nuclear magnetic resonance, X-ray diffraction, electron microscopy, and/or high performance liquid chromatography, and detected them in cell lysates by liquid chromatography coupled to mass spectrometry. We evaluated their binding to estrogen receptor-alpha (ERalpha, their effect on the transcriptional activity mediated by ERalpha (gene reporter assays), and the proliferation of cancer cells (MCF-7 and cells from a cervical cancer primary culture). Structural studies demonstrated the expected identity of the molecules. All PCTDs did bind to ERalpha, one of them induced ERalpha-mediated transcription while two others inhibited such genomic action. Accordingly, PCTDs were detected in cell lysates. PCTDs inhibited cell proliferation, noteworthy, two of them displayed higher inhibition than tamoxifen. Structure-activity analysis suggests that PCTDs permanent positive charge and the length of the aliphatic chain might be associated to the biological responses studied. We suggest genomic effects as a mechanism of action of PCTDs. The experimental approaches here used could lead to a better design of new therapeutic molecules and help to elucidate molecular mechanisms of new anticancer drugs., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Due to recent use of short-chain ceramides in preclinical studies, we characterized C6-ceramide metabolism in cancer cell lines and assessed metabolic junctures for enhancing efficacy. MDA-MB-231 breast cancer cells decreased the amount of C6-ceramide metabolized to C6-sphingomyelin (C6-SM) and increased the amount metabolized to C6-glucosylceramide (C6-GC) in response to increasing concentrations. A similar trend was seen in DU-145 (prostate cancer), PANC-1 (pancreatic cancer), and LoVo (colorectal cancer) cells. KG-1 leukemia cells favored C6-SM synthesis at low (0.6muM) and high-dose (12muM) C6-ceramide. Partnering C6-ceramide with tamoxifen, a P-glycoprotein antagonist that impedes ceramide glycosylation, was an effective regimen for enhancing cytotoxicity in cells. Experiments to assess the mechanism of cell death using KG-1 cells showed that tamoxifen inhibited synthesis of C6-GC and C6-SM from C6-ceramide by 80% and 50%, respectively, which was accompanied by enhanced apoptosis. Radiolabeling of KG-1 cells with [(3)H]palmitic acid produced a 2-fold increase in (3)H-long-chain ceramides when unlabeled C6-ceramide was added and a 9-fold increase when C6-ceramide and tamoxifen were added. The increase in (3)H-palmitate radiolabeling of long-chain ceramides was blocked by inclusion of a ceramide synthase inhibitor; however, inhibiting synthesis of long-chain ceramide did not rescue cells. These studies show that tamoxifen enhances the apoptotic effects of C6-ceramide. The proposed mechanism involves blocking short-chain ceramide anabolism to favor hydrolysis and generation of sphingosine. We propose that use of tamoxifen and other P-glycoprotein antagonists can be an effective means for enhancing cytotoxic potential of short-chain ceramides in the treatment of cancer., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

The microsomal antiestrogen binding site (AEBS) is a high-affinity target for the antitumor drug tamoxifen and its cognate ligands that mediate breast cancer cell differentiation and apoptosis. The AEBS, a hetero-oligomeric complex composed of 3beta-hydroxysterol-Delta8-Delta7-isomerase (D8D7I) and 3beta-hydroxysterol-Delta7-reductase (DHCR7), binds different structural classes of ligands, including ring B oxysterols. These oxysterols are inhibitors of cholesterol-5,6-epoxide hydrolase (ChEH), a microsomal epoxide hydrolase that has yet to be molecularly identified. We hypothesized that the AEBS and ChEH might be related entities. We show that the substrates of ChEH, cholestan-5alpha,6alpha-epoxy-3beta-ol (alpha-CE) and cholestan-5beta,6beta-epoxy-3beta-ol (beta-CE), and its product, cholestane-3beta,5alpha,6beta-triol (CT), are competitive ligands of tamoxifen binding to the AEBS. Conversely, we show that each AEBS ligand is an inhibitor of ChEH activity, and that there is a positive correlation between these ligands' affinity for the AEBS and their potency to inhibit ChEH (r2=0.95; n=39; P<0.0001). The single expression of D8D7I or DHCR7 in COS-7 cells slightly increased ChEH activity (1.8- and 2.6-fold), whereas their coexpression fully reconstituted ChEH, suggesting that the formation of a dimer is required for ChEH activity. Similarly, the single knockdown of D8D7I or DHCR7 using siRNA partially inhibited ChEH in MCF-7 cells, whereas the knockdown of both D8D7I and DHCR7 abolished ChEH activity by 92%. Taken together, our findings strongly suggest that the AEBS carries out ChEH activity and establish that ChEH is a new target for drugs of clinical interest, polyunsaturated fatty acids and ring B oxysterols.

Membrane receptors for steroid hormones are currently a subject of considerable debate. One approach to selectively target these putative receptors has been to couple ligands to substances that restrict cell permeability. Using this approach, an analogue of the estrogen receptor ligand 4-hydroxytamoxifen was attached to fluorescent dyes with differing degrees of predicted cell permeability. The conjugates bound to estrogen receptor in vitro, but all three conjugates, including one predicted to be cell-impermeable, inhibited estradiol-induced transcriptional activation. Fluorescence microscopy revealed cytoplasmic localization for all three conjugates. We further characterized a 4-hydroxytamoxifen analogue conjugated to a BODIPY fluorophore in breast cancer cell lines. Those experiments suggested a similar, but not identical, mode of action to 4-hydroxytamoxifen, as the fluorescent conjugate was equally effective at inhibiting proliferation of both tamoxifen-sensitive and tamoxifen-resistant breast cancer cell lines. While these findings point to significant complicating factors in designing steroid hormone mimics targeted to the plasma membrane, the results also reveal a possible new direction for designing estrogen receptor modulators.

A mixture of the (Z)- and (E)-isomers of 4-hydroxy-N-desmethyltamoxifen was conveniently prepared in four steps. These geometrical isomers were then neatly separated by semi-preparative Reverse Phase High Performance Liquid Chromatography (RP-HPLC) using specified conditions. Additionally, the isolated E-isomer could be equilibrated in aqueous strong acid in acetonitrile or trifluoroacetic acid/dichloromethane to give a clean 1:1 mixture of Z/E isomers that was re-subjected to HPLC separation. In this way, most of the undesired (E)-isomer could be readily converted to the desired (Z)-isomer providing quick access to over 200mg quantities of pure endoxifen (Z-isomer), a potent antiestrogenic metabolite of tamoxifen traditionally used in breast cancer treatment., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The aim of this work was to investigate the mechanism of action of ferrocifen (Fc-OH-TAM), the ferrocenyl analog of 4-hydroxy-tamoxifen (OH-TAM), which is the active metabolite of tamoxifen, the drug most widely prescribed for treatment of hormone-dependent breast cancers. Fc-OH-TAM showed an anti-proliferative effect on the six breast cancer cell lines tested, 3 ERalpha positive (MCF-7, T-47D, ZR-75-1) and 3 ERalpha negative (MDA-MB-231, SKBR-3, Hs578-T) whatever their ER (estrogen receptor) status. However, the mechanism of action of the ferrocenyl derivative appeared to differ depending on the status of the ERalpha. Analysis of cell cycle distribution revealed that Fc-OH-TAM first recruits cells in the S phase in both ERalpha positive and ERalpha negative cells. In the presence of ERalpha, Fc-OH-TAM allowed cell cycle progression, with a subsequent blockade in G0/G1, whereas in the absence of ERalpha, cells remained in the S phase. Significant production of ROS was observed only in the presence of Fc-OH-TAM in both ERalpha positive and negative breast cancer cell lines. Within our experimental conditions, this ROS production is associated with cell cycle arrest and senescence rather than apoptosis. In the presence of ERalpha, Fc-OH-TAM seems to mainly act in the same way as OH-TAM but also induces an additional cytotoxic effect not mediated by the receptor. Our data suggest that this cytotoxic effect of Fc-OH-TAM is expressed via a mechanism of action distinct from the non-genomic pathway observed with high doses of OH-Tamoxifen.

Background, Aim, and Scope: The occurrence and fate of pharmaceuticals in the aquatic environment is recognized as one of the emerging issues in environmental chemistry and as a matter of public concern. Existing data tend to focus on the concentrations of pharmaceuticals in the aqueous phase, with limited studies on their concentrations in particulate phase such as sediments. Furthermore, current water quality monitoring does not differentiate between soluble and colloidal phases in water samples, hindering our understanding of the bioavailability and bioaccumulation of pharmaceuticals in aquatic organisms. In this study, an investigation was conducted into the concentrations and phase association (soluble, colloidal, suspended particulate matter or SPM) of selected pharmaceuticals (propranolol, sulfamethoxazole, meberverine, thioridazine, carbamazepine, tamoxifen, indomethacine, diclofenac, and meclofenamic acid) in river water, effluents from sewage treatment works (STW), and groundwater in the UK., Materials and Methods: The occurrence and phase association of selected pharmaceuticals propranolol, sulfamethoxazole, meberverine, thioridazine, carbamazepine, tamoxifen, indomethacine, diclofenac, and meclofenamic acid in contrasting aquatic environments (river, sewage effluent, and groundwater) were studied. Colloids were isolated by cross-flow ultrafiltration (CFUF). Water samples were extracted by solid-phase extraction (SPE), while SPM was extracted by microwave. All sample extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the multiple reaction monitoring., Results and Discussion: Five compounds propranolol, sulfamethoxazole, carbamazepine, indomethacine, and diclofenac were detected in all samples, with carbamazepine showing the highest concentrations in all phases. The highest concentrations of these compounds were detected in STW effluents, confirming STW as a key source of these compounds in the aquatic environments. The calculation of partition coefficients of pharmaceuticals between SPM and filtrate (observed partition coefficients, Kobsp, Kobsoc), between SPM and soluble phase (intrinsic partition coefficients, Kintp, Kintoc), and between colloids and soluble phase (Kcoc) showed that intrinsic partition coefficients (Kintp, Kintoc) are between 25% and 96%, and between 18% and 82% higher than relevant observed partition coefficients values, and are much less variable. Secondly, Kcoc values are 3-4 orders of magnitude greater than Kintoc values, indicating that aquatic colloids are substantially more powerful sorbents for accumulating pharmaceuticals than sediments. Furthermore, mass balance calculations of pharmaceutical concentrations demonstrate that between 23% and 70% of propranolol, 17-62% of sulfamethoxazole, 7-58% of carbamazepine, 19-84% of indomethacine, and 9-74% of diclofenac are present in the colloidal phase., Conclusions: The results provide direct evidence that sorption to colloids provides an important sink for the pharmaceuticals in the aquatic environment. Such strong pharmaceutical/colloid interactions may provide a long-term storage of pharmaceuticals, hence, increasing their persistence while reducing their bioavailability in the environment., Recommendations and Perspectives: Pharmaceutical compounds have been detected not only in the aqueous phase but also in suspended particles; it is important, therefore, to have a holistic approach in future environmental fate investigation of pharmaceuticals. For example, more research is needed to assess the storage and long-term record of pharmaceutical residues in aquatic sediments by which benthic organisms will be most affected. Aquatic colloids have been shown to account for the accumulation of major fractions of total pharmaceutical concentrations in the aquatic environment, demonstrating unequivocally the importance of aquatic colloids as a sink for such residues in the aquatic systems. As aquatic colloids are abundant, ubiquitous, and highly powerful sorbents, they are expected to influence the bioavailability and bioaccumulation of such chemicals by aquatic organisms. It is therefore critical for colloids to be incorporated into water quality models for prediction and risk assessment purposes.

Estrogens can potentially be classified into planar (class I) or nonplanar (class II) categories, which might have biological consequences. 1,1,2-Triphenylethylene (TPE) derivatives were synthesized and evaluated against 17beta-estradiol (E2) for their estrogenic activity in MCF-7 human breast cancer cells. All TPEs were estrogenic and, unlike 4-hydroxytamoxifen (4OHTAM) and Endoxifen, induced cell growth to a level comparable to that of E2. All the TPEs increased ERE activity in MCF-7:WS8 cells with the order of potency as followed: E2 > 1,1-bis(4,4'-hydroxyphenyl)-2-phenylbut-1-ene (15) > 1,1,2-tris(4-hydroxyphenyl)but-1-ene (3) > Z 4-(1-(4-hydroxyphenyl)-1-phenylbut-1-en-2-yl)phenol (7) > E 4-(1-(4-hydroxyphenyl)-1-phenylbut-1-en-2-yl)phenol (6) > Z(4-(1-(4-ethoxyphenyl)-1-(4-hydroxyphenyl)but-1-en-2-yl)phenol (12) > 4-OHTAM. Transient transfection of the ER-negative breast cancer cell line T47D:C4:2 with wild-type ER or D351G ER mutant revealed that all of the TPEs increased ERE activity in the cells expressing the wild-type ER but not the mutant, thus confirming the importance of Asp351 for ER activation by the TPEs. The findings confirm E2 as a class I estrogen and the TPEs as class II estrogens. Using available conformations of the ER liganded with 4OHTAM or diethylstilbestrol, the TPEs optimally occupy the 4OHTAM ER conformation that expresses Asp351.

Protein kinase C (PKC) plays a major role in regulation of both pre and postsynaptic neurotransmission. Excessive activation of PKC results in symptoms related to bipolar disorder. Tamoxifen, a widely used breast cancer drug is known to inhibit PKC and demonstrate antimanic properties in human. We describe herein the synthesis of endoxifen, a tamoxifen active metabolite and compared its PKC inhibitory activity with that of tamoxifen. Endoxifen exhibited fourfold higher potency compared to tamoxifen., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Background: Different preparation methods for the production of lipid micro- and nanoparticles as controlled release formulations have been widely developed. Novel techniques are attracting increasing attention for their preparation., Method: The objective of the present investigation was to produce solid lipid-based micro-nanospheres using the electrohydrodynamic atomization (electrospraying) and to evaluate whether it is a suitable method to prepare drug-loaded particles., Results: Narrowly dispersed spherical particles lower than 1 mum, easily internalized in cells, were obtained using stearic acid and ethylcellulose in a 4.5:0.5 (w/w) ratio. Tamoxifen, as model drug, was encapsulated with good entrapment efficiency. The in vitro release, after an initial burst effect, showed a prolonged drug release., Conclusion: The electrospraying method might be proposed to prepare in a single-step monodisperse lipid-based micro- and nanoparticles in powder form for drug delivery.

We have implemented a noninvasive optical method for the fast control of protein activity in a live zebrafish embryo. It relies on releasing a protein fused to a modified estrogen receptor ligand binding domain from its complex with cytoplasmic chaperones, upon the local photoactivation of a nonendogenous caged inducer. Molecular dynamics simulations were used to design cyclofen-OH, a photochemically stable inducer of the receptor specific for 4-hydroxy-tamoxifen (ER(T2)). Cyclofen-OH was easily synthesized in two steps with good yields. At submicromolar concentrations, it activates proteins fused to the ER(T2) receptor. This was shown in cultured cells and in zebrafish embryos through emission properties and subcellular localization of properly engineered fluorescent proteins. Cyclofen-OH was successfully caged with various photolabile protecting groups. One particular caged compound was efficient in photoinducing the nuclear translocation of fluorescent proteins either globally (with 365 nm UV illumination) or locally (with a focused UV laser or with two-photon illumination at 750 nm). The present method for photocontrol of protein activity could be used more generally to investigate important physiological processes (e.g., in embryogenesis, organ regeneration and carcinogenesis) with high spatiotemporal resolution.

UDP-glucuronosyltransferase (UGT) 1A4-catalyzed glucuronidation is an important drug elimination pathway. Although atypical kinetic profiles (nonhyperbolic, non-Michaelis-Menten) of UGT1A4-catalyzed glucuronidation have been reported occasionally, systematic kinetic studies to explore the existence of multiple aglycone binding sites in UGT1A4 have not been conducted. To this end, two positional isomers, dihydrotestosterone (DHT) and trans-androsterone (t-AND), were used as probe substrates, and their glucuronidation kinetics with HEK293-expressed UGT1A4 were evaluated both alone and in the presence of a UGT1A4 substrate [tamoxifen (TAM) or lamotrigine (LTG)]. Coincubation with TAM, a high-affinity UGT1A4 substrate, resulted in a concentration-dependent activation/inhibition effect on DHT and t-AND glucuronidation, whereas LTG, a low-affinity UGT1A4 substrate, noncompetitively inhibited both processes. The glucuronidation kinetics of TAM were then evaluated both alone and in the presence of different concentrations of DHT or t-AND. TAM displayed substrate inhibition kinetics, suggesting that TAM may have two binding sites in UGT1A4. However, the substrate inhibition kinetic profile of TAM became more hyperbolic as the DHT or t-AND concentration was increased. Various two-site kinetic models adequately explained the interactions between TAM and DHT or TAM and t-AND. In addition, the effect of TAM on LTG glucuronidation was evaluated. In contrast to the mixed effect of TAM on DHT and t-AND glucuronidation, TAM inhibited LTG glucuronidation. Our results suggest that multiple aglycone binding sites exist within UGT1A4, which may result in atypical kinetics (both homotropic and heterotropic) in a substrate-dependent fashion.

This communication reports on the first electrochemical study of the human flavin-containing monooxygenase 3 (hFMO3) either absorbed or covalently linked to different electrode surfaces. Glassy carbon and gold electrodes gave reversible electrochemical signals of an active hFMO3. The midpoint potential measured for the immobilized enzyme on a glassy carbon electrode was -445 +/- 8 mV (versus Ag/AgCl). A monolayer coverage was obtained on gold functionalized with dithio-bismaleimidoethane that covalently linked surface accessible cysteines of hFMO3. A structural model of the enzyme was generated to rationalize electrochemistry results. The turnover of the active enzyme was measured with two specific drugs: tamoxifen and benzydamine. For tamoxifen, 1.7 and 8.0 microM of its N-oxide product were formed by the enzyme immobilized on glassy carbon and gold electrodes, respectively. In the case of benzydamine, a K(M) of 44 +/- 5 microM was measured upon application of a -600 mV bias to the enzyme immobilized on the glassy carbon electrode that is in good agreement with the values published for microsomal hFMO3 where NADPH is the electron donor.

The cytochome P450 enzyme system is responsible for the metabolism of xenobiotics, including > 75% of commonly prescribed medications. Many of the cytochrome enzymes exhibit polymorphic genotypes, partly accounting for the variations observed in individual drug responses. Recent advances in the understanding of functional alleles of cytochrome P450 enzymes have changed the use of medications. Improvements in drug efficacy and the prevention of toxicity, as well as improvements in clinical drug dosing, have enhanced pharmacotherapy decisions in medical practice. In addition to personalizing medicine, the identification and quantification of genotypic differences in cytochrome P450 metabolism has the potential to facilitate population-based personalized medicine in countries without the resources to perform genotypic tests at the point of care. This review provides an update on the utility of genotype-guided therapy when treating breast cancer, malaria and coagulation disorders. Also discussed are advancements made in diagnostic tests for cytochrome genotypes and the need for future research in the area of diagnostic tests.

Background: Tamoxifen (Tam) is used for the treatment and prevention of estrogen-receptor-positive human breast and other cancers. Its use in ovarian cancer has not been well studied., Method: We formulated and characterized a water-soluble Tam-2-hydroxylpropyl-beta-cyclodextrin (HPbetaCD; 1:2 M) complex., Results: The differential scanning calorimetery of Tam-HPbetaCD indicated the transition of Tam from crystalline to amorphous form on addition of HPbetaCD. (1)H-nuclear magnetic resonance nuclear overhauser effect cross-peaks between phenyl moieties of Tam and HPbetaCD, and downfield shifts in H-3 (0.26) and H-5 (0.29) protons of HPbetaCD suggested the inclusion of Tam in HPbetaCD cavity. Transmission-electron microscopy studies of HPbetaCD and the Tam-HPbetaCD complex revealed the formation of aggregated nanoassembly at 60-180 nm. Dimethyl thiazol diphenyltetrazolium bromide assay demonstrated 7.37 +/- 2.32% cell survival of OAW-42 cells with 3 microg/ml Tam concentration., Conclusion: The Tam-HPbetaCD nanoassembly entered the cell owing to enhanced permeability and retention property of tumor cell and antiestrogenic Tam and, therefore, resulted in excellent anticancer efficacy in the ovarian cancer cell line.

The breast cancer treatment drug tamoxifen has been widely administered for more than three decades. This small molecule competes with 17beta-estradiol for binding to estrogen receptor, a hormone receptor upregulated in a majority of breast cancers, subsequently initiating programmed cell death. We have synthesized a thiol-PEGylated tamoxifen derivative that can be used to selectively target and deliver plasmonic gold nanoparticles to estrogen receptor positive breast cancer cells with up to 2.7-fold enhanced drug potency in vitro. Optical microscopy/spectroscopy, time-dependent dose-response data, and estrogen competition studies indicate that augmented activity is due to increased rates of intracellular tamoxifen transport by nanoparticle endocytosis, rather than by passive diffusion of the free drug. Both ligand- and receptor-dependent intracellular delivery of gold nanoparticles suggest that plasma membrane localized estrogen receptor alpha may facilitate selective uptake and retention of this and other therapeutic nanoparticle conjugates. Combined targeting selectivity and enhanced potency provides opportunities for both multimodal endocrine treatment strategies and adjunctive laser photothermal therapy.

Background: Microparticles are used for controlled drug delivery. With the aim of improving both bioavailability and tamoxifen selective toxicity, the activity of tamoxifen embedded in calcium alginate/chitosan microparticles was studied., Materials and Methods: Tamoxifen alone and embedded in microparticles prepared with sodium alginate from Kelco (62% mannuronic acid and 38% guluronic acid) and from Fluka (30% mannuronic acid and 70% guluronic acid) was added to MCF-7 and Vero cultures and evaluated for antiproliferative activity by the MTT test., Results: The use of Kelco or Fluka alginate resulted in different LD(50) values on Vero and MCF-7 cultures, showing a higher cytotoxicity toward Vero cells treated with tamoxifen embedded in Kelco microparticles (25 microM vs. 48 microM on MCF-7 cells) but a selective toxicity with Fluka microparticles (25 microM and 10 microM on Vero and MCF-7 cells respectively)., Conclusion: Microparticle formulation may improve selective toxicity according to the alginate employed: differences in the chemical alginate composition can dramatically change both drug activity and toxicity.

Tamoxifen citrate is an antiestrogen for peroral breast cancer treatment. The drug delivery encounters problems of poor water solubility and vulnerability to enzymatic degradation in both intestine and liver. In the current study, tamoxifen citrate self-nanoemulsifying drug delivery systems (SNEDDS) were prepared in an attempt to circumvent such obstacles. Preliminary screening was carried out to select proper ingredient combinations. All surfactants screened were recognized for their bioactive aspects. Ternary phase diagrams were then constructed and an optimum system was designated. Three tamoxifen SNEDDS were then compared for optimization. The systems were assessed for robustness to dilution, globule size, cloud point, surface morphology and drug release. An optimum system composed of tamoxifen citrate (1.6%), Maisine 35-1 (16.4%), Caproyl 90 (32.8%), Cremophor RH40 (32.8%) and propylene glycol (16.4%) was selected. The system was robust to different dilution volumes and types. It possessed a mean globule size of 150 nm and a cloud point of 80 degrees C. Transmission electron microscopy demonstrated spherical particle morphology. The drug release from the selected formulation was significantly higher than other SNEDDS and drug suspension, as well. Realizing drug incorporation into an optimized nano-sized SNEDD system that encompasses a bioactive surfactant, our results proposed that the prepared system could be promising to improve oral efficacy of the tamoxifen citrate.

With the goal of discovering a selective agonist of estrogen-related receptor gamma (ERRgamma) with enhanced potency, we designed a series of small-molecule ligands derived from a known ERRgamma agonist, GSK4716, that can substantially potentiate the transcriptional activity of ERRgamma. Individual compounds among a 30-member library of acyl hydrazones were pre-evaluated through in silico docking studies on the receptor cavities of ERRgamma LBDs using X-ray crystal structures cocrystallized with GSK4716 and 4-OHT. This rational approach to achieve the enhanced potency in ERRgamma transcriptional activity with selectivity over ERRalpha/beta enables us to complete the construction of a focused library by carrying out microwave-assisted parallel synthesis with excellent yields and purities. Finally, we identified a more potent ERRgamma agonist, E6, with excellent selectivity over ERRalpha/beta.

Genetic variability in drug-metabolizing enzymes and signaling pathways affects chemotherapy-related toxicity and treatment outcome in cancer. In breast and colorectal cancer, polymorphisms in metabolic enzymes involved in tamoxifen and irinotecan therapies has led the U.S. Food and Drug Administration to address genetic factors relevant to patient consideration of treatment with these compounds. Tamoxifen therapeutic failure in breast cancer has been associated with reduced CYP2D6 activity due to inefficient activation of tamoxifen. Irinotecan toxicity in colorectal cancer is more common in patients with reduced-activity UGT1A alleles, resulting in excessive exposure to the potent SN-38 metabolite. In colorectal and lung cancers, somatic mutations in the epidermal growth factor receptor and downstream signaling molecules have been associated with the therapeutic outcome of epidermal growth factor receptor-directed therapies. This review discusses the current knowledge regarding the utility of single gene-UGT1A1, CYP2D6, EGFR, and KRAS-or multigene analysis, for optimizing breast, colorectal, and lung cancer therapy. Current advances in these areas highlight how pharmacogenetics help personalized decision-making for patient management.

Microglial cells play an important role during neuroinflammation in the central nervous system. Among other factors, activated microglia produce nitric oxide (NO), which is toxic to neurons and excessive microglial activation and NO production contribute to the pathology of neurodegenerative diseases. Chloride channels have previously been shown to participate in microglial activation. Here we investigate the effects of established chloride channel blockers with different chemical structures on interferon-gamma (IFNgamma)-induced activation of the murine microglial cell line, BV2. IFNgamma-induced NO production was effectively reduced by NPPB, IAA-94, tamoxifen, NS3728 and NS1652, with NS1652 being the most potent. In contrast, DIDS reduced NO production only at cytotoxic concentrations. Furthermore, NS1652 reduced the protein and mRNA levels of inducible nitric oxide synthase (iNOS), without altering STAT1 phosphorylation. These observations suggest a microglial chloride conductance as a critical permissive factor downstream in the IFNgamma-induced iNOS cascade. The nature of the underlying channel is unknown, but the pharmacological profile appears incompatible with the involvement of the volume activated anion conductance (VRAC).

The present study deals with the preparation and characterization of an injectable and in situ forming drug delivery system based on photocrosslinked poly(epsilon-caprolactone fumarate) (PCLF) networks loaded with tamoxifen citrate (TC). Networks were made of PCLF macromers, a photoinitiation system (comprising initiator and accelerator) and the active ingredient N-vinyl-2-pyrrolidone (NVP) as a crosslinker and reactive diluent. Shrinkage behavior, equilibrium swelling and sol fraction ratios of photocrosslinked PCLF gels were determined as functions of NVP content. It was shown that the crosslinking is facilitated up to a certain concentration of NVP and most of NVP remained unreacted above this value. In vitro drug release, biocompatibility evaluation and activity against MCF-7 breast cancer cell line were also investigated. Accurate but simple bipartite expressions were also derived that enable rapid determination of effective diffusion coefficients of TC in photocrosslinked PCLF/NVP disks. Cytotoxicity assay showed that while the photocrosslinked PCLF network with optimum NVP content exhibits no significant cytotoxicity against MCF-7 and L929 cell lines, 40-60% of the MCF-7 cells were killed after incubation with TC-loaded devices.

The previously reported reactions of cis and trans-hydroxytamoxifen drug derivatives, 1 and 2, with [Cp*Rh(L)(3)](2+) complexes (L = H(2)O, CH(3)OH), initially provided the kinetically controled eta(1)-N complexes, 4(OTf, CH(3)OH) and 5(OTf, CH(3)OH), which underwent a novel, intramolecular, regioselective N-pi rearrangement to provide the eta(6) complexes, 6 and 7. A dramatic solvent effect was also observed on the rate of this N-pi rearrangement in CH(3)OH or CH(2)Cl(2). Therefore, a DFT study was conducted that provided further mechanistic and thermodynamic data on this N-pi rearrangement. The preferred structures of both the eta(1)-N and eta(6) complexes in the two solvents were determined, and a thorough analysis of their geometries and electronic structures has been provided. The influence of the solvent on the N-pi rearrangement was studied by including the solvent both implicitly using a PCM model, and explicitly by introducing the counterion and/or the solvent molecules into the inner and outer coordination spheres of the complexes. It was shown that the triflate (OTf(-)) counterion was strongly bound in the inner coordination sphere of the eta(1)-N complexes, 4(OTf) and 5(OTf), and in the outer sphere of the coordinatively saturated eta(6) complexes, 6 and 7, especially in non-polar media. The cleavage of the ionic Cp*Rh-OTf bond was found to be the rate-limiting step in the N-pi rearrangement. The thermodynamic results suggested that the eta(6) complexes were more stable than the eta(1)-N complexes in CH(2)Cl(2) and in CH(3)OH at elevated temperatures. The opposite relationship for the stabilities of the eta(1)-N complexes was found in CH(3)OH at room temperature, thus corroborating the experimental results that the N-pi rearrangement did not occur, under these conditions. A plausible mechanistic pathway for the N-pi rearrangement was proposed from our extensive DFT studies, that included several important intermediates and transition states, and provided a unique view of this novel transformation.

Estrogen plays vital roles in human health and diseases. Estrogen mediates its actions almost entirely by binding to estrogen receptors (ER), alpha and beta which further function as transcription factors. Selective estrogen receptor modulators (SERMs) are synthetic molecules which bind to ER and can modulate its transcriptional capabilities in different ways in diverse estrogen target tissues. Tamoxifen, the prototypical SERM, is extensively used for targeted therapy of ER positive breast cancers and is also approved as the first chemo-preventive agent for lowering breast cancer incidence in high risk women. The therapeutic and preventive efficacy of tamoxifen was initially proven by series of experiments in the laboratory which laid the foundation of its clinical use. Unfortunately, use of tamoxifen is associated with de-novo and acquired resistance and some undesirable side effects. The molecular study of the resistance provides an opportunity to precisely understand the mechanism of SERM action which may further help in designing new and improved SERMs. Recent clinical studies reveal that another SERM, raloxifene, which is primarily used to treat post-menopausal osteoporosis, is as efficient as tamoxifen in preventing breast cancers with fewer side effects. Overall, these findings open a new horizon for SERMs as a class of drug which not only can be used for therapeutic and preventive purposes of breast cancers but also for various other diseases and disorders. Major efforts are therefore directed to make new SERMs with a better therapeutic profile and fewer side effects.

Tamoxifen citrate (TAM)-loaded polyacrylonitrile (PAN) fibers were prepared using an improved wet-spinning technique. TAM was used as a model drug to evaluate the potential application of the loaded fiber system for drug delivery. PAN was first homogeneously dissolved in the N,N-dimethylacetamide (DMAc) solution containing TAM and then the co-dissolving solution was solidified to prepare the fibers using a wet-spinning method. Chemical, morphological and mechanical property characterizations were carried out, as well as the studies of the drug release properties. TAM was successfully encapsulated into a monofilament fiber, and this system was stable in terms of high loading capacity and effectiveness in release. The diameter of drug-loaded fiber was in the range of 40-60 microm. The best values of the tensile strength at 2.968 cN/dtex and breaking elongation at 14.9% of drug-loaded fibers were obtained when the drug loading content was 23.1 wt.%. These characteristics were suitable for the weaving process. The in vitro release experiment indicated that constant drug release from the fiber was observed for a long duration of time. Kinetic studies demonstrated that the system followed the Higuchi kinetics. These findings demonstrate that controlled release of drugs from PAN fibers could be potentially useful in drug delivery systems.

Chemotherapy of brain tumors remains a big challenge owing to the low drug transport across the blood-brain barrier (BBB), multidrug resistance (MDR), and poor penetration into the tumor tissue. We developed a novel dual-targeting liposomal carrier that enabled drug to transport across the BBB and then target the brain tumor. In the dual-targeting liposomal carrier, tamoxifen (TAM) was incorporated into the lipid bilayer membrane of liposomes and wheat germ agglutinin (WGA) was conjugated to the liposomes' surface. Topotecan was then loaded into the above liposomes. In vitro, topotecan liposomes modified with TAM and WGA were applied to the glioma cells, BBB model, and avascular C6 glioma spheroids, respectively. In vivo, they were systemically administered via vein to brain C6 glioma-bearing rats. In view of the microtiter tetrazolium (MTT) results, topotecan liposomes modified with TAM and WGA exhibited a significant inhibitory effect compared to unmodified topotecan liposomes, suggesting that TAM plus WGA contributed strong drug delivery effects into the brain tumor cells after direct drug exposure. In the experiments of drug transport across the BBB model following drug exposure to tumor cells, topotecan liposomes modified with TAM and WGA exhibited the most robust dual-targeting effects: crossing the BBB and then targeting brain tumor cells. Similar strong activity was found in the reduction of C6 glioma tumor spheroid volume and in the apoptosis of the spheroids. In the brain tumor-bearing rats, the dual-targeting effects of topotecan liposomes modified with TAM and WGA could be evidently observed, resulting in a significant improvement in the overall survival of the brain tumor-bearing rats compared with free topotecan and topotecan liposomes. Moreover, results from an extended treatment group indicated that the survival could be further significantly enhanced, indicating that an extended chemotherapy with topotecan liposomes modified with TAM and WGA would be beneficial for treatment. The dual-targeting effects in vivo of topotecan liposomes modified with TAM and WGA could be related to an enhanced effect by TAM via inhibiting efflux of MDR proteins in the BBB and the brain tumor, and an enhanced effect by WGA via endocytosis in the BBB and in the brain tumor. In conclusion, topotecan liposomes modified with TAM and WGA significantly improve topotecan transport across the blood-brain barrier and the survival of brain tumor-bearing animals, showing dual-targeting effects. These findings would encourage further developments of noninvasive therapy for brain tumor.

The metadynamics method has been shown to be a valuable tool to study the mechanism of molecular recognition in atomistic detail [Gervasio, F. L.; et al. J. Am. Chem. Soc. 2005, 127, 2600]. However, it requires an a priori knowledge of all slow degrees of freedom relevant to the docking/undocking mechanism. Here we investigate a combination of docking/clustering with metadynamics performed with a subset of the necessary degrees of freedom (coarse metadynamics), and show that it provides a full mechanistic insight on the protein-ligand docking mechanism. Moreover, the proposed protocol is able to clearly distinguish between crystallographic and noncrystallographic poses of protein-ligand complexes, and also to find the transition state of the full undocking mechanism, thus giving an indication on the binding free energy.

The entrapment of two hydrophobic molecules, benzophenone and tamoxifen, into self-assembling cyclodextrin (CD)-based nanogels has been studied. These nanogels formed spontaneously upon the association of a hydrophobically modified dextran (MD) and a cyclodextrin polymer (pbetaCD). The interactions of benzophenone and tamoxifen with MD and pbetaCD were investigated using phase solubility studies, circular dichroism, and isothermal titration calorimetry. Both hydrophobic molecules were included into the CD cavities of the pbetaCD and were also solubilized by MD into its hydrophobic microdomains. We took advantage of these interactions to form benzophenone- and tamoxifen-loaded nanogels. The highest benzophenone loadings were obtained by solubilizing it in both pbetaCD and MD solutions before mixing them to form nanogels. These studies open new possibilities of applications of the nanogels, mainly in the cosmetic field, as sun screen carriers prepared by a simple "green" technology.

A label-free and time-resolved biosensor based on reflectometric interference spectroscopy (RIfS) has been developed to evaluate the agonistic or antagonistic effects of potential ligands with unknown behavior. The biosensor utilizes the specific interaction between the estrogen receptor alpha (ER alpha) and short specific peptides. The unique feature of these peptides allows the investigation of the behavior of ligands and the discrimination between the agonistic and antagonistic effects caused by conformational changes of the receptor. Thus, this developed biosensor allows not only the differentiation between ligands and nonligands of a receptor, but also the potential of these ligands to influence conformational changes in the receptor, leading to activation or inhibition of the receptor-dependent pathways. Owing to the robustness of the direct optical detection principle used, the biosensor is applicable to complex biological matrices, even crude cell extracts. Moreover, the reliability of the biosensor, including regeneration steps when performing subsequent measurements, has been verified.

Tamoxifen microcapsules and drug loaded medicated fabrics were investigated. The microcapsules were prepared using a complex coacervation procedure involving gelatin B and acacia gum. The morphology, particle size, drug loading capacity and in vitro release characteristics of the drug microcapsules were optimized for coating tamoxifen microcapsules onto the cotton fabrics. Infrared (IR) spectra and SEM were used to characterize the medicated fabrics and air permeability and laundering testing were undertaken to determine the efficiency and effectiveness of the system. Results showed that optimum condition for the microcapsules was at drug/polymer ratio 1:4, polymer concentration 3%, and rate of stirring 1000 rpm. In vitro release assays demonstrated that the tamoxifen was liberated over 10h after an initial bust rate period. SEM images illustrated that the tamoxifen microcapsules were spherical in shape and were also tightly fixed on to the cotton fabrics fast. These observations demonstrate that we have designed and fabricated a medicated system that potentially could be applied within a transdermal drug delivery system and so act in a system for the treatment of breast cancer.

This paper presents the optimization of instrumental and solution parameters for determination of tamoxifen in urine and plasma and formulation by fast Fourier transform square wave voltammetry (SWV) using a gold microelectrode in flow-injection system. The samples are subjected by the same buffer solution and are injected in the flow-injection apparatus. By applying a novel square wave voltammetry method to perform as a sensitive method the voltamograms are recorded. The method used for determination of tamoxifen by measuring the changes in admittance voltammogram of a gold ultramicroelectrode (in 0.05 mol L(-1) H(3)PO(4) solution) caused by adsorption of the tamoxifen on the electrode surface. The best sensitivity was achieved using a frequency of 600 Hz and a medium composed of 0.05 mol L(-1) phosphate buffers at pH 2.0. The best performance was obtained with the pH value of 2, pulse amplitude 25 mV, frequency 600 Hz, accumulation potential of -100 mV and accumulation time of 0.5s. Furthermore, signal-to-noise ratio has significantly increased by application of discrete fast Fourier transform (FFT) method, background subtraction and two-dimensional integration of the electrode response over a selected potential range and time window. Calibration plots are given for solutions containing 1.0 x 10(-11) to 3.0 x 10(-6)mol L(-1) of tamoxifen. The detection limit is calculated to be 3.0 x 10(-12)mol L(-1) ( approximately 2 pg mL(-1)). The relative standard deviation at concentration 2.0 x 10(-8)M is 6.1% for five reported measurements.

We report here the synthesis and cell-proliferation properties of derivatives of the breast cancer drug tamoxifen, in which the -O(CH(2))(2)N(CH(3))(2) side chain, responsible for the drug's antiestrogenic properties, has been modified by a ferrocenyl moiety. We recently reported the diphenol compound 5, in which this amino chain had been replaced with an acyl-ferrocenyl (-O(CH(2))(2)C(O)[(eta(5)-C(5)H(4))FeCp]) group, and which showed antiproliferative effects against both the hormone-dependent MCF-7 and -independent MDA-MB-231 breast cancer cell lines. We now report the results of a structure-activity relationship (SAR) study, in which the lateral chain length has been varied, the ketone group has been omitted, and the number of phenol groups has been varied. Compounds 1-4, with a side chain lacking the carbonyl function (-O(CH(2))(n)[(eta(5)-C(5)H(4))FeCp], n = 1-4) and which show a decreasing affinity for ERalpha (ER = estrogen receptor) with increasing chain length, act as estrogens on MCF-7 cells, and mild cytotoxics on PC-3 prostate cancer cells, with IC(50) values around 10 microM. The two monophenolic derivatives of 2, 2 a and 2 b, which show a reduced affinity for ERalpha compared to 2, are also estrogenic, but are only slightly cytotoxic. Finally, we have reexamined compound 5 and discovered that its antiproliferative effect against the MCF-7 cell line does not arise from antiestrogenicity as we had originally suspected, but by means of a cytotoxic pathway. This compound is also sensitive to the number of phenol groups as cell death is diminished when one of the hydroxyl groups is omitted (5 a and 5 b). Molecular modeling studies of the ligand-ERalpha binding stability are broadly consistent with the experimental binding affinity results for compounds 2, 2 a, 2 b, 5, 5 a, and 5 b. Electrochemical experiments show that 1-4, 2 a, and 2 b are stable to oxidation on the electrochemical timescale, unlike 5, 5 a, and 5 b, and that cytotoxicity is related to less positive phenol oxidation potentials. The SAR study shows that the presence of a ketone group and two phenol groups is necessary for strong receptor binding and cytotoxic effects, and that all compounds are estrogenic, despite the presence of a bulky side chain.

Tamoxifen is a nonsteroidal estrogen-receptor modulator widely used in the treatment of breast cancer. Apoptosis has been reported to be a major mechanism for its antitumor effect. In the current studies, an endeavor was made to investigate the efficacy of vesicularly encapsulated tamoxifen on human breast cancer MCF-7 cells. Phospholipid-based vesicular systems viz. conventional liposomes and elastic-membrane liposomes were employed to encapsulate the drug. The MTT colorimetric assay was used to determine the efficacy of the tested formulations. The results demonstrated composition-dependent strong inhibition in the viability of MCF-7 cells with encapsulated tamoxifen vis-à-vis free drug. The encouraging findings from the current work construe immense potential of the lipid-based vesicular systems in the treatment of breast cancer.

Cytochrome P450s (CYPs) exhibit a large plasticity and flexibility in the active site allowing for the binding of a large variety of substrates. The impact of plasticity and flexibility on ligand binding is investigated by docking 65 known CYP2D6 substrates to an ensemble of 2500 protein structures. The ensemble was generated by molecular dynamics simulations of CYP2D6 in complex with five representative substrates. The effect of induced fit, the conformation of Phe483, and thermal motion on the accuracy of site of metabolism (SOM) predictions is analyzed. For future predictions, the three most essential CYP2D6 structures were selected which are suitable for different kinds of ligands. We have developed a binary decision tree to decide which protein structure to dock the ligand into, such that each ligand needs to be docked only once, leading to successful SOM prediction in 80% of the substrates.

Although calmodulin (CaM) interaction with estrogen receptor alpha (ERalpha) has been known for more than two decades, it is only recently that the molecular mechanism of CaM-mediated regulation of ERalpha is beginning to emerge. Others and we have identified a putative calmodulin binding site (P(295)LMIKRSKKNSLALSTADQMVS(317)) in ERalpha, at the boundary between the hinge and the ligand binding domain. ERalpha mutations affecting its association with CaM have been reported to generate high basal, estrogen-independent transactivation activity, indicating that the P(295)-T(317) sequence has an inhibitory function. Moreover, we found that a synthetic peptide (ERalpha17p: P(295)-T(311)) containing residues crucial for CaM binding exerts estrogenic effects on breast carcinoma cells. Finally, computer-aided conformational studies revealed that the CaM binding site might associate with a region located downstream in ERalpha (the beta turn/H4 region), this association likely resulting in an auto-inhibitory folding of the receptor. Thus, we propose as a hypothesis that CaM acts as a positive regulator by relieving this ERalpha auto-inhibition.

The main objective of this study was to evaluate the ability of folic acid-functionalized diblock copolymer micelles to improve the delivery and uptake of two poorly water-soluble anti-tumor drugs, tamoxifen and paclitaxel, to cancer cells through folate receptor targeting. The diblock copolymer used in this study comprised a hydrophilic poly[2-(methacryloyloxy)ethyl phosphorylcholine] (MPC) block, carrying at the chain end the folate targeting moiety, and a pH-sensitive hydrophobic poly[2-(diisopropylamino)ethyl methacrylate] (DPA) block (FA-MPC-DPA). The drug-loading capacities of tamoxifen- and paclitaxel-loaded micelles were determined by high performance liquid chromatography and the micelle dimensions were determined by dynamic light scattering and transmission electron microscopy. Cell viability studies were carried out on human chronic myelogenous leukaemia (K-562) and colon carcinoma cell lines (Caco-2) in order to demonstrate that drug-loaded FA-MPC-DPA micelles exhibited higher cytotoxicities toward cancer cells than unfunctionalized MPC-DPA micelles. Uptake studies confirmed that folate-conjugated micelles led to increased drug uptake within cancer cells, demonstrating the expected selectivity toward these tumor cells.

Stable nanocolloids of insoluble drugs with very high drug content (up to 90% wt) can be easily and reproducibly prepared through the application of the layer-by-layer (LbL) technology, alternate adsorption of oppositely charged polyelectrolytes on the surface of drug nanoparticles produced by ultrasonication of larger drug crystals. Such polymeric coating prevents drug nanoparticle aggregation and creates a firm polymeric shell on their surface. Drug release rate from such nanocolloidal particles can be easily controlled by assembling multilayer shells with variable shell density and thickness. Various additional functions, such as specific targeting ligands, can be easily attached to the surface on nanocolloidal particles of poorly soluble drugs by using a polymer with free reactive groups for the "outer" coating. This may represent a novel approach to preparing convenient dosage forms of poorly soluble drugs.

Several recent studies from the author's laboratory have shown that tamoxifen, at higher concentrations than used for breast cancer and given i.v., can substantially prevent tissue infarct and behavioral deficits in reversible and permanent rat focal stroke models for up to at least 14 days after initiation of ischemia. Longer times and purely i.p. or oral administration have not yet been tried. Its marked effectiveness may be because it has several neuroprotective modes of action including free radical scavenging and, being highly lipid soluble, readily crosses the blood-brain barrier. Plus, it has a three hour therapeutic window. Thus it meets many of the STAIR criteria and should be a promising candidate for clinical use. However, a number of its positive effects were exhibited by the free radical trapping agent NXY-058, which also functions as a free radical scavenger. In two recently completed clinical trials (SAINT 1 and 2) NXY-058 had marginal positive effects and did not meet treatment criteria, respectively. Differences that may make tamoxifen still desirable and the problem of predicting clinical efficacy from successful animal studies are discussed.

The estrogens play important role in the homeostatic maintenance of several target tissues including those in the mammary gland, uterus, bone, cardiovascular system, and brain. Most of estrogen's action is thought to be mediated through its nuclear estrogen receptors, ERalpha and ERbeta, which are members of the nuclear receptor superfamily that act as ligand-induced transcription factors. Acting via its receptors, estrogen also plays an essential role in the development and progression of human breast cancer. The ER and progesterone receptor (PR), which are regulated by estrogen via ER, have been used as prognostic markers in the clinical management of breast cancer patients. However, the prognosis of a patient with ER+/PR+ breast cancer can be highly variable and a significant proportion of hormone receptor positive breast cancers does not respond to endocrine therapy. The identification of estrogen receptor target genes may improve our understanding of the role played by estrogens in breast cancer making it possible to better tailor hormone treatments and improve a patient's response to hormonal therapy. In this review, we explore the literature for data regarding the identification of estrogen receptor-regulated genes in breast cancer cell lines and breast tumor biopsies using high throughput technologies such as serial analysis of gene expression (SAGE) and cDNA microarrays.

This study examined the effect of morin on the bioavailability and pharmacokinetics of tamoxifen and its metabolite, 4-hydroxytamoxifen, in rats. A single dose of tamoxifen was administered to rats intravenously (2 mg/kg) or orally (10 mg/kg), with or without morin (3 or 10 mg/kg). The presence of morin significantly altered the pharmacokinetics of the orally administered tamoxifen. Compared with the oral control group (given tamoxifen alone), the total body clearance (CL/F) of tamoxifen in the presence of morin was significantly reduced (by 35.9-40.8%, p<0.01). The area under the plasma concentration-time curve (AUC(0-infinity)) and the peak plasma concentration (Cmax) of tamoxifen significantly (p<0.05 for 3 mg/kg of morin, p<0.01 for 10 mg/kg of morin) increased by 50.6-68.9% and 65.1-80.9%, respectively. Consequently, the absolute bioavailability (AB) of tamoxifen in the presence of morin was 37.4-40.5%, which was enhanced significantly (p<0.05) compared with the oral control group (23.9%). The relative bioavailability (RB) of tamoxifen was 1.56 to 1.68 times higher than the control group. The increased bioavailability of tamoxifen is likely to be due to the decrease in the first-pass metabilism by the intestines and liver. Morin at a dose of 10 mg/kg significant increased the AUC(0-infinity), of 4-hydroxytamoxifen (by 50.9%, p<0.05) but the metabolite:parent ratio (MR) of 4-hydroxytamoxifen was not altered significantly, suggesting that the formation of 4-hydroxytamoxifen is not affected considerably by morin. The increased bioavailability of tamoxifen in the presence of morin should be taken into consideration for dosage regimens due to potential drug interaction.

A novel modification of tamoxifen [(Z)-2-[4-(1,2-diphenyl-1-butenyl) phenoxy]-N,N-dimethylethylamine] citrate, tamoxifen hemicitrate hydrate was prepared. The crystalline form was identified and characterized by powder and single crystal X-ray diffractometries, differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), and hot-stage microscopy, and its physicochemical stability was also evaluated. The results of an elemental analysis, a single crystal X-ray analysis, and the TGA suggested that the molar ratio of tamoxifen:citric acid:water was 2:1:3 indicating it to be tamoxifen hemicitrate sesquihydrate. Simultaneous XRD-DSC measurements also indicated that two hydrates, sesquihydrate and hemihydrate, and an anhydrous form would exist during heating. The physicochemical stability of tamoxifen citrate forms A and B suspended in water and of form A during kneading and drying suggested that tamoxifen citrate was transformed into tamoxifen hemicitrate hydrate in water within 24 h, whereas tamoxifen citrate in a mixture with microcrystalline cellulose was quite stable during kneading. These results suggested that water and a mixture of water and organic solvent should be used for the manufacturing process with special attention paid to the transformation to tamoxifen hemicitrate sesquihydrate, because it showed a different stoichiometry from the active ingredient, tamoxifen citrate.

For the first time, two organometallic triphenylethylene compounds (Fc-diOH and DFO), with strong antiproliferative activity in breast cancer cells, but insoluble in biological fluids, were incorporated in two types of stealth nanoparticles (NP): PEG/PLA nanospheres (NS) and nanocapsules (NC). Their physicochemical parameters were measured (size, zeta potential, encapsulation and loading efficiency), and their biological activity was assessed. In vitro drug release after high dilution of loaded NPs was measured by estradiol binding competition in MELN cells. The influence of the encapsulated drugs on the cell cycle and apoptosis was studied by flow cytometry analyses. Notwithstanding potential drug adsorption at the NP surface, Fc-diOH and DFO were incorporated efficiently in NC and NS, which slowly released both compounds. They arrested the cell cycle in the S-phase and induced apoptosis, whose activity is increased by loaded NS. A decrease in their antiproliferative activity by the antioxidant alpha-tocopherol indicated that reactive oxygen species (ROS) may be involved. Therefore, nanosystems, containing for the first time a high load of anticancer organometallic triphenylethylenes, have been developed. Their small size and delayed drug release, combined with their enhanced apoptotic potential, are compatible with an increased persistence in the blood and a promising antitumour activity.

Hydroxyferrocifen compounds are a new and promising class of ferrocifen-type breast-cancer drug candidates. They possess both endocrine-modulating properties and cytotoxic activity, which come from the tamoxifen skeleton and the presence of a ferrocene moiety, respectively. However, they suffer from reduced solubility in water, which presents a problem for their eventual therapeutic use. Herein, we examined the interactions of hydroxyferrocifen compounds with cyclodextrins (CDs) to evaluate whether or not their electron-transfer oxidation pathways were affected by their inclusion. It has been demonstrated that these inclusion complexes are soluble in pure water, which shows that CDs can be used to deliver these biologically active molecules. Therefore, a series of these compounds has been investigated by cyclic voltametry in various media in the presence of CDs (beta-CD and Me-beta-CD). In methanol, the hydroxyferrocifen compounds exhibited a weak interaction with the CD cavity. These interactions became stronger as the amount of added water increased. The complexation effect between the hydroxyferrocifen compounds and beta-CD was found to be stronger if the CD was partially methylated, which is probably due to hydrophobic effects between the cyclopentadienyl ring and/or the aromatic rings and the methoxy groups. Moreover, it appears that the structure of the hydroxyferrocifen compounds affects both their solubility and their complexation dynamics. Investigations in the presence of pyridine show that the base kinetically favors the dissociation of the ferrocifen-CD complex during the electron transfer step, but does not affect the follow-up reactivity of the electrogenerated ferrocenium cation, which leads eventually to the corresponding quinone methide, as reported in the absence of CD. Accordingly, the cytotoxicity of these beta-CD-encapsulated organometallic complexes in hormone-independent breast-cancer cells (MDA-MB231) were confirmed to be similar to those obtained in the absence of cyclodextrin.

Two new synthetic pathways to the anti-cancer agent tamoxifen and its derivatives were developed. The first route involved the aldol reaction of benzyl phenyl ketone with acetaldehyde followed by Friedel-Crafts substitution with anisole in the presence of Cl(2)Si(OTf)(2) to produce 1,1,2-triaryl-3-acetoxybutane, a precursor of the tamoxifen derivatives. The second one utilized the novel three-component coupling reaction among aromatic aldehydes, cinnamyltrimethylsilane, and aromatic nucleophiles using HfCl(4) as a Lewis acid catalyst to produce 3,4,4-triarylbutene, that is also a valuable intermediate of the tamoxifen derivatives. The former strategy requires a total of 10 steps from the aldol formation to the final conversion to tamoxifen, whereas the latter needs only three or four steps to produce tamoxifen and droloxifene including the installation of the side-chain moiety and the base-induced double-bond migration to form the tetra-substituted olefin structure. This synthetic strategy seems to serve as a new and practical pathway to prepare not only the tamoxifen derivatives but also the other SERMs (selective estrogen receptor modulators) including estrogen-dependent breast cancer and osteoporosis agents.

Tamoxifen undergoes sequential metabolism to N-desmethyltamoxifen and N,N-didesmethyltamoxifen. Whereas N-desmethyltamoxifen is a major metabolite in humans, nonhuman primates, and rats, appreciable concentrations of N,N-didesmethyltamoxifen are formed in humans and nonhuman primates but not in rats. This difference in the extent of N,N-didesmethyltamoxifen formation may be important because it has been proposed that N,N-didesmethyltamoxifen inhibits the cytochrome P450 (CYP)-catalyzed alpha-hydroxylation of tamoxifen and resultant tamoxifen-DNA adduct formation. To test this hypothesis directly, we compared the extent of tamoxifen-DNA adduct formation in rats co-administered 27micromol N,N-didesmethyltamoxifen per kg body weight and either 27micromol tamoxifen per kg body weight or 27micromol alpha-hydroxytamoxifen per kg body weight daily for 7days. Female Sprague-Dawley rats treated with N,N-didesmethyltamoxifen had a 44% decrease (p >0.05) in CYP 3A2 content (the CYP isoform responsible for tamoxifen alpha-hydroxylation), an 18% decrease (p =0.010) in CYP 3A activity, and higher blood levels of tamoxifen and N-desmethyltamoxifen compared to rats treated with solvent. Total tamoxifen-DNA adduct levels were 4.1-fold higher (p <0.001) in rats given alpha-hydroxytamoxifen as compared to tamoxifen. N,N-Didesmethyltamoxifen treatment caused a 1.2-fold increase in total tamoxifen-DNA adduct levels with both tamoxifen and alpha-hydroxytamoxifen, a difference that was not significant. These results indicate that, with this experimental model, N,N-didesmethyltamoxifen does not impair the metabolism of tamoxifen to a reactive electrophile.

Tamoxifen (TAM) is an antiestrogen that has been widely used in the treatment and prevention of breast cancer in women. One of the major mechanisms of metabolism and elimination of TAM and its major active metabolites 4-hydroxytamoxifen (4-OH-TAM) and 4-OH-N-desmethyl-TAM (endoxifen; 4-hydroxy-N-desmethyl-tamoxifen) is via glucuronidation. Although limited studies have been performed characterizing the glucuronidation of 4-OH-TAM, no studies have been performed on endoxifen. In the present study, characterization of the glucuronidating activities of human UDP glucuronosyltransferases (UGTs) against isomers of 4-OH-TAM and endoxifen was performed. Using homogenates of individual UGT-overexpressing cell lines, UGTs 2B7 approximately 1A8 > UGT1A10 exhibited the highest overall O-glucuronidating activity against trans-4-OH-TAM as determined by Vmax/K(M), with the hepatic enzyme UGT2B7 exhibiting the highest binding affinity and lowest K(M) (3.7 microM). As determined by Vmax/K(M), UGT1A10 exhibited the highest overall O-glucuronidating activity against cis-4-OH-TAM, 10-fold higher than the next-most active UGTs 1A1 and 2B7, but with UGT1A7 exhibiting the lowest K(M). Although both N- and O-glucuronidation occurred for 4-OH-TAM in human liver microsomes, only O-glucuronidating activity was observed for endoxifen; no endoxifen-N-glucuronidation was observed for any UGT tested. UGTs 1A10 approximately 1A8 > UGT2B7 exhibited the highest overall glucuronidating activities as determined by Vmax/K(M) for trans-endoxifen, with the extrahepatic enzyme UGT1A10 exhibiting the highest binding affinity and lowest K(M) (39.9 microM). Similar to that observed for cis-4-OH-TAM, UGT1A10 also exhibited the highest activity for cis-endoxifen. These data suggest that several UGTs, including UGTs 1A10, 2B7, and 1A8 play an important role in the metabolism of 4-OH-TAM and endoxifen.

In this work, we introduce a four-step scoring and filtering procedure, furnishing target specific virtual screening (TS-VS), which serves to minimize false positives resulting from conformational artifacts of the docking process and is optimized to converge on novel chemotypes of estrogen receptor alpha (ERalpha). As a proof of concept, VS of a commercial compound database was undertaken (SPECs database release: Aug 2005, 202 054 compounds in total), resulting in the identification of both previously known and novel putative ER scaffolds. Application of distance constraints within TS-VS allowed facile identification of three novel active ligands with ERalpha binding affinities (IC50) of 1.4 microM, 57 nM, and 53 nM. Importantly, they all exhibited ERalpha over ERbeta selectivity, with the most selective being 17-fold. The ligands also displayed low micomolar antiproliferative activity (7-15 microM) in the human MCF-7 breast cancer cell line.

A new approach to the treatment of cancer is suggested, based on the innate overproduction of hydrogen peroxide in cancer cells. Hydrogen peroxide serves as a prodrug in the presence of transition metal ions, such as iron delivered by ferrocene. Under the effect of ferrocene, hydrogen peroxide is split into hydroxyl anions and highly reactive hydroxyl radicals. The latter cause oxidative DNA damage, which induces apoptosis, leading to elimination of cancer cells. Tamoxifen, a drug that interacts with oestrogen receptors, was used as a carrier to deliver ferrocene to breast cancer cells. For this aim tamoxifen conjugated to ferrocene (Tam-Fer) was synthesized. We have shown that the frequency of apoptotic events in MCF-7 breast cancer cells treated with Tam-Fer is significantly higher than in cells treated with tamoxifen or ferrocene separately. The increase of apoptosis correlates well with the rise in generation of reactive oxygen species in cancer cells. These results show that the hydrogen peroxide overproduced in tumour cells can serve as a prodrug for the treatment of cancer.

Macromolecular conjugates of tamoxifen could perhaps be used to circumvent some of the limitations of the extensively used breast cancer drug. To test the feasibility of these conjugates, a 4-hydroxytamoxifen analogue was conjugated to a diaminoalkyl linker and then conjugated to activated esters of a poly(methacrylic acid) polymer synthesized by atom transfer radical polymerization. A polymer conjugated to the 4-hydroxytamoxifen analogue with a six-carbon linker showed high affinity for both estrogen receptor alpha and estrogen receptor beta and potent antagonism of the estrogen receptor in cell-based transcriptional reporter assays. These results suggest that the conjugation of 4-hydroxytamoxifen to a polymer results in a macromolecular conjugate that can display ligand in a manner that can be recognized by estrogen receptor and still act as a potent antiestrogen in cells.